MICROBIOLOGY

1. Origins of Microbiology (ms7)

Micro small,
bios life,
logos science
Science studies and deals with microorganism and their activites
Medical microbiology is the study of interactions between human organisms & the microorganisms
which they co-exists.
Also include the study of the mechanisms of infection and the methods of specific therapy and
prophylaxis of infectious diseases.
History of microbiology
- The English scientist Robert Hooke is the first person to use a microscope for academic study in
the early 1660's. Hooke studied plant sections, in particular cork and he drew what he saw, which
was a matrix of tiny cylindrical-like structures he called cells
- Antonie van Leeuwenhoek in the Netherlands, was using microscopes to look at animal and plant
tissue. He examined a drop of rainwater and noticed it contained tiny creatures,which he called
"animalicules" or little eels. These were in fact bacteria and so van Leeuwenhoek became the first
person to study bacteria.
- in 1745, John Needham shows that boiled broth that cools down overnight becomes richly
contaminated with microorganisms. He forcefully argues the microbes must be borne from the broth.
He publishes a formal presentation of the Theory of Spontaneous Generation
- Lazzaro Spallanzani disputed the theory by showing that boiled broth would not give rise to
microscopic forms of life. He found that boiling broth would sterilise it and kill any microorganisms
in it. He also found that new microorganisms could settle only in a broth if the broth was exposed to
the air
- Louis Pasteur expanded upon Spallanzani's findings by exposing boiled broths to the air in vessels
that contained a filter to prevent all particles from passing through to the growth medium. By boiling
the broth beforehand, Pasteur ensured that no microorganisms survived within the broths at the
beginning of his experiment. Nothing grew in the broths in the course of Pasteur's experiment. This
meant that the living organisms that grew in such broths came from outside, as spores on dust, rather
than spontaneously generated within the broth. Thus, Pasteur dealt the death blow to the theory of
spontaneous generation and supported germ theory instead.
- Ferdinand Julius Cohn was a German biologist. His classification of bacteria into four groups
based on shape (sphericals, short rods, threads, and spirals) is still in use today
- In 1876, Robert Koch established that microbes can cause disease. He found that the blood of
cattle who were infected with anthrax always had large numbers of Bacillus anthracis. Koch found
that he could transmit anthrax from one animal to another by taking a small sample of blood from the
infected animal and injecting it into a healthy one, and this caused the healthy animal to become sick.
He also found that he could grow the bacteria in a nutrient broth, then inject it into a healthy animal,
and cause illness.
- Martinus Beijerinck (one of the founders of virology) made two major contributions to
microbiology: the discovery of viruses and the development of enrichment culture techniques. While
his work on the Tobacco Mosaic Virus established the basic principles of virology, it was his
development of enrichment culturing that had the most immediate impact on microbiology by
allowing for the cultivation of a wide range of microbes with wildly different physiologies.

- 1890 Sergei Winogradsky (the founders of general microbiology) performs the definitive work on
the microorganisms responsible for nitrification in nature. He was the first to develop the concept of
chemolithotrophy and to thereby reveal the essential role played by micro-organisms in geochemical
processes. He was responsible for the first isolation and description of both nitrifying and nitrogenfixing bacteria.
Modern medical microbiology become an extensive science, which is subdivided into
Bacteriology the science of bacteria, the causative agents of a number of infectious
diseases
Virology the science of viruses, non-cellular living systems capable of causing infectious
diseases in man
Immunology the science which is concerned with the mechanisms of body protection
against pathogenic microorganism, & foreign cells and substances
Mycology the study of fungi pathogenic for man
Protozoology deals with pathogenic, unicellular animal organisms
Ecology is the study of microbial flora for human body, infection, immunity

2. Taxonomy and classifications of microbes (ms8-9)

Taxonomy is the science of classification.
Classification is an orderly arrangement of bacteria in groups.
Identification is the practical use of classification to isolate and distinguish desirable organism from
undesirable ones.
Nomenclature is the means through which the characteristics of a species are defined and
communicated among microbiologists.
Methods/principles of classification of microorganisms
1) Adansonial/numerical classification
Method which large number of biochemical, morphological, and/or cultural
characteristics are used to determine the degree of similarity between microorganisms.
o Scoring large number of phenotypic characteristics, possible to estimate similarity
coefficient when shared positive characteristics are considered.
2) Molecular/genetic classification
Based on degree of genetic relatedness of different organisms
o Parameters are used to determine DNA relatedness - genome size, guanine plus
cytosine (G + C) content, DNA relatedness under conditions optimal for DNA
reassociation, thermal stability of related DNA sequences and DNA relatedness
under supraoptimal conditions for DNA reassociation.
o G + C content for any species is relatively fixed/within very narrow range (basis
for classification)
No methods universally accepted.
The most widely adopted from Bergeys Manual of Determinative bacteriology.
Bacteria in kingdom of Prokaryotae & divided into 35 groups on basis of morphology,
Gram reaction, oxygen requirement, spore formation, metabolic pattern.
35 parts are further divided into classes. Orders, families, genera, species, subspecific
categories (biotypes, serotypes, phage types, colicin types)
Linnaean principles
Developed by Swedish scientist, Carl Linnaeus
Microorganisms are named according to standard rules of biological taxonomy and
nomenclature
Species is designated by Latin binomial, the first term is the genus
eg: Escherichia (genus) coli (species)
other term
pure culture : a population of bacteria consisting from the microbes of one and the same species
clone : the cells derived from single cell
strain : a population of bacteria derived from a particular source (a patient)

3. Morphology of microorganisms (bacteria, actynomyces, spirochaetes, rickettsiae,

fungi). Major characteristics of Eukaryotes and Prokaryotes (ms16-17, 10-11)
Microorganisms are living forms that are microscopical in size and relatively simple (unicellular) in
str.
3 main kingdoms
1- Vira : study viruses (no cellular organization, are particles, has several classification)
2- Prokaryotes : cellular organism, no isolated nucleus (absent nuclear membrane)
3- Eukaryote
: cellular microorganism, isolated nucleus, more complex str
GROUPS OF PROKARYOTES
Actynomyces
Belong to group 22-29 of Bergeys Manual (BM)
Usually form branching filaments (D = 0.5 1.0 m), which may fragment into irregular
sized elements/remain stable & produce arthrospores
Spores : usually nonmotile, some genera produce flagellate spores
Visualized by dark field microscopy, in stained smears (by simple, Gram, Ziehl-Neelsen
methods)
Gram + , acid fast
Some are pathogenic to humans
Cause mycetoma
Spirochetes
Belong to group 1 of BM
Helically shaped, motile bacteria. 0.1-3.0 x 5-250 m
Unicellular, Gram
Outmost str is a multilayers outer membrane,
- refer as outer sheath/outer cell envelope
- completely surrounds protoplasmic cylinder
Protoplasmic cylinder
- consists of cytoplasmic & nuclear regions
- surrounding cytoplasmic membrane-cell wall complex
Periplasmic flagella (motile move in corkscrew fashion)
- around the protoplasmic cylinder
- periplasmic : component of motility apparatus of the cell
- permanently wound around cell body & entirely endocellular, being enclosed by outer
sheath
Visualized by dark field microscopy, in stained smears (silver impregnation by Morosov,
Romanovsky-Giemsa, Burry methods)
Three genera (Treponema, Borrelia, Leptospira) are responsible for human disease
- T. pallidum : causative agents of syphilis
- B. recurrentis : causes epidemic relapsing fever
- L. interrogans : leptospirosis
Rickettsia
Belong to group 9 of BM
Obligate intracellular parasites (survive only inside body)
Multiply by binary fission
Cell walls contain muramic acid
Mainly rod-shaped, coccoid, pleomorphic (ability to alter their shape/size in response to
environmental conditions) bacteria, small Gram , lack flagella (nonmotile)

 Visualized by Zdrodovsky stain

Parasitic species are associated with the reticuloendothelial & vascular endothelial cells /
eryhtocytes of vertebrates, various organs of athropods which acts as vectors/primary hosts
(grow only in eukaryotic cells; laboratory cultivation requires the use of tissue culture cell
lines, yolk sacs of embrionated eggs/experimental hosts (guinea pigs/mice)
Human is accidental hosts
Cause epidemic thypus (R. prowazekii), murine thypus (R. typhi), Q fever (Coxiella
burnetii), Rocky Montain spotted fever (R. rickettsii)
Treatment : tetracyclines/chloramphenicol (both are rickettsiastatic & allow ppts immune
system to respond & control infection)
Prophylaxis : live vaccine for epidemic typhus
GROUP OF EUKARYOTE
Fungi (ms 99-101)
Exist as saprophytes, parasites,commensals
Eukariotic prostista & different from bacteria,other prokaryotes in many ways
Has rigid cell walls contain chitin,mannan,other polysaccharides
Cytoplasmic membrane has sterols
Has mitochondria, Golgi apparatus, ribosomes bound to endoplasmic reticulum, a
cytoskeleton w microtubules,microfilaments,intermediate filaments
Has true nuclei w nuclear membrane & paired chromosomes
Divided asexually, sexually,or both
May be unicellular/multicellular
Depend on morphology, divide into
o Yeast
- unicellular fungi, reproduced by simple budding, Cryptococcus neoformans is the
only pathogenic yeast
o Yeast-like fungi
- grow partly as yeast & partly as elongated cells resemble hyphae, form
pseudomycelium, Candida albicans is pathogenic
o Moulds/filamentous fungi
- form true mycelia, reproduced by formation of different types of spores,
Dermatophytes are patogenic
o Dimorphic fungi
- occur as filaments (in soil,culture at 22C)/as yeasts (in host tissue/culture at 37C)
depend on conditions of growth, cause systemic infections
Cause fungal infections (mycoses)
- classified by areas of body (superficial (epidermis), cutaneous (deeper in epidermis),
subcutaneous (in dermis & subcutaneous tissues), systematic (infect internal of
body)mycoses)
Major characteristics of Eukaryotes and Prokaryotes
Characteristics
Major groups
Size (approx..)
Nuclear structures
Nucleus
Chromosomes
Ploidy
Cytoplasmic structures
Mitochondria

Eukaryotes
Algae, fungi, protozoa,
plants, animals
> 5 m

Prokaryotes
Bacteria
0.5 to 3 m

Classic membrane
Strands of DNA
Diploid genome

No nuclear membrane
Single, circular DNA
Haploid genome

Present

Absent

Golgi bodies
Endoplasmic reticulum
Ribosomes (sedimentation
coefficient)
Cytoplasmic membrane
Cell wall
Reproduction
Movement
Respiration

Present
Present
80S (60S + 40S)
Contains sterols
Absent/composed of chitin

Absent
Absent
70S (50S + 30S)

Does not contain sterols

Complex str. Contain protein,
lipid, peptidoglycan
Sexual, asexual
Asexual (binary fission)
Complex flagellum, if present Simple flagellum, if present
Via mitochondria
Via cytoplasmic membrane

4. Study of morphology of bacteria, methods of microscopical examination. Simple

and complex staining methods (ms 18-20)
Bacteria

: unicellular prokaryotic microorganism without chlorophyll

: depend on shape, classified into
1. Coccus (spherical/oval shape)
-according to arrangement
: micrococcus (single)
: diplococcus (pairs)
: streptococcus (chains)
: staphylococcus (irregular groups)
: tetrococcus (group of 4)
: sarcina (packet-like of 8,16,32)
2. Rods
: bacilli produces spore
: bacteria non-sporing bact
3. Spiral shape
-subdivided into number of spiral
: vibrio (one-comma shape)
: spirillum (few)
: spirochetes (many)

Morphological study of bacteria requires the use of microscopes.

TYPES OF MICROSCOPES
1) Brightfield/ light microscopy
- consist of light source : to illuminate specimen on a stage,
- condenser : to focus the light on the specimen,
- two lens systems (objective & ocular lens) : to magnify the image of the specimen
-specimen is visualized by transllumination, with light passing up through condenser to the
specimen
- total magnification of the image is the product of magnifications of the objective and ocular
lens
- best LM have resolving power of approx.. 0.2m, which almost most bacteria, not viruses to be
visualized
- organisms must be stained with a dye to be observed
- examination of stained smears indicates the shape, arrangement, approx. size of the cells
(sufficient to identify bacterium)
2) Darkfield/darkground microscopy
- use reflected light
- darkfield condenser illuminate the object with a cone of light without letting any ray of light fall
directly on the objective lens which may cause object appears self-luminous against a dark
background
- contrast increase the resolution, so very slender organisms (spirochetes) can be clearly seen
-its resolving power is improved in 10x compared with LM
3) Phase contrast microscopy
- enables internal details of living microbes to be examined

- use parallel beams of light to pass through objects of different densities, the beam moving
through the more dense material is retarded more than the other beam
- annular rings in condenser and objective lens are used to amplify the differences in phase so
that in phase light appears brighter than out of phase light
- create 3D image which permit more detailed analysis of the internal structures
- may demonstrate motility, spores, intracellular granules
4) Fluorescent microscopy
- fluorochromes absorb short wavelength ultraviolet/ultrablue light and emit energy at a higher
visible wavelength
- involve staining organisms with fluorescent dyes and then examining them with a specially
designated fluorescent microscope
- microscope uses a high pressure mercury,halogen/xenon vapor lamp that emits by LM
- a series of filters are used to block heat generated from the lamp, eliminate infrared light, select
appropriate wavelength for exiting the fluorochrome
- light from fluorochrome is magnified through objective & ocular lenses
- specimens stained with fluorochromes appear brightly illuminated against a black background
5) Electron microscopy
- use electron beam, which is focused by circular electromagnets
- object in the path of beam scatters the electrons & produces an image on a fluorescent-viewing
screen
- can see individual viral particles as resolving power is 0.1nm
- 2 types : transmission EM (particles pass directly through specimen), scanning EM (particles
pass through specimen at an angle & produce 3D picture)
- used for visualizing of viruses & fine structure of bacterial cell
Methods of microscopical examination
1. Unoil object-plate w soap
2. Anneal bacterial loop & transfer 2-3 drops of physiological sol on glass
3. Anneal bacterial loop, burn the mouth of test tube,take microorg from culture medium surf
4. Introduce microbe into physiological sol & spread it on glass surf
5. Dry the smear
6. Fix it above burner flame (for attachment to glass, for killing pathogenic microbes, for best
staining)
Microbes are colourless, thus need to stain
STAINING METHODS
1) Simple staining
- impart the same colour (single dye is used) to all bacteria & other biological material
- apply watery solution of basic dye (methylene blue, gentian violet,basic fuchsin)
- coloured body correspond to cell protoplasm only, cell wall is not stained by ordinary methods
- see microbes, their form, dimensions, cells location
- certain bacteria not colour with simple stains
-method : dye is put on for 1-2 min, then wash w water, dry up, microscope
2) Negative/background staining
- a rapid method for simple morphological study of bacteria
- use Indian ink/nigrosine, yields a dark background in which the organisms stand out as bright,
unstained objects (Burry method)
3) Silver impregnation methods
- to stain spirochetes especially in tissues
- slender cells are thickened by a dark deposit of silver on their surface

4) Complex staining methods

- impart distinctive colour only to certain types of bacteria
- contain several stages & apply different dyes, decolourizing agents & other reagents
- for study bacterial cell surf
- 2 groups of methods :
* differential staining rxn, which distinguish one groups of bacteria from another (Gram
stain/acid fast stain (Ziehl-Neelsen method))
* methods for detection of different structures of bacterial cell
- spores are visualized by modified acid fast stain (Oscheshko method),
- capsule of bacteria by Burry-Gins stain,
- bacterial nucleus by Romanovsky-Giemsa method,
- volutin granules by Neissers method
* Neissers method
- in Diphtheria bact to stain volutin granules
-1. stain smear w acetic methylene blue for 1 min
2. pour off & wash w water
3. flood w Lugols iodine for 30 sec
4. stain w vesuvin for 1-3 min
5. wash & dried up

Volutin are stained w blue/black & cytoplasm w yellow

* Burri-Gins method (for spirochetes)

-1. a drop of Indian ink on object-plate
2. introduce microbe w sterile loop & mix w dye
3. distribute layer to thin layer w help of 2nd glass
4. dry it & flood w 1% of muriatic alcohol solution for some seconds
5. after burning of alcohol above flame, conterstain smear w fuchsine for 1-3min
6. washed w water, dried & microscoped
- red rods surrounded by colourless capsules are visible against dark background

* Ziehl-Neelsen stain
- for differentiating acid-fast bact (bacilli of tuberculosis, leprosy, actinomycete) & spores
1. cover smear w carbol fuchsin(red), heat w flame until it streams & keep it stream for 510min
2. treat w 3-5% sulphuric acid sol/acid alcohol (3% HCl in 95% ethanol/5% H2SO4) & leave
for 5-10min
3. wash w water (acid-fast bact retain original stain-red)
4. counterstrain w methylene blue (blue) for 3 min
5. wash w water & dry
- Acid-fast bact & spores retain red stain while non-fast bact /vegetative part of bacilli are stained
blue
- Acid-fastness in bact is related to presence of large amount of lipid,waxes,arid oxyacid

*Grams method
- depend on cell wall composition
1. stain smear w methyl violet for 60 sec
2. then, w Lugols iodine sol for 30 sec in excess
3. decolorized w alcohol for 30 sec (gram lose violet color)
4. counterstain w fuchsin for 60 sec
5. wash w water & dry
- Gram + (staphylococci, streptococci) are violet due to peptidoglycans. Gram (gonococci,
meningococci, brucella, E.coli, salmonella, cholero vibrio) are red

5. Bacterial ultrastructure. Chemical composition of bacteria (ms11-15)

CELL WALL
Rigid str surround bacteria
Responsible for form of bacteria cell & place of fundamental role in live activities of bacteria
10-25nm in thickness, 20-30 of dry weight
Chemical nature in Gram + , bacteria

Gram

Cell wall : thinner, monolayer, 2-3nm

thick
Contain inner layer of peptidoglycans (110%)
Outer layer link together by B-lipoprotein
o Composed of lipopolysaccharides
(mainly, endotoxin), protein,
phospholipids
o Protein (outer membrane protein)
embedden in phospholipid bilayer
which is attached to
lipopolysaccharides
o No teichoic acids
o Present periplasmic space (space
btween cell membrane (inner) &
cell wall (outer))
stain w red
F(x)

Gram +

Cell wall : 16-18nm thick, multilayer

Component is mucopeptide/ peptidoglycans
Compose of acetylmuromic acid &
acetylglucosamine link together by 1-4
linkage
Peptidoglycans is major component (50-80%)
associated with polysaccharide & special
class of polymers (teichoic acids -consist
major surface antigen in Gram + bacteria)
stain w violet

: give rigidity & shape to cell

: provide mechanical support to cell membrane
: plays role in virulent & immunity (target site for antibiotic)
: help to maintain osmotic pressure
: protect cell against damage
: lipopolysaccharides (LPS) (for Gram )
- have endotoxic activity/antigen specificity
- lipid A of LPS have endotoxic act like pyrogenesity (temp. rxn), lethal
effect, tissue necrosis, antitumor activity

: provide site phage absorption

: take part in cell division
*Demonstration of cell wall
Grams method of staining to study cell wall (due to diff composition)
o + > stain with violet
o > stain with red
*LPS consist of 3 structural sections Lipid A (for endotoxic activity), Core polysaccharide (for LPS
str, same for a species of bact), O antigen (distinguish serotypes of bacterial sp)
CELL MEMBRANE (cytoplasmic membrane)
Thin, elastic, 5-10nm, semipermeable layer, link with inner surface of cell wall
Typical unit membrane str compose of phospholipids & proteins molecules
Contain small amount of carbohydrates but no steroles & carrier molecules
Enzymes situated inside : oxydase, polymerase, permease
F(x)

: as semipermeable membrane & control inflow & outflow of metabolites

: selectively allow passage of nutrient inside & waste products outside cell
: as center of respiration activity to generate ATP because contain cytochromes &
other enzymes necessary for respiration (TCA cycle)
: participate in synthesis of cell wall component pepsidoglycans
CELL ENVELOPE
Cell wall + cell membrane
To demonstrate
o Use plasmolysis experiment
o When bacteria cell placed in hypertonic solution of salt & cytoplasm shrinks by lose
water while cell envelope return original shape & size
o Stain by simple method (fuchsin, methyl violet)
CYTOPLASM
Colloidal system contain variety organic & inorganic solutes in a disposed watery solutions
Contain ribosomes, mesosomes, inclusion granules, vacuoles
Absent endoplasmic reticulum, mitochondria
Ribosome
Globular str composed of RNA & proteins
Made up of 2 subunits : larger (50S), smaller (30S)
10-15nm in size w sedimentation coefficient of 70S
F(x) : site for protein synthesis
Mesosome
Complex in folding of cell membrane
Compose of invaginative cell membrane w many vesicles/tubules filling the invagination
Chemically similar to cytoplasmic membrane w few other properties
More prominent in Gram + & also found in Gram
2 types :
o Septal
Attach to bacterial chromosomes
Involve in DNA segregation & in formation of binary fission (cross walls)
o Lateral
F(x) : center of respiratory activity

- possess respiratory enzymes (ATP-ase, hydrogenase, cytochrome)

- as mitochondria absent
: take part in cell division
Inclusion granules
Non-living bodies deposited in cytoplasm
Non-permanent & essential str when large amount of nutrient present & disappear under
condition of starvation
Eg : lipid granules stain by Sudan (red)
: volutin granules stain by Nessers ( eg:Metaphosphate &
polyphosphatin
nature)
: starch (blue), glycogen (brown) stain by iodine solution
: inorganic granules (eg:sulphur)
F(x)
: store energy & present in some bacteria
: have diagnostic significant
Nucleoid
Not well develop
Without nuclear membrane, without nucleolus
Contain single circular molecule of double-stranded DNA
Free from basic proteins such as histones
1m in length
F(x)
:control frowth & metabolism of cell
: multiplication of cell
Plasmids/episomes
Extranuclear genetic elements
Consist circular double stranded DNA
Encoded non-essential str for life (may present/absent in cell)
confer on certain properties like drug resistance and toxigenecity.
Can be transmitted from one bacteria to another by conjugation or by bacteriophage
5 types
: r-plasmid (resistance to antibiotic or poisons)
: f-plasmid (fertility of bacteria)
: Col plasmid (produce bacteriocins, proteins that can kill other
bacteria)
: Degradative plasmids (for digestion of unusual substances, e.g.
toluene and salicylic acid)
: Virulence plasmids (turn bacterium into a pathogen)
CAPSULE
- types : macro : has width > 0.2m, demonstraded by LM
: micro : has width < 0.2m, only by EM
- chemical nature
: water is main component (98%)
: solid (1-2%) carbohydrates, polysaccharide
- f(x) : enhances bacterial virulence by inhibiting phagocytosis
: acts as protective covering against antibacterial action of substances (lysozyme,
bacteriophage, antibodies, antibiotic)
: acts as antigen, helps in identification & types of bacteria
- demonstration : by negative staining using Indian ink & Burigins method
FLAGELLA
- thread-like str arise from cytoplasm & extend out through out cell wall
- f(x) : movement

- types : monotricad single flagella at one end

: amphitricad single flagellum at both ends
: lophotricad have tough flagella at one end
: peritricad flagella around cell
- chemical nature
: consist of protein flagiden
: organ of locomotion
: has antigen significant because flagella is Hantigen
- demonstration by EM by special staining by silver impregnation & by dark film microscopy & by
Heigen
PILI (FIMBRIAE)

-hair like appendages projecting from cell surface as straight filaments

-types : general/regular
- organs of adhesion/attachment
: sex
- transfer genetic material cell from donor by conjugation
-protein in nature & consist protein pilin
-usually Gram negative bacteria
-demonstration:EM, haemagglutination
SPORES
Dehydrated, multishelled str, that protect & allow bact to exist in suspended animation
Contain complete copy of chromosome, essential protein,ribosome. High conc of calcium bond to
dipicolinic acid
Spore has inner membrane, 2 peptidoglycan layers, outer-keratin-like coat
The str protect genomic DNA from desiccation, intense heat, radiation, enzyme & chemical attack
Acid fast staining
Use Oscheshko stain (modified acid fast stain) to examine vegetative cells & spores
Spores are bright red, vegetative part of bacilli are blue

6. Physiology of microorganisms : nutrition of microbes, microbial growth,

bacterial metabolism. Respiration of bacteria (ms22-31)
NUTRITION OF MICROBES
The process by which chemical substances (nutrients) are obtained from surrounding environment &
used for metabolic activity & growth of cell
Bacteria require 2 categories of essential nutrients
1. Macronutrients
- required in large quantity & play important role in cell str & metabolism
2. Micronutrients
- required in small quantity for functioning of certain enzyme systems
For optimum growth, bacteria require : water, source of carbon, source of energy, inorganic salts,
growth factors
Water
- most important requirement & constitute 80% of total weigth
- as vehicle for entry of all nutrients into cells * for elimination of cell waste products
-participate in metabolic rxn
Form integral part of protoplasm
Source of carbon
- according to it, divide bacteria into
1. Autotrophs draw carbon from CO2 (inorganic compounds)
2. Heterotrophs carbon from organic compounds
- saprophytes : live on dead organic matter
- parasites
: use nutrients from living host & cause damage
: all pathogenic microorg. are parasites
3. Mixotrophic carbon from both organic & inorganic compounds & by fixing CO2
Energy source
- phototrophs : use energy of sunlight
- chemotrophs : obtain energy from oxidation-reduction of external chemical
compounds (chemical process)
These requirements can be combined:
- photoautotrophs
: light energy,carbon from inorganic compounds
- photoheterotrophs : light energy,carbon from organic compounds
- chemoautotrophs
: energy from chemical compounds, carbon from CO2
- chemoheterotrophs : energy from chemical compounds, carbon from organic
compound
Inorganic salts
1.Essential elements
- for synthesis of bacterial structural compounds (carbohydrate, lipid,
protein,
nucleic acid) hydrogen, oxygen, carbon, nitrogen, phosphorus, sulphur
2.Trace elements
- incorporated into structure of active groups of some enzymes potassium,
calcium, sodium, magnesium, iron, manganese
- potassium : exert catalytic action, activate enzyme system

- calcium : participate in nitrification, nitrogen fixation by soil microorg, in production of

gelatinase
- phosphorus, sulphur, magnesium, iron : great importance in life process of bacteria; iron in
respiratory enzymes & acts as catalyst
Growth factors
- essential factors (bacteria cant synthesize by themselves)
- not grow in their absence
- eg : nutrients vitamins, purines, pyrimidines, amino acids
- penetration can be passive (w/o energy, by ordinary/facilitated
diffusion) / active (w help of enzyme permease)
: physical requirements environment fctors (moisture & desiccation,
gases (O2,CO2), tempt, pH, light)
Moisture & desiccation
- absolutely required
- to survive in dry environment
- Gonococci, T.pallidum dry quickly in dry cond. , Staphylococcus aureus, Tubercle
bacilli survive in dry
Oxygen requirements
- obligate aerobes : must presence air (21% O2)
- microaerophilia bacterium : grow well in low conc. Of O2 (3.5%), killed by higher
conc. of O2
- facultative anaerobe : perform both fermentation & aerobic respiration, can survive
in O2 presence
- obligate anaerobe : not carry out oxidative phosphorylation, killed by air, lack
certain enzymes cytochrome & cytochrome oxidase, catalase, peroxidase,
superoxidase dismutase
CO2
- bacteria require small amount of CO2
- obtain from atmosphere/CO , produced endogenous by metabolism
- need additional CO2 (capnophilic bacteria)
Temperature
- pathogenic bact grow best in body temp (37C)
- optimum temp is higher (42C ampylobacter jejuni), lower (30C Yersinia
pestis)
- subdivided into
- psychrophiles
- optimal 15-20C
- not grow above 20C
- most : soil & water saprophytes
- mesophiles
- moderate tempt (20-40C)
- majority : pathogenic organism
- thermophiles
- high tempt (45-80C)
- most : spore forming (Bacillus, Clostridia) live in soil,
water, pathogenic to human
- extremely thermophilic bact grow at 250C
pH

- affect growth & multiplication of bact

- optimum pH (7.2-7.6). poor pH (6.0 or above 7.8)
- stops at pH below 5.0, above 9.0
- some bact grow at acidic pH (Lactobacillus sp) known as acidophilic bact
- Vibrio cholerae are sensitive to acid but tolerate w alkali
Light
- grow well in dark
- sensitive to Uv rays, radiation
- photosynthetic bact (phototrops) require light
- photochromogenic mycobacteria produce pigment only when expose to light
Osmotic effect
- due to mechanical strength of cell wall, able to tolerate osmotic
variations but sudden exposure to hypertonic solution cause plasmolysis
(shrinkage of protoplasm)
- sudden transfer to distilled water cause plasmoptysis (swelling &
rupture)
MICROBIAL GROWTH
- Bacterial cells are metabolically active.
- When growth reaches a critical mass, cell division (binary fission) occurs
- Mode : binary fission
- Bacterial cell division
- replication of chromosome
- cell wall extension
- septum formation
- membrane attachment of DNA pulls into new cell
- Bacterial growth curve : when bacterium is added to suitable liquid medium & incubated, its
growth follows definite course (4 phases)
: Lag phase
- no increase in number, but increase in size of cell
: Log/exponential phase
- cell start dividing, number increase expotentially
: Stationary phase
- cell division stops due to depletion of nutrients & accumulation of toxic
subst
- equilibrium exist between dying cells & newly formed cells, so viable
count remain stationary
: Decline phase
- population decrease due to death of cells by autolytic enzymes,
nutritional exhaustion, toxic accumulation

2 types of growth curve acc. to measurement of cell numbers

1. Total count
-based on number of cells present, irrespectively they are living/not
2. Viable count
-measures only those cells capable of growing & produce a colony on a
suitable growth medium
-express as colony-forming units, not absolute numbers of bacteria
Generation time
- interval time between 2 cell divisions
- time required for bacterium to give rise to 2 daughter cells under optimum condition
- also call population doubling time
- eg: Escherichia coli (20min), Mycobacterium tuberculosis (20hours), Mycobacterium leprae
(20days)
BACTERIAL METABOLISM
- Bacterial metabolism is a complex of all reactions realized in bacterial cells.
- The main goal of all these biochemical reactions is the yield of energy and building material.
-All microorg require constant supply of ATP energy (derived from controlled breakdown of various
organic substrate) to survive
* catabolism : process of substrate breakdown & conversion into usable energy
* anabolism : process of energy produced is used to synthesize cellular constituents
* metabolism : combination of catabolism & anabolism, which are interrelated
- energy primarily produced by fermentative, respiratory, autotrophic metabolism
1. Fermentative metabolism
- use organic compounds as both electron donors, acceptors
- major pathway is glycolytic/Embden-Meyerhof, which converts glucose to pyruvate (1
glucose:2 pyruvates)
- glycolysis yields 2 ATP molecule per 1 glucose molecule
- pyruvate is used in secondary fermentation processes (substrate-level phosphorylation) that
result in production of CO2, H, or methane gas during breakdown of pyruvate
- simplest secondary fermentation process is lactic fermentation, which convert pyruvate to
lactate
- in alcoholic fermentation, pyruvate is converted to CO2 & ethanol
- in propionic fermentation, pyruvate is converted to oxaloacetate w addition of a CO2 and
eventually, to propionic acid
- mixed acid fermentation is a combination of lactate, acetate, formate production, which
produce CO2, H, and ethanol
- alternative pathway to metabolize glucose:Entner-Doudoroff pathway (major hexosedegrading pathway in pseudomonas)

2. Respiratory metabolism
- use O2 as electron donor
- in aerobic resp,pyruvate is converted to CO2 & acetyl-CoA
- complete respiration yields 38 ATP for 1 glucose
- many bacteria lack the enzymes needed for complete oxidation, so they obtain energy from
incomplete oxidation
- incomplete oxidation is facilitated by electron transport syst (ETC), where cytochrome ,
flavoproteins, ubiquinones(coenzyme Q) as major elements
- energy is used to extrude a pair of protons (H+) across an insulating membrane, thus create
electrochemical gradient
- return of protons through membrane reverse ATPase activity and refer as oxidative
phosphorylation

3. Autotrophic metabolism
- use variety of alternative inorganic sources for energy & reducing power
- eg: Autotrophic bacteria uses carbon dioxide as the main source of carbon.
- Energy is obtained in these microorganims by the oxidation of inorganic compounds or
from sunlight (most common pathways: reductive pentose phosphate cycle, reductive tricarboxylic
acid cycle, and acetyl-CoA pathway)
- Autotrophic behaviour depends on the ability of the cell to carry out photosynthetic or
aerobic respiratory metabolism, which are the only processes able to deliver sufficient energy
to maintain carbon fixation.
- in anaerobic resp, bacteria use inorganic subst as terminal electron acceptors in place of
oxygen (TCA cycle)
RESPIRATION OF BACTERIA (ms 23)
- bacteria have wide range of responses to oxygen
* strict aerobes
: must have oxygen to grow
: eg Mycobacterium tuberculosis
* facultative anaerobes
: medically important can grow whether or not O2 is present
: eg E.coli
* strict/obligate anaerobes
: cant grow in presence of O2
: eg Clostridium ssp, Bacteroides ssp, Peptococcus ssp
* microaerophilic
: grow best in presence of trace (5%) free O2, often prefer an
increased concentration CO2
: eg Campylobacter
- difference in response to O2 show the way bacteria oxidase substrates to obtain energy
* strict aerobes carry out respiration only, final electron acceptor in oxidation-reduction series is
molecular O2
* strict anaerobes carry out fermentation, final H acceptor is organic molecule (pyruvate reduce to
lactate in lactic acid fermentation & then to alcohol in ethanol fermentation
* facultative anaerobes are capable of either form of metabolism, depend on O2 present/absent
(respire in its presence & ferment in its absence)
- toxicity of O2 results from its reduction by enzymes in cells (flavoprotein) to hydrogen peroxide &
more toxic free radical superoxide
- aerobes & facultative anaerobes are protected by presence of superoxide dismutase & catalase
- strict anaerobes lack catalase, peroxidase, superoxide dismutase

7. Principles of isolation and identification of microorganisms. Media for bacterial

growth. Culture of anaerobic microbes. Methods of isolating pure cultures.

Bacteriological method.
A. Isolation and identification for:
1) Aerobes and facultative anaerobes
Consist of 3 stages;
i)
ii)
iii)

Inoculation of agar plate; the streak plate is used primarily for isolating microorganism in pure
culture from specimens containing a mixed flora. Incubation.
Obtaining isolated colonies on permits a study of cultural characteristics. Each type of isolated
colony should be stained for studying cellular morphology (Gram Method) and inoculated on
solid agar slant. Incubation.
Identification of isolated pure culture is made by examining morphology of microorganism and
studying their morphological, staining, cultural, biochemical, antigenic, and virulent properties
and susceptibility to phages, chemical substrates, antibiotics etc.

2) Strict anaerobes *grow only in the absence of oxygen

Methods of achieving anaerobiosis:
i)
Physical. Cultivation in vacuum was tried by incubating culture in vacuum dessicator or
displacement of oxygen with gases like hydrogen, nitrogen, helium or carbon dioxide.
ii)
Chemical. Absorption of oxygen can be achieved by pyrogallic acid and sodium hydroxide.
Eg:
GASPAK (method consisting of on envelope and jar)
-

Envelope is placed inside the jar. The envelope contains 3 tablets, one each of citric acid, sodium
carbonate and sodium borohydrate.

McIntosh and Flides anaerobic jar

-

Consist of glass or metal jar with metal lid (can be clamped airtight with screw)
Inoculated culture plates are placed inside jar.
Reduced methylene blue is used for verifying anaerobic condition in jar

B. Media for bacterial growth

Culture media gives artificial environment simulating natural conditions necessary for growth of bacteria
(adequate temperature, pH, moisture, Posm, light, oxidation-reduction potential, sterility)
Classification of media:

- By their consistence;
i)
ii)
iii)

Solid media
Liquid media
Semisolid media

-By their nature;

i)
ii)
iii)

Nature
Artificial
Synthetic

-By their application;

i)
ii)
iii)
iv)

Simple or basal media (nutrient broth, nutrient agar)

Enriched media (blood agar, serum agar)
Selective media (salt agar,bile broth, alkaline peptone water)
Differential media (Endos medium, Levinas medium, Hisss media)

-Anaerobic media

Natural media; from natural products (milk, eggs, serum)

Artificial media; from digested or extracted natural products: meat extract broth, digest broth,
peptone, yeast extract.
Synthetic media; from pure chemical substances and the extract composition of medium
Nutrient broth; consist of meat extract peptone, sodium chloride and water.
Nutrient agar; addition of 3% agar in nutrient broth
Enriched media; like blood serum or egg is added to basal medium
Selective media; contain subs that inhibit or poison all but few types of microorganism.
Differential media; contain subs enabling them to bring out differing characteristic of bacteria to help
distinguish between them.

C. Culture of anaerobic microbes

The use of a battery of nonselective, selective, and enrichment media is recommended (eg: CDC
Anaerobe Blood Agar and Schaedler Agar)
When supplemented with selective agents, the media allow the isolation and differentiation of
specific bacteria or groups of anaerobes.
The addition of kanamycin and vancomycin provide media selective for obligately anaerobic, gramnegative bacteria (Bacteriodes and Fusobacterium spp.)
The used of laked sheep blood improves pigmentation of B. melaninogenicus.
The use of colistin and nalidixic acid for isolating of anaerobic gram-positive cocci (Peptococus,
Peptostreptococcus)
Bacteroides Bile Esculin Agar is for primary isolation of Bacteroides fragilis group.
Reinforced Clostridial Agar and TSN Agar for isolation of clostridia from clinical specimen.
A backup liquid enrichment medium (Enriched Thioglycollate Medium or Chopped Meat Glucose
medium supplemented with hemin and vit.K1.

As the plates and tubes are inoculated, they should be incubated under anaerobic conditions in
GASPAK jar at least 48h before the jar is opened. After observation, primary isolation media should
be reincubated for at least to 7 days
Isolated colonies should be examined by cultural characteristics, Prepared the stained smears and
each of colony should be inoculated on selective media in two petri dishes. The oxygen tolerance of
each colony should be determined:
i)
One anaerobe agar plate to be incubated anaerobically
ii)
Second anaerobe agar plate to be incubated aerobically
Identification of isolated pure culture should be done by biochemical characterisation of anaerobes
by 50 different tests.

D. Bacteriological method
Consist in;
-

Inoculation of an investigated material on culture mediums

Isolation of a pure culture of an originator with its subsequent identification (morphological,
tinctorial, cultural, biochemical, antigenic, pathogenic properties)

Is carried out in 4 stages;

-

Day 1 (stage 1)
A drop of an investigated material is applied on the medium surface with loop.
A dish is covered, marked and placed into a thermostat at a temperature of 37C for 18-24hrs
A loop are burned above the burner flame
Day 2 (stage 2; the inoculated dishes are got out from a thermostat and inoculum are studied)
1. Study of the cultural properties. On the dense mediums bacteria forms colonies which will be
characterized acc. to : dimensions, form, pigment-dependent colour, consistence, surface,
boundary
2. Study of morphological and tinctorial properties. A smear is made and it is carried out the Gramreaction and microscopy (the location of form will be marked)
stage3
For isolation and accumulation of pure culture
The material is taken from same colony with a loop and it is subcultured into a separate testtube with the slant beef-extract agar. The inoculum is placed into a thermostat at 37C for 1824hrs
Day 3 (stage4; study the identification of pure culture)
1. Study of morphological and tinctorial properties. The smear is made, stained and microscoped. If
culture is pure, other properties are studied.
2. Study of biochemical (enzymatic) properties.
For detection of microorganisms ability to ferment carbohydrates it is carried out inoculation
of a pure culture on the Hisss media with carbohydrates
As an indicator the Andrades reagent is added into all the media.
Inoculation is made and a loop by the injection method
To detect the proteolytic enzymes a pure culture is inoculated by the injection in gelatin and
beef extract broth (BEB). The inoculums in gelatin are incubated at 20-28C during several
days. If the proteolytic enzymes are present, bacteria dissolve gelatin.
The test-paper with reagents are placed into inoculated test tube with BEB
i)
Indole test; indole formation, the paper impregnated by solution of oxalic acid is
coloured pink
ii)
Hydrogen sulphide test; if hydrogen sulphide is present, the test-papers impregnated
by solution of lead acetate becomes black.
iii)
Ammonia test; ammonia is determined with he help of litmus paper will turn in its
presence.
3. Study of antigens properties of bacteria with the help of immune area

4. Lysotypy is carried out with the help of bacteriophages

5. The determination of pathogenicity features and virulence degrees
6. The estimation of antibiotic sensitivity

8. Sterilization and disinfection; physical agents, chemical agents, filtration (mechanical agents)
Sterilization; removal of the microorganism including spores and viruses (sterile items is free of microbes
including endospores and viruses
Disinfection; elimination of most or all pathogens
-

Some liable microbes may remain

Disinfectants used on inanimate objects (biocides, bacteriocides)
Antiseptics used on living tissues
Bacteriacidal: preparation to kill bacteria, bacteriostatic: inhibit bacteria growth

Methods of Sterilization
i)
ii)
iii)
iv)

Physical
Chemical
Mechanical
Biological

A.Physical

Heat
Factor of affecting ; nature of heat (dry/moist), temperature and time, no. of organism, characteristic
of organanism, type of material.
Type of heat
Dry heat: kills by oxidation, protein,denaturation and toxic effect of elevated levels of electrolyte
o Type of processes: flaming, inceneration, hot air oven
o Flaming:
250-300C
point of forceps and inoculation loops; heat in Bunsen frame till red hot
slow passage through the flame to destroy the vegetative bacteria on suface of scalpel blade,
glass glide, mouth of test tube.
o Incineration:
870-950C
Complete burning to ashes
Used for soiled dressings, animal carcasses, pathological material, disposables, non-reusable
soiled bedding.

o Hot air oven:

160C for 1.5hrs
Used for glassware, forceps, swabs, water, impermeable oils, waxes and powders
Before placing in hot air oven; dry glassware completely, plug test tube wih cotton wool,
wrap glassware in Kraft papers.
Dont overload in the oven, allow free circulation of air between the material
Sterilization controls: to check whether the equipment is working properly
Chemical control (Brownes tubes) ; colour change from red to green
Thermocouples
Biological methods controls; paper stripes containing 10 spores of Clostridium
Tetani
Place stripes in oven along with other material for sterilization
Later culture the stripes in thioglycollate brth at 37C for 5 days
Growth in medium indicates failure of sterilization
-

Moist heat: lethal effect due to denaturation and coagulation of protein

o Below 100C:
Pasteurization
63C : 30mins (holder method)
73C : 15 20sec (Flash method)
132C : 1sec (Ultra High temp.)
Vaccine baths (60C : 60min) ; for vaccins of non-sporing bacteria
Water-bath (56C : 60min, 3 days) ; for serum/body fluids containing coagulable proteins
Inspissation (80-85C : 30min, 3 days) ; for media containing egg or serum- LJ,L8S
o At 100C:
Boiling (100C : 10mins)
Kills all vege. bacteria
Water should be soft, deionized or distilled
2% sodium bicarbonate promotes the process
Kilss vege. Bacteria, hepatitis virus and some spores
Steaming (free steam) : 30 60min in Arnold/Koch Steamer, for heat labile media
Tyndallisation (intermittent sterilization) : 56 - 58C, 60min, 5 days

Nutrients media and media containing sugars or gelatin

1 day all vege. bacteria are killed; on 2 and 3 day spores that germinate are killed.

o Above 100C
Autoclave (stern under pressure; psi)
111C : 20min, 5psi
121C : 15 20min, 15psi
131C : 15min, 30psi
Used for rubber articles, dressigs, sharp instruments, infectious medical waste, culture media
Sterilization control;
Thermocouples
Brownes tube (red-green), Bowie and Dick Tape (white brown)
10C spore of B stearophilus. Incubate at 55C for 5 days
o Low temperature

Low temperature storage

Refrigeration inhibits growth of pathogens and soilage organisms by showing or
stopping enzyme reaction
Freezing pressure by stopping all microbial growth. Some microbial cells killed by ice
crystal formation but many survive and can grow once thawed.
Dessication (reducing available water)

Accomplished by salting, adding sugar, or drying food

Addition of salt, sugar increase environmental solutes, causes of cellular polymolysis
(water exits bacterial cells)
Some bacteria grow in high salt environment (staphylococcus aureus)
Drying of ton supplemented by salting
Lyophilisation (freezing drying) foods; coffee, milk, meats, fruits, vegetables
drying stop microbial growth but does no reliably kill

Radiation
Types:

non-ionising; infrared radiation (rapid mass sterilization of syringes etc), UV radiation

(encloses area), microwaves
UV radiation destroys microbes directly
Damage DNA, used to destroy microbes in air, water, and on surface
Poor penetrating power (cannot kill microbes in solid or turbid lipids)
Must be carefully used since damaging to the skin
Microwaves kill by generating heat not directly (microwaves oven heat food
unevenly, so cells can survive)
ionising; gamma, xray, cathode ray (plastics, syringes, oil material foils)
can remove electrons from atoms
destroy DNA
damage cytoplasmic membrane
reacts with oxygen to produce reactive oxygen sp.
Xrays and gamma-rays important forms
used to sterilize heat-sensitive materials
generally used after packing
approved for use on foods, although consumer resistance has limited used
sterilization controls : dosimeter: measures the radiation dose
coloured discs
M radiodurans B pomilus

B.Mechanical methods

Filtration
Aqueous fluid may be sterilized by forced passage through a filter of porosity small enough to retain
any microorganism present in them
Used to sterilized serum, carbohydrates solution, filtrates of toxins and bacteriophages in water
bacteriology, in examination of Schistosoma eggs.
Types of filtration :
o Eathernwave candle
- Unglazed ceramic and diatomaceous earth filters
- Eg: Chamberland, Doulton.
o Asbestos filter (Seitz, Carlson, Stenmat)

o Sintered glass filter

o Membrane filters (cellulose nitrate, cellulose acetate, polycarbonate, polyester filters)
- Pure size 0.15-12m
- HEPA filters ; for large volume of air
o Sterilization control
- Bubble pressure test

C.Chemical method

Disinfection.
- The process of freeing an article or a surface from all or some of the pathogenic
microorganism but not necessarily bacterial spores
- Strong disinfectant for inanimate object
- Mild disinfectant (antiseptic) superficial application on living tissue
Factors affecting disinfection
- Concentration of disinfectant
- Time of action
- pH of medium
- temperature
- nature and number of organism
- presence of extraneous material
- others; hardness of water, relative humidity
Categories:
o Alcohol
ethanol, isopropyl alcohol
- skin antiseptics at 70%
- less sporicidal and virucidal activity
- denaturate bacterial protein
- isopropyl alcohol better fat solvent, more bactericidal and less volatile
- methyl alcohol; to treat cabinets/incubators affected by fungal spores
- others; benzyl alcohol, chlorbutal, phenyl ethanol
o aldehyde
formaldehyde; 10% used
- in aqeous solution is viracidal, bactericidal, sporicidal.
- Used to fumigate wards, sick rooms, labs
- Expose to ammonia to remove residual formaldehyde
- Has pungent smell, irritant to skin, eyes, mucus membrane and toxic when
inhaled
Glutaraldehyde; less toxic, less irritant
- Endotracheal tubes, metal instruments, polythene tubing
B prypiolactane (BPL); condensation product of lactone and formaldehyde
- More efficient for fumigstion but is carcinogenic
- 0.2% used
Ethylene oxide
- Highly inflammable, mixed with inert gases
- Especially for heat lung machine, respirators, sutures, syringes, dental
equipment
Dyes
- Combine with nucleic acid
- Aniline dyes; brilliant green, malachite green, crystal violet.
- Acridine dyes; proflarine, acriflavine, euflavine, aminacrine

- Skin and wound antiseptics

- Bacteriostatic, more active against gram positive bacteria
Halogens
- Kills bacteria by oxidation
- Iodine 2.5% in75% alcohol, skin antiseptic
- Iodophones (iodine+non-ionic surface active agent), betadine (non-staining,
less irritant, less toxic)
- Chlorine; disinfect water supplies, swimming pools
- Sodium hypochlorite; 1% for HIV
- Organic chloramines; antiseptic for wound dressing
Phenolics
Carbolic acid 2.5%
- Powerful microbicidal, very corrosive
- General purpose disinfectant in hospital
Cresol, Lysol
Chloroxylenol, chlorophenol
Hexachlorophene
- Less toxic, less irritant, less active, more readily inactivated by organic matter
Surface active agent
Disrupt cell membrane (4 main groups)
- Anionic surfactants; strong detergent action, weak antimicrobial action
- Non-ionic surfactants
- Cationic surfactants; quaternary, ammonium compound; cetrimide,
benzalkonium chloride; bacteriostatic
- Amphoteric surfactant; both detergent and antimicrobial properties (tego
components)
Metallic salts
Mercuric salts: ointments
Siler salts: AgNO3; to prevent infection of burns, opthalmia neonatorum
Copper salts: antifugal, antialgae; water reservoirs, swimming pools
Diguanides
Chorhexidine: burns, skin disinfection
Pidoxydine: hospital equipment, floors
Amides
Propamide
Dibromopropamide
Antiseptic cream, eye ointments
Sterilant gases
Ethylene oxide
Formaldehyde
Betapropiolactone

9. The major groups of antimicrobial agents: antibiotics; classification; mechanisms of

action. Antibiotic sensitivity tests. Mechanisms of resistance to antimicrobial agents.

Atibiotics:. Large groups of drugs

Selectively inhibit growth of bacteria, fungi or tumor cell, without causing serious damage to host
Can induce human defense mechanism

Classification of antibiotics

Bacteriostatic/bactericidal
- Bacteriostatic: inhibit bacterial growth but does not kill
- Bactericidal: kill bacteria
Spectrum of activity/action
1. Narrow (limit) spectrum
- Antibiotics are active against one or few types of microorganism
- Benzyl-penicillin is highly active against many gram positive but has little activity
enteric gram negative bacilli
2. Broad spectrum
- Antibiotics are active against several type of organism
- Broad spectrum agents inhibit both gram positive and gram negative species
(tetracyclines, cephalosporins)
Sources of antimicrobial agents
1. Natural
- Bacteria (polymyxin)
- Streptomycetaceae (streptomycin, tetracycline)
- Fungi (penicillin)
- Plant (phytoncydes)
- Animal and fish cells (ectricide)
2. Chemically modified (semisynthetic; molecular manipulation of natural antibiotics)
- Oxacillin, anipicillin
3. Synthetic (chemically synthesized. Antimicrobial agents)
- Sulphonamides

Chemical structues
- B-lactam antibiotics
- The amino glycoside antibiotics
- The macrolide antibiotics
- Antibiotics in fused ring system: Lynocymycine
Polygenes antibiotics (antifugal)
- Polypeptide antibiotics
- Melasifide antibiotics

Mechanism of antibiotics action

1. Inhibition of cell wall synthesis
- Most bacteria possess of cell wall to protect from osmotic pressure
- Microbes divides; needs to create a new cell wall
Interrupt this leads to new microbes being susceptible to external influences
Cell ruptures to death microbes
- Eg: penicillin, encephalosporins
2. Disruption of microbial cell membrane
- Affect cell membrane transportation in and out
- Increase permeability of membrane
External influences have greater effect
Microbes death
- Polymycin, collistin
3. Inhibition of protein synthesis
- Proteins vital for growth and repair
- Act at
Site of protein synthesis (ribosomes)
Within the nucleus by inhibiting synthesis of nucleic acid (DNA replication, DNA
synthesis)
- Eg: tetracyclines, aminoglycosides and microlides (erythromycin)
- Exploit structural differences between microbial and human cell (high doses to toxicity)
4. Interferences with metabolic processes
- Agents similarly to paraaminobenzoic acid (PABA) ; component of folic acid
Essential for nucleic acid synthesis, without it microbe cannot produce the proteins
for growth
Exploit: microbes need to create their own folic acid, whilst get it from diet
Eg: saphonamides, thrimetophin.
Antibiotic sensitivity tests.
1) Diffusion test
- Prepre emulsion from microbes in physiological solution
- Put 1ml of emulsion on the MEA and inoculated it with spatula
- Put disc paper with antibiotic
- Incubation in t= 37C, 24hr
- Results:
In the diameter of 3ml oh inhibition is smaller thsn 15mm which is small sensitivity
If diameter between 15mm-25mm it is middle sensitivity
If more than 25mm which is has high sensitivity
If microbes grow around the disc, its resistance to antibiotics
2) Dilution test
- Put 1ml of MEB into 7 tubes. Any antibiotics, dilute it in MEB (from 50mg/ml to 1,5mg/ml)

Put emulsion of Staphylococcus (investigation microbe) into 7 test tubes with different
dilutions
Incubation t= 37C, 24hrs
Result : for the 1st four test tubes are transparent (no growth), in the last three are muddy
(microbial growth)
Inoculate material from transparent dilutions in MEA. Incubate.
MIC (minimal inhibitor concentration) = 6.2 mg/ml
- Lowest concentration of drug that inhibits growth of the cell
MBC (minimal bactericidal concentration) = 12.5 mg/ml
- Lowest concentration of drug that kills the bacteria.

Mechanisms of resistance to antimicrobial agents.

Altered permeability
o Altered influx: mutation in a transporter necessary to import antibiotics can lead to resistance
o Altered efflux: acquire transporter gene that will pump the antibiotics out (tetracycline)
Activation of antibiotics
o B-lactamase
o Chloramphenicol acetyl transferase
Mutation in the target site
o Penicillin binding protein (penicillin)
o DNA polymerase (infampin)
o 30s ribosome (streptomycin)
Replacement of sensitive enzyme with a resistant enzyme
o Plasmic mediated acquisition of a resistant enzyme (sulphonamide trimethopin)

10. Bacteriology of water, air and soil.

A.Soil
1. Soil is unfavourable habitat for most pathogenic species of bacteria, rickettias, viruses, fungi and
protozoa.
2. Soil factor of transmitting a number of causative agents of infectious disease
3. Important (impotent) epidemiological role in spreading some infectious disease of animal s and man
4. Habitat for various animal rodents which carriers of causative agents of plague, tularemia, viruses of
mosquito fever, haemorrhagic fever, encephalitis etc.
5. The cyst of intestinal protozoa (amoeba, balantidium etc) spend a certain stage in soil
6. Soil is important in transmitting worm invasion (hook worm, nematode)
7. Some fungi live in soil enter body they cause fusmic toxicosis, asperigillosis)
8. Index of sanitary condition of soil depends on
- Presence of microfora of human intestines (colibacillus and related bacteria)
- Thus indicates fecal contamination
9. Microorganism indicating sanitary conditions
- Sanitary microbes of the soil
- The presence of E.coli and Str.fecalis show fresh fecal pollution (survive in soil for several
days)
- Discover bacteria genus (enterobacter; indicate not fresh fecal contamination which survive
for several weeks)
- Clostridium perfringens is indicated old fecal contamination which survive for several years.
Sanitary indicators of soil
Sanitary:. Hygienic importance can be valued by calculating quantity of E.coli and claustridium perfringens
in soil.

Condition for sanitary indicator:

Permanent presence in intestine of human and animals

Not having the ability to multiply in soil
Live in soil about the same time as pathogenic intestinal microorganisms and
anaerobic infectious
Presence of quantity; in soil characterized the degree of fecal contamination

Evaluation of fecal contamination

Coli-index: number of E.coli cells in 1gm of soil.

Perfringens index: number of clostridium perfringens cells in 1gm of soil
Coli-titer: mass of soil (in grams), containing 1 cell of E.coli
Per titer: mass of soil containing 1 cell of Clostridium perfringens
Microbial no.: total no. of bacteria in 1gm of soil

Microbial investigation of soil

i)

Collection of soil
The sample of soil taken from the different places
Average btwn 100-200g
Put sample of soil in sterile tanks, mark and deliver to laboratory
Microbial no. of soil
Soil specimens (1gm) dilute in an isotonic solution of NaCl (1:10, 1:100, 1:1000 etc)
Plate 1ml of each specimen into special media for aerobic and anaerobic microbes
After incubation at optimal temperature count the colonies on the plates

ii)

Total no. of bacteria in 1gm of soil (x)

X= no. of colonies x degree of dilution
B.Water
natural water: saprophytic bacteria (micrococcus, vibria, acinetobacter)
anaerobic bacteria rare in water
important factor for transmission of a no. of infectious disease (enteric fever, cholera, dysentery,
leptospirosis)
due to sanitary epidemiological role of water in relation to intestinal group of diseases, its
necessary to indicate coli bacillus and pathogenic
the micro flora of rivers depends of
- degree of pollution
- quality of purification of sewage waters
microorganism are widespread in waters of seas and oceans found in different depth (3700-9000m)
degree of contamination of water with organisms expressed as saprobity
zones of water
1. polysaprobic zone
- strongly polluted water, poor in oxygen, rich in organic compounds
- no. of bacteria in 1ml reaches > 1000 000
- quantity of colli-bacilli few tens, hundreds in 1ml
2. mesoprobic
- moderate pollution, the mineralization of organic substances with intense oxidation and
marked nitrification takes place

total no. of bacteria in 1ml of water hundreds -> thousands

there is a marked decrease in no. of colibacilli (few tens per ml)

3. oligosaprobic
- pure water
- no. of microbes is low (in 1ml there are 100)
- this zone is devoid of colibacillus
depending on the degree of pollution pathogenic bacteria can survive in water
- salmonellae of enteric fever : 2days-3months
- shigellae of enteric fever : 5-9days
- leptospirae of enteric fever : 7-150days
- cholera, vibrio in water : many months
- Causative agent of tularaemia : few days-3months

Microorganism indicating sanitary conditions

The quality of water determined by the presence of E.coli and its variants
E.coli : normal inhabitant of intestinal tract of humans. Its presence in water indicates that the water
is polluted with intestinal watses and contain disease-producing organisms

Sanitary indicator of water.

Sanitary: hygienic importance can be valued by calculating the quantity of E.coli in water
To choice E.coli in quality sanitary indicator microorganism are in the following:
I.
Permanent presence of E.coli in intestine of human being and animals
II.
Not having the ability to multiply in water
III.
Live in water about the sametime as pathogenic intestinal microorganism
- In this way presence of quality of E.coli in water characterized the degree of fecal
contamination.

Why E.coli is used as sanitary indicator of water?

Testing for microbes that cause disease (eg: Salmonellae typhymurium and Vibrio cholera) can be
expensive and if the bacteria are present in low numbers, they may escape detection. Instead, other
more numerous bacteria provide an indication of fecal pollution of water
E.coli has been used as an indicator of fecal pollution (for decades)
o Present in intestinal tract in hygiene
o It is more numerous that the disease-causing bacteria and viruses
o The chances of detecting is better than the actual disease causing microorganism
o Advantage: not capable to grow and reproduce in water. Thus the presence of the bacterium
in water is indicator of recent fecal pollution
o Can be detected easily and unexpensively
Degree of fecal pollution of water
Estimated by coli-titre: smallest amount of water in mm in which one E.coli bacillus is
found
Coli-index: no. of individual of E.coli found in 1ml of water
Sanitary index (std) for normal drinking water:o Coli-titre 300ml
o Coli-index 3 (per 1L)
o Microbial number 100

Bacteriological examination of water

Microbial no. of water
I.
II.
III.

Inoculation of water sample (1ml) on the meat-peptone agar

Incubation at T=37C for 1-2 days
Calculation of colonies on the meat-peptone agar

Quantity of colonies in 1ml = microbial no. of water

Examination of coli-titre and coli-index
1. Membrane filtration method
o A measured volume of water (100ml, 200ml, 300ml) is filtered through a membrane filter. All
bacteria E.coli are retained on the surface of filter
o Membrane filter is placed on Endos media and incubated at T= 37C
- Colonies that develop on the surface of membrane filter are counted
- Calculate no. of E.coli per 1L (coli index)
- The smallest amount of water which contain one E.coli (coli-litre)
2. Eijkmans method
o 333ml of water is inoculated into the Eijkmans media
o Incubate at T= 37C for 24hr
o Subculture onto the Endos media
o Incubate at T- 37C
o Result in the Eijkmans table

C.Air
-composition lf microbe of air variable
-factors: contamination with mineral and organic suspension
On the temperature
Rainfall locality
-unfavourable medium for microbe
-absence of nutrient substances
-presence of moisture
-optimum temperature
-lethal activity of sunlight
-dessication (do not create for keeping microbes viable and most of them perish
- factor for transmission of pathogenis bacteria and viruses from sick to healthy person, cause extensive
epidermis of diseases such as influenza
-more dust, smoke and soot, great no. of microbes
-each particle of dust etc able to adsorb on its surface number of microbes
-rarley found on the surface of mountain in seas of arctic lands, snows
-micro flora of air consist different species which enter it from soil, plants, and animal organisms
-pigmented saprophytic bacteria (micrococci various sarcinae), B-cereus
-no. of microbe in air varies from few specimens to many tens of thousands per 1 cu m
-for eg. the air of arctic contains 2-3 microbes per 20 cu m
-in industrial cities, large no. of bacteria found in per 1ml of air
-in forests, few microbes because the some plant substances

-1gm of dust contains up to 1 million bacteria

-pathogenic sp. of microbes (eg: anthrax bacili) may be found in surrounding of sick animals and human,
infected arthropod and insects and in dust.
-Microbes spread by air currents, aerial dust and aerial droplets
-sneezing, coughing, and talking, sick person expel pathogenic bacteria with droplets of mucus and sputum
within a radius of 1.0-1.5ml of air
-microorganism contained in air remain in 3 phases of bacterial aerosol
:droplets, droplets-nuclei, dust
-an aerosol: physical system of solid or liquid particles suspended in environment
-greatest amount of bacteria discharged during sneezing, coughing, talking
-with each sneeze a man expels from 10 000-1 000 000 droplets
-in 1 cough from 10-1000 droplets containing bacteria are discharged into the environment
-no. of microbe is associated with the sanitary hygienic conditions of the building. Poor ventilation and
natural lighting the no. of microbes increases
-the causative agents of influenza , measles, scarlet fever, diptheria, whooping cough, meningococcal
infections, tonsilitis, tuberculosis, smallpox, pneumatic plague, and others can be transmitted through the air
with droplets of mucus and sputum during sneezing, coughing and talking.
-the total amount of microbes in an operating room before operation should not exceed 500 per 1 cu m of air,
and after the operation not more than 1000
-there should be no pathogenic staphylococci and streptococci in 250 L of air
-in operating rooms for maternity hospitals before work the number of saprophytic microflora colonies
isolated from the air by precipitating microbes on meat-peptone agar within 40mins should not exceed 20.
Microorganism indicating sanitary condition of air
Streptococcus viridans and haemolytic staphylococci indicate sanitary condition of air
sanitary indicator in air
-sanitary: hygienic importance can be valued by calculating the quantity of streptococcus viridans and
haemolytic staphylococci in air
-to choice both bacteria as sanitary indicator in air are following conditions:i) permanent presence of them in upper respiratory tract or human being
ii) not having the ability to multiply in air
iii) live in air about the same time as pathogenic respiratory microorganisms
-in this way presence of both bacteria in air characterized the degree of respiratory discharged continuation.
microbial number of air: total number of bacteria per 1 cu m of air
-the laboratory investigation of air is carried out to determine the qualitative and quantitative composition of
its microflora by Koch's and Krotov's methods
Koch's (sedimentation) method
- set open petri dishes with meat-peptone agar (MPA) in a room for 5 mins (for cocci 40min, blood agar)
-Place the dishes into 37C incubator for 24hr and then incubate for 24hr at room temperature (18-20C)
-study colonies, count them, and isolate pure culture of difference microbes
-according to omeliansky data on a surface of medium by 100cm per cube sedimentate in 5mins as so many
microbes, as they present in 10L of air.
-for eg., on the dish surface with MPA after 5mins exposure 32 colonies have grown, it is necessary to
calculate amount of microbes which are present in 1m per cube of the air, applying the omeliansky's
formula.
-the plate has 78.5cm per square (S=r^2= 3.14x5^2= 78.5 cm^2)
-thus quantity of microbes (x) would grow at given exposure on surface medium in 100cm per square.

X= (32x100): 78.5x40
-this quantity of microbes contains in 10L of the air, and in 2m per square (1000L) there will be (40x100) :
10 = 4000
Krotov's (aspiration) method
-Krotov's apparatus is used for this purpose
-pass 50-100L of air with a speed of 25L per min through clinoid chink of Krotov's apparatus above the open
dishes with MPA
-then dish is incubatedin thermostat at 37C for 18-24hr
-calculate amount of microbes which are present in 1m per cube of air
-eg: 250 coloniesare revealed on the surface of dish after 2mins exposure with a 25L/min speed. Thus the
number of microbes (x) in 1L of air is
X = (250x1000) : 50 = 5000

11. Normal microbial flora of human body. Formtion of normal flora. Dysbios.
Preparation of dysbios.
- nose and throat (upper respiratory system), eyes, mouth, skin, large intestine, urinary nd genital system
(lower urethra in both sexes and vagina in females)
-population of microorganism that inhabit the skin and mucous membranes of healthy person
Symbiosis
-living together
-interaction that occur between 2 dissimilar organism (usually 2 different species) that live together or are in
close association with one another
Neutralism
-relationship which neither symbiont is affected by the relationship
-both are infected
Commensalism
-one symbiont benefits and other species is not affected (neither harm nor help)
Eg: propionobacterium
Many species in this genera live on the skin and are thought tonneither hurt nor
Host: an organism that harbor another organisms
Mutualism
-relationship that is beneficial to both symbionts

help human

Eg: tremotes and protozoa

Lichens
Some species of our microflora (E.coli)
Parasitism
-one symbiont is benefited and other is harmed
Eg: sheep liver fluke
Opportunistic (pathogen of our normal flora)
Normal flora
-mixture of microorganism (bacteria and fungi) regularly found at any anatomical site of human body like
-such as: skin, eyes (conjuctiva), nose, mouth, ears, ugenital tract, elementary tract
sterile tissue
-internal tissue: blood, brain, muscle, cerebrosppinal, normally free of microorganism
Normal flora:
1) resident flora
-acquired rapidly during and after birth
-reflects age of person
-changes continuously through out life
-reflects nutrition of person
-reflects genetics of person
-reflects environment of person
fixed type of microorganism
regularly found in given area at given ageq
if disturbed, it promptly restablishes itself
2) transient flora
-live temporaril
-often 'pick up' from our daily routines
non-pathogenic/potentially pathogenic microorganism
inhabit the skin or mucous membrane for hours, days/weeks
derived from the environment
does not established itself permanently on the surface
membranes of transient flora are little importance and long as normal resident flora remains intact
if resident flora disturbed, transient microorganism may colonize proliferate
Imporatnce of normal flora
Advantages
1)constitue a protective host defense mechanism by occupying ecological niches
2)produce vitamin B and vitamin K in intestine
3)contribute to immunity by inducing low levels of circulating and secretory antibodies that may cross react
with pathogens
4)exert microbial antagonism against non indigenous species by production of inhibitory folic acids,
peroxides, bacteriocins etc (pathogen are inhibited)
5)may may antagonize other bacteria through the production of substances which inhibit/kill non indigenous

Disadvantages
1)cause disease:

-when individual becomes immunocompromised or debiliated

-when they change their usual anatomic location
2)may harm the host since some of these bacteria are pathogens/opportunistic pathogens
Estimation of normal flora
Normal flora human body:
-10^12 bacteria on the skin
-10^10 bacteria in the mouth
-10^14 bacteria in GIT
1) normal flora in the skin
: axilla, groin, areas between toes
-consist of bacteria and fungi
-most are anaerobes, some anaerobes
-anaerobes live in the deeper layers of the skin hair follicles, sweat and sebaceous glands
-no. and variety of microorganism depends on the
:amount of moisture present, pH, temperature, salinity, presence of chemical wastes (such as urea and folic
acid), presence of other microbes (which may produce toxic substances)
-resident bacteria: staphylococcus epidermis, staphylococcus saprophyticus, micrococcus species and
corynecbacteri
-transient microbes: s.aureus, candida
2) normal flora in the ears and eyes
i. Ears
-middle ear & inner ear sterile, outer ear & auditory canal contain same types of microorganism as are found
on the skin
-external contains avariety of microorganism: staphy. epidermis, staphy. saprophyticus, corynebacterium sp.,
staphy. aurens.
ii. Eyes (conjuctiva)
-contain some microorganism
-tears, mucus and sebum produced in and around the eye greatly reduce many microorganism
-microorganism: staph. epidermis, staph. saprophyticus, corynebacterium sp., neissera sp., hemophilus
influenza
-pathogens which do infect the conjuctiva: neissera gonorrhoeae, chlamydia trachomatis
3) normal flora of respiratory tract
i. Upper respiratory tract
-nasal passages and throat
-have many species of microorganism
-provide moist, warm environment
-many are harmless
-some are opportunistic pathogens
-some people are carriers of virulent pathogens

ii. Lower
-usually microbes free
A. Nostrils: staphy. epidermis, corynebacterium, staphy aureus, neissera sp., haemophilus sp.,
B. Upper respiratory tract: non-haemolytic streptococci, neissera sp., streptococcus pneumoniae,

streptococcus pyogenes,mneissera meningitis

C. Lower respiratory tract:
-usually sterile
-the individual may become susceptible to infection by pathogens descending from nasopharynx
-eg: hemophilus influenza, streptococcus pneumoniae
4) normal flora of urogenital tract
: anterior urethtra, mycoplasma sp., candida, mycobacterium smegmatis
-staphy. epidermis, corynebacterium sp. enterococcus faecalis, streptococci, some enteric bacteria (eg: E.coli
proteus sp.), Acinobacter sp.
i. Vagina: lactobacillus acidophilus, corynebacterium sp., staphylococci, nonpyogenic streptococci,
escheridia coli, flavobacterium sp., clostridium sp., other enterobacteria.

5) normal flora of GIT

-digestion of food, absorption of nutrients, elimination of undigested material
-includes: oral cavity and throat, esophagus, stomach, s.intestinal, large initestines, anus.
-GIT flora influenced by:
-age, diet, cultural conditions, the use of antibiotics
-at birth: the entire intestinal tract sterile, but bacteria enter with the first feed,
The initial colonizing bacteria vary with the food source of infant
-breast-feed: bifidobacteria, lactobacili, enterococci, bacteroides
-bottle-fed infants: enterococci, candida, lactobacilli, bacteroides, staphylococcus
-oral cavity of adult: viridans streptococci, lactobacilli, corynebacterium sp., bacteroides sp., streptococcus
mutans, actinomyces sp.
GIT:-proximal small intestine: lactobacilli, bifidobacteria, bacteroides, enterococcus faecalis, coliforms (E.coli)
-colon: lactobacilli, bacteroides, bifidobacteria (bifidum), E.coli, clostridia, streptococci sp., proteus sp.,
klebsiella
Dysbiosis
-an imbalance of resident to transient bacteria
-can occur from many factors: antibiotics use, contraceptive (the pill), steroids, altered gastric secretion,
spastic colitin, constipation, diarrhoea, radiation, diet including sulphur, excessive protein, excess refined
carbohydrates, lack of fibre, excess, decreased immune status (esp. low secretory IgA)

Main symptoms
-abdominal bloating: diarrheoa, constipation, disturbed bowed reaction
-excess flatulence
-fatigue and weakness
-halitosis or bad breath
-nausea and sometimes vomiting
Treatment
-prebiotic: substansis that selectively enhance the growth and metabolic activity of normal bacterim
(lactulose)
-biotherapeutic agents (BTA) and probiotic (live bacteria; lactobacilli, bifidobacteria)

-symbiotic: mixture of prebiotic and probiotics

-dietary supplements: vitamin, minerals, herbs, amino acids
-functional foods: yogurts, milk preparation

Q.12 INFECTION
INFECTION: it is a physiological & pathological rxn which arise and develop in microorganism in case of
penetrating of pathogenic microorganisms.
INFECTION DISEASES: one of the extreme degree of manifestation of infectious process.
Differ from other diseases:
-

They are caused by microbes

Contagious
Production of immunity
Have some stages.

Stages of disease:
i.
ii.
iii.
iv.
v.

Incubation: infection up to 1st symptoms may/may not be variable.

Prodromal: short period of early mild symptoms malaise.
Period of illness: clear signs fever & chills, swollen lymph node.
Period of decline disease: signs& symptoms subside susceptible to 2 infections.
Period of convalescence: regain strength & recovery but maybe reservoir.

Classification of infection:
1. Primary: initial infection with organism in host.
2. Secondary: when in a host, whose resistance is lowered by preexisting inf. dis., a new org. may set
up.
3. Reinfection: subsequent infection by some org. in a host.

4.
5.
6.
7.
8.
9.

Cross: patient suffering from disease & new inf. is set up from another host/ext source.
Nosocomial: cross infections occurs in hospital.
Iatrogenic: physician induced infections resulting from investigation, therapeutic.
Inapparent/ subclinical: where clinical effects are not apparent.
Atypical: typical clinical manifestation of the particular infectious disease are not present.
Latent: some parasite may remain in tissue in latern/hidden form proliferating & produce clinical
disease when host resistance is lowered.

Depends on no. of causative agents:

1. Mono infection: caused by one species of agent eg Cholera
2. Mix infection: caused by several species of microbes, viruses, protozoa.
Depends on the prolongation of development:
1. Acute: short incubation period & fast development of clinical symptoms eg influenza.
2. Chronic: prolonged of microbe development, several weeks/months eg chronic TB.
Depends on the localization of infectious diseases:
1. Localized: infection at localized sites eg appendix/tonsillitis.
2. Generalized:
i.
Bacteremia: circulation of bacteria in the blood.
ii.
Septicemia: bacteria circulate& multiply in blood, form toxic poducts.
iii.
Pyemia: pyogenic bacteria produce septicemia with multiply abcesses in intenal organs eg
liver.
Depends on spreading of infectious disease in community:
1. Endemic: spreads rapidly, involving many persons in an area at the same time.
2. Pandemic: an endemic that spreads thru many areas of the world within a short period.
SOURCE OF INFECTION
Endogenous: from hosts own body.
Exogenous: from external source.
Source of exogenous infection
1) By human (Antroponosis)
a. Patient
b. Healthy carrier: person harboring pathogenic org. without causing any disease to him.
c. Convalescent carrier: one who has recovered but continue to harbor pathogenic in his body.
2) By animals (Zoonosis)
a. Bacterial: plaque from rat, anthrax from cattle.
b. Rickettsial: murine thypus from rodent or Q fever from cattle.
c. Viral: rabies from dog.
d. Protozoal: leishmaniasis from dogs
e. Fungal: zoophilic dermatophytes from cats & dogs.
f. Helmintic: hydadid cyst from dog.
3) Insects
- Anthropod borne disease: diseases caused by insects
- Vector: mosquitoes, fleas, lice.
a. Mechanical: transmission of dysentery/ S. typhi by housefly.
b. Biological: if pathogen multiplies in the body of vector (anopheles mosquito in malaria).
c. Reservoir host: vectors act as reservoir host (ticks in relapsing fever & spotted fever).

4) By environment (Sopronosis)
a. By soil: spores of tetanus bacilli, fungi like Histioplasma capsulatum.
b. By water: vibrio cholera, hepatitis A.
c. By food: salmonella spp., E.coli, C.botulinum.
Based on their relationship with the host, microorganism can be:
Saprophytes

Parasites

- free living org., which live on

decaying organic matter.

-org. that can establish

themselves & multiply in
hosts.

- fail to multiply on living

tissue so not impt in infectious
disease.

- may be commensals or
pathogens
- Commensals: live in a host
w/o causing any disease.

Classification of bacterial pathogens

Primary (true)
- Capable of establishing
infection & causing disease in
previously healthy individuals
with intact immunological
defenses.
- may be readily cause disease
in invidual with impaired
defenses.

Strict intracellular

Facultative intracellular

Extracellular

- Pathogens: produce disease

in host.
Oppurtunistic
- cause disease only to people
with preexisting disease or
condition that enhance their
susceptibility.
- Eg: Pseudomonas Aeruginosa
infect burn patients & the
lungs of patients with cystic
fibrosis.

cannot be cultivated in broth

medium.
can replicate only in vivo/ in tissuecultured cells in vitro.
eg: Chlamidia, Rickettsia
can be grown in vitro on culture media
used to infect tissue culture
eg: M.Tuberculosis, M.leprae, S.thypi
grow freely in environment.
eg: B.pertusis, T.pallidum, C. diphtheriae.

Transmission of pathogens:
1.
2.
3.
4.
5.
6.

Direct contact: plague & anthrax

Indirect contact: formites in the transmission of diphtheria & trachoma.
Inhalation: influenza, paainfluenza, TB, whooping cough,measles.
Ingestion: cholera, food poisoning, dysentery.
Inoculation: tetanus, rabies, hepatitis A.
Insects: mechanical vector dysentery & enteric fever
Biological vector tick-borne infection
7. Congenital: syphilis, rubella.

Pathogens

8. Iatrogenic & lab infection: as result of exchange transfusion, dialysis, and injections.
If certain pathogen enter the wrong portal, they will not be infectious.
PATHOGENICITY: ability of microbial species to produce disease.
VIRULENCE: degree of pathogenicity.
Doses of virulence
-

Successful infections require that an adequate no. of bacteria should gain entry in host.
Dosage may be:
Minimum infecting dose (MID) = min no. of bacteria required to produce clinical evidence
of infection.
Minimum lethal dose (MLD) = min no. of bacteria required to produce death 80% of
animals tested under std. conditions.
ID50 & LD50 = are used, as the dose required to infect/ to kill 50% of animals tested under
std. conditions.
DCL = dose required to kill 100% of animals tested under std. conditions.

Factors of pathogenicity:
1)
-

Adhesiveness
Attachment of bacteria to body surfaces.
It is initial event in the pathogenesis of many infection by specific rxn.
Adhesive structures:
Fimbriae (bacterial projection)
Capsules (bacterial coating)
Spikes (viral particles)
Hooks (treponema) / flagella (salmonella)
2) Colonization
- Ability of microorganism to multiply and spread in host.
Exotoxin
-produced inside Gram +ve as part of
their growth then released into
surrounding medium
- heat labile proteins
- diffuse readily into ext medium
- their action is enzymatic
- toxic in small quantities
- can be partially denatured to
generate toxoids (anatoxin)
- polypeptide composition
- no fever
- high specificity
- unstable at 60
- Example:
> cytotoxin: kill host cells by
distrupting protein synthesis. Eg: C.
diphetheria.
> neurotoxin: act on nerve cells.
Eg: clostridium tetani & C.botulinum.
>enterotoxins: binds to villus cells
causes excess loss of water&

3) Invasiveness
- Ability of microorganism to penetrate tissues of organism
by releasing the degradative enzymes.
- Eg: hyaluronidase, protease, mucinase, phospholipase.
4)
-

Antiphagocytic factors
Help bacteria to resist phagocytosis.
Mechanism: production of extracellular capsule
Eg of capsulated bacteria: N.meningitis, S.pneumonia,
E.coli.
- Capsule are polysaccharide, which reduce the efficiency
of phagocytosis due to hydrophilic nature of this
structure.
- Produce thrombin-like enzyme coagulase, which prevent
phagocytosis by forming a fibrin barrier around the
bacteria.
5) Toxigenicity
- Ability to produce toxin.
- TOXIN: bacterial components that directly harm tissue/
trigger destructive biological activities.

Endotoxin
-part of the outer membrane of Gram
ve. Released when bacteria die & cell
wall breaks.
- heat stable protein
- not diffuse into the medium
- has no enzymatic action
- toxic only in large quantities
- it cant be toxoided (anatoxin cant
be prepared)
- lipopolysaccharide composition
- cause fever
- low specificity
- stable at 60
Example:
>Escherichia coli,
>Salmonella,
>Shigella,
>Neisseria,
>Bordetella pertussis

Q.13 BIOLOGICAL METHOD

- Biological study consist of infecting animals for the purpose of isolating the culture of the causative agent
and their subsequent examination for pathogenicity and virulence.
- Most frequently used are: rabbits, guinea pigs, mice, rats, dogs, monkeys and also gnotobiotes (without
micro flora) animals.
Stages for Biological Experiments
1. Selection of animals :
- Experiments are conducted only on healthy animals with smooth lustrous (shining) coat that are
active and feed vigorously. Before inoculation, details of animals recorded (weight, growth, sex,
age), marking on animals and fixation of animals.
2. Infecting the animals:
- By subcutaneous, intramuscular, intraperitoneal, intravenous and per os (orally) inoculation.
- All inoculations are performed with observation aseptic and antiseptic.
3. Observation of clinical disease:

Observe the changes in : Body temperature

Behavior
Clinical symptoms
Diarrhea
Vomiting
Common cold
Coughing
Necrotic change in skin
Formation of toxin
4. Autopsy of animals:
- Autopsy of dead animal is done in order to observe changes in the internal organs and to prepare
smear , stained by gram technique and fix it in slide.
5. For introduction of pure culture, agent have to do test in part of infected organs in nutritive medium.
To study the properties of microbes.
6. Before autopsy, the body of animals is soaked with disinfectant solution to prevent inoculation of the
operators and contamination of the surrounding objects. Instrument are washed in disinfectant
solution and sterilized.
Aims
1. Reproduction of infectional process, to study pathogenicity and effective treatment.
2. Introduction of pure culture in microbes, if the growth is not good in nutritive medium.
3. To determine the pathogenicity and virulence of microbes.
Virulent can be determine in doses: DLmin ( Dosis Letalis Minimalis)
- Minimum number of microbes cause death of 85% experimental animals.
DL50 (Dosis Letalis 50)
- Minimum number of microbes cause death of 50% experimental animals.
DcL (Dosis Cerma Letalis)
- (absolutely death cause). Minimum number of microbes, cause death of 99% experimental
animals.
Blood agar (5%) added red colour due to erythrocytes.
Hemolysin cause destruction of or give toxic to erythrocytes.
To determine whether the microorganism is pathogenic or saprophytes (not cause infection).

Q.14 IMMUNITY. TYPES AND FORMS OF IMMUNITY. THE HUMAN IMMUNE

SYSTEM, INNATE IMMUNITY. NONSPECIFIC RESISTANCE
In biology, immunity is the state of having sufficient biological defenses to avoid infection, disease, or other
unwanted biological invasion. It is the capability of the body to resist harmful microorganisms or viruses
from entering it.
-

TYPES AND FORMS

PASSIVE IMMUNITY
Production
Received passively by the host.
No participation by the hosts immune
system.
Induced by

ACTIVE IMMUNITY
Produced actively by the hosts immune
system.

Conferred (given) by introduction of

readymade antibodies naturally or artificially
in the body.

Effectivity
Protection transient and less effective.
Latent period
No latent period.
Immunity effective immediately.

Secondary response
No immunological memory; subsequent
administration of antibodies less effective due
to immune elimination.
Negative phase
No negative phase, as antigens are not
injected.

Duration
short lasting
Applicability in immunodeficient
individuals.
Applicable

Induced by infection or by contact with

immunogens (vaccines, allergens, etc).
In response to antigens introduced into the
body.

Provide durable and effective protection.

Immunity effective only after a lag or latend

period.
(time required for generation of antibodiesvaries from 4 days to 4 weeks).

Immunological memory present; subsequent

challenge more effective (booster effect).

Negative phase may occur during which the

immunity is transiently lowered. This is due
to antigen combining with pre-existing
antibodies and lowering their level.

Long lasting
Not applicable

- INNATE (NATIVE) IMMUNITY

Definition:
-the resistance to infections, which an individual possesses by his genetic and constitutional make up.
-not affected by initial contact with microorganisms or immunization.
-nonspecific bcoz it indicates a degree of resistance to infections in general.
-the differences in innate immunity exhibited by different individuals known as INDIVIDUAL
IMMUNITY.
- Factors influences: - age, hormonal disorders and nutrition.
Classification of Factors of innate immunity and their MAO (mechanism of action):1. Mechanical barriers
- Intact skin and mucous membranes of body afford a high degree of protection against pathogens
- Skin is resistant barrier bcoz its outer layer consisting mainly of keratin ( indigestible by most
microbes).
Both characteristics are inhibitory or lethal to many microbes.
- Dry condition of skin.
- High concentration of salt in drying sweat.
2. Surface secretions

Sebaceous secretions and sweat of skin:

contain bactericidal & fungicidal fatty acids.

Sticky mucus covering the resp tract:

acts as trapping mechanism in inhaled particles.

Cilia:
sweep the secretions containing foreign material towards oropharynx so it is swallowed.

Acid secretions of stomach and bile of intestine:

destroy most of microbes.

Nasal secretion & saliva:

contain mucopolysaccharides capable to block some viruses.

Tears, mucous secretion of resp, alimentary, urogenital tracts :

Contain lysozyme which is active against some Gram +ve bacteria.
Particles trapped in mucus of intestinal tract and form small masses, which propelled by
peristalsis.
3. Humoral defence mechanisms
Antibacterial substances in blood and tissues include :
-

Normal antibodies : synthesized by host without any antigenic stimuli;

Complement system & properdin;
Lysozyme : mycolytic enzyme, splitting sugars of peptidoglycan of call wall of many Gram +ve
bacteria thus cause lysis;
-lysin : thermostable subs against B.antracis & Bacillus species;
Basic polypeptides : leukins from leukocytes
Plakins from platelets;
Acidic subs: lactic acid found in muscle tissue & in inflammatory zones;
Lactoperoxidase in milk;
Acute-phase proteins : C-reactive protein
(limit spread of
1-antitrypsin
infectious agent
2-macroglobulin
& stimulate host
fibrinogen
response)
serum amyloid A protein
Lactoferrin : an iron binding protein, which take away the free iron needed for growth of
microbes;
Interferons : antiviral substances.

Some of these microbicidal subs are secreted:

- Constitutively (lysozyme)
- In response to infection ( acute-phase protein & interferons).
COMPLEMENT SYSTEM
-is a group of serum proteins present in normal serum.
-present in inactive form but can be activated to form an enzyme cascade.
-consist 9 fxtional components C1-C9
2 pathways:
Classical : antigen-antibody complexes dependent
Alternative : antigen-antibody non-dependent
Both pathways lead to same physiological consequences:
Opsonization

Cellular activation
Lysis

Classical pathway

Alternative pathway

4. Cellular factors
-are phagocytic cells (micro and macrophages) & natural killer cells.
-

Microphages
polymorphonuclear leukocytes (neutrophils, eosinophil & basophils)

Macrophages
Mononuclear phagocytes
In circulating blood called monocytes
In tissues differentiated into macrophages such as:

Alveolar macrophages in lungs

Dendritic cells in skin
Kupffers cells in liver
Histiocytes in CT
Microglial cells in brain
Mesangial cells in kidney
Sinus-lining macrophages in lymph node & thymus

3 basic fxs:i.
Phagocytosis
ii.
Antigen presentation to T cells to initiate specific immune response
iii.
Secretion of lymphokines to activate & promote immune & inflammatory responses.

Phagocytosis of bacteria involves 4 steps:1) Chemotaxis

- Process of movement of phagocyte to site of infection.
- Chemotactic factors:
Products of injured tissue
From blood (C3a & C5a)
Subs produced by neutrophils and mast cells (histamine, leukotriene)
Bacterial products (formyl-methionine peptides)
Neutrophils respond first and move faster than monocyte.

2) Attachment
- Mediated by
receptors for bacterial carbs (lectin)
receptor for opsonin (complement C3a & C4b)
fibronectin receptor
Fc receptor for antibody.
- After attachment, particle surrounded by plasma membrane , form a phagocytic vacuoles
(endosome / phagosome).
- This vacuoles fused with 1 lysosomes(macrophages) or granules (polymorphonuclear
leukocytes) forming phagolysosomes.
- Ingested microorganism is killed and digested by various enzyme.
3) Ingestion or phagocytic killing
Oxygen-dependent
- Accompanied by glycolysis & increase in synthesis of proteins & membrane phospholipids.
- There is Respiratory burst consisting a steep rise in O2 consumption.
- Increase activity of enzymes
- Leads to reduction of ROS( reactive O2 species microbicidal activity) :
Superoxide, O2 Hydrogen peroxide, H2O2
Hydroxyl radical, OH.
Oxygen-independent
- Present within phagocytes in azyrophilic granules
- Able to destroy ingested material and can damage membranes.
- Eg lysozyme & elastase
- Both attack peptidoglycan of bacterial cell wall.

Lactoferrin has antimicrobial prop. An iron binding protein, which take away the free iron needed
for growth of microbes.

4) Digestion
Complete
- Once killed, most microbes are digested and solubilized by lysosomal enzymes.
- The degradation products then released to exterior.
Incomplete
- Bacteria not digested by macrophages.
- Their capsule is slimy which is hard to grasp and tears away when grabbed by phagocyte.
- Bacteria can avoid phagocytic killing in variety ways: Produce enzyme capable of lysing phagocytic cells (streptolysin by S.pyogenes or toxin by
C.perfringens).
Inhibit phagocytosis or block intracellular killing.
Develop mechanisms to protect them from intracellular killing by:
Their ability to prevent phagolysosome fusion ( M.TB);
To resist being killed by bactericidal lysosomal enzymes(Salmonella);
Adaptation of some pathogens to cytoplasmic replication(Rickettsia ssp.);
Produce catalase , which makes the myeloperoxidase system less effective( Staphyllococci).
Macrophages impt link btw innate and acquired immune mechanisms.
It secrete IL-1, IL-6, IL-12 and TNF in response to bacterial.
NATURAL KILLER
- Recognizes changes on virus-detected cells & destroy them by an extracellular killing
mechanism.
- After bind to target cell, NK cells produce molecules that damage membrane then lead to cell
lysis.
- NK cells present without initial exposure to infectious agent.
- Implicated in host defense against cancer.
- Natural killing enhanced by IFN.
5. Microbial antagonism
- Of resident bacterial flora from skin and mucous surfaces.
- Prevent colonization by pathogens.
- Uses up niche( a protein) that cannot be used by pathogens.
- They compete for nutrients & produce metabolites that can inhibit growths of other organism.
- Alteration of normal resident flora lead to invasion by extraneous microbes.
- Importance of normal bacterial flora proved by extreme susceptibility of germ free animals to all
types of infection.

Q.15 ANTIGENS AND ANTIBODIES

ANTIGENS
- any substance capable of provoking the immune system of the host to respond by generation an immune
reaction directed at the inducing substance.
- The response is not to entire molecule but to individual chemical groups.

- the specificity of the response to these antigenic determinants or epitopes is impt characteristic of immune
response.
- the reaction of host to cantact with antigen, called acquired immunity, takes 2 forms :
1) humoral antibody response;
2) cell-mediated response.
- antibody directed against an epitope of an antigen will react against only with this determinants.
- even minor changes in the conformation of epitope will reduce the original antibody to react with the
altered material.
- antigens (bacteria,viruses) carry many diff types of epitopes, presenting an antigenic mosaic.
- Two main attributes of antigenicity are:
1) induction of the immune response(immunogenicity);
2) specific reaction with antibodies or sensitized cells.
- HAPTENs are substances which can react with antibodies but incapable of inducing antibody formation
by themselves. It becomes immunogenic (capable of inducing antibodies) when combine with a lager
molecule carrier (a protein).
- only antigens which are foreign to individual (nonself) induce an immune response. In general,
antigenicity of a subs is related to the degree of its foreignness.
Factors of degree of antigenicity
i.
Species
- Ag from other individuals of the same species are less antigenic than from those from other species.
ii.

Biological origin

- most naturally occurring Ags are : protein & polysaccharides.

- Lipids & nucleic acids less antigenic.
iii.

Molecular size

- very large molecules are highly antigenic.

- low molecular weight (< 10 000) are nonantigenic. But they can become antigenic by absorbing them on
large inert paticles.
ANTIBODIES
Def: humoral subs (gamma globulin) produced in response to an antigenic stimulus.
-

Ab bind to Ag and then mediate several other activities or fx in the interaction with other cells &
molecules of immune system.
Serve as protective agent against microbes.
Ab found in serum, lymph and other body fluids.
Sera having high Ab levels called immune sera.
Most Ab are present within the gamma-fraction of serum separated electrophoretically.

Immunoglobulins
-

Are glycoproteins.
Synthesized by plasma cells and lymphocytes.
5 distinct classes (the H chains are structurally & antigenically diff for each class) :
IgG - gamma

IgA - alpha
IgM mu
IgD - delta
IgE epsilon
Aminoterminus

Kappa/
Lambd
a

Chains:
L (light)
- Similar in all classes of Ig.
- Either kappa/ lambda.
- Molecular weight 25,000.
- 214 aminoacids make up L chain.
- 107 constitute carboxyterminal half occur only in constant region.
H (heavy)
- Are structurally & antigenically diff for each class.
- , , m, , .
- Molecular weight 50,000.
Regions:
C (constant)
- Also called carboxyterminal portion.
- 107 constitute carboxyterminal half of L chain occur in this region.
V (variable)

- Constitute of aminoacid sequences in aminoterminal (N) half of chains.

- This determines immunological specificity of Ab.
Hypervariable
- Highly variable zones numbering 3 in L and 4 in H chains.
- Involved with the formation of Ag binding sites.

Fragments:
Fc ( 1 fragment, crystallized)
- Composed of the carboxyterminal portion of H chains.
- Interacts with cell surface receptors and some proteins of complement system.
- Does not possess Ag combining activity but determines the biological prop. of Ig molecules such
as complement fixation, opsonisation.
Fab (2 fragments, antigen-binding)
- Region of an Ab bind to Ag.
- Composed of 1 constant & 1 variable domain of each L and H chain.
Classes of each Ig:
% of total
serum
Molecular
weight
Half-life
Classes
Normal serum
[ ]
Form
Main fx

Participates in
immunologica
l rxns

IgG
75%

IgA
10%

IgM
5-10%

IgD
0.25%

IgE
0.05%

150,000

160,000

900,000

180,000

190,000

23 days
IgG 1,2,3,4
5-7 mg/ml

6-8 days
IgA 1, 2
0.7-5.0

5 days
IgM 1, 2
0.7-2.0

3 days
0.03

2 days
0.0003

monomer
Only Ig can
pass thru
placenta &
provides
natural
immunity to
newborn.
Precipitation,
complement
fixation,
neutralizatio
n of toxins &
viruses.

dimer
Found in
colostrum,
tear, bile,
saliva, nasal
secretions.

pentamer
Appears
earlier in
primary
response
compare to
IgG.

monomer
Responsible
for allergic
reaction.

Alternative
complement
pathway,
phagocytosis,
intracellular
killing of
organisms.

Fixes
complement,
agglutination,
cytolytic,
cytotoxic rxn.

monomer
As mutually
reacting Ag
receptor for
the control of
lymphocyte
activation &
suppression.
B cell
activation

Has affinity
for surface of
tissue cells
(mast cell) of
same species.

Q.16 SEROLOGICAL REACTIONS IN DIAGNOSIS OF INFECTION DISEASE.

AGGLUTINATION REACTION, PRECIPITATION REACTION, COMPLEMENT
FIXATION TEST, IMMUNOFLOURESCENT TESTS.

SEROLOGICAL REACTIONS IN DIAGNOSIS OF INFECTION DISEASE

-Used to: 1-Identify the infecting agent that are difficult to isolate & to grow in lab
2-Evaluate the course of an infection
3-Determine the nature of infection - primary or reinfection
- acute or chronic
1 - Serological data provided by: Antibody type and titer
Identity of antigenic targets
- TITER: - the relative antibody concentration
- The patients specific IgG, IgA, IgM or IgE can be evaluated
separately using direct immunofluorescence, ELISA, RIA.
2- The time course of an infection determined by present of SEROCONVERSION.
- SEROCONVERSION: occurs when antibody is produced in response to 1 infection.
- IgM found during the first 2-3 weeks of 1 infection.
3 - To determine stage of acute or chronic infection:
- by the basis of the presence of Ab to specific Ag.
- The first Ab to be detected are those directed against Ag most available to immune system (on
virion, on infected cell surfaces).
- Later when cell have been lysed, Ab directed against intracellular proteins & enzymes are
detected.
AGGLUTINATION REACTION
Def: the reaction of Ab with an insoluble Ag or Ag-coated particle resulting in the formation of clumps of
Ag-Ab complexes.
1) Slide agglutination test
- A routine procedure for identification of many bacterial isolates from clinical specimens.
- A drop of antiserum (the test) and a drop of physiological solution (the control) are put on an
object plate.
- Culture of investigated microbe added to each of the plates.
- A +ve result: clumping together of the particles. There are floccules in the drop with the
antiserum.

2) Tube agglutination
- A standard quantitative method for the measurement of an Ab.
- 1ml of physiological sol. Poured into 6 test tubes (5 trial & 1 control).
- 1ml of investigated serum with dilution 1:50 added into 1st test tube.
- 1ml from 1st test tube then transferred into 2nd.
- Then 1ml from 2nd transferred into 3rd, and so on up to 5th test tube.
- So the following serum dilutions received 1: 100, 1:200, 1:400, 1:800 & 1: 1600.
- Serum not added into control test tube.
- 2 drops of diagnosticum(suspension of killed bacteria) added to all test tubes.
- All test tubes then placed into thermostat at 37 for 2hours.
- Ultimate result considered in 18hous of incubation at room temp (20)
The intensity of agglutination rxn is marked as follows:
Intensity
++++
+++
++

Agglutination
Complete
Incomplete
Partial

Sediment
Well-marked
Well-marked
Small

Fluid
Full clarification
Weak opalescence
Cloudy

+
Very small
Absence
*rxn is +ve if there is clear agglutination (not less ++)

Opaque

3) Hemagglutination inhibition test

- performed by preventing the agglutination of Ag-coated red blood cells by
homologous Ag.
- allows detection of Ag that are soluble in serum or other biological fluids.
- Erythrocytes (sensitized by Ag) introduced in the dilutions of patients serum ( 1:50, 1:100, 1:200).
- results in 2hours of incubation at 37C.
-ve: Erythrocytes sediment in form of a compact paint or thick ring.
+ve: Erythrocytes sediment in form of loose with an irregular edge (umbrella like).
PRECIPITATION REACTION
Def: the test when a soluble Ag combines with its Ab in the presence of electrolytes (NaCl) at suitable temp
& pH.
- Ag-Ab complex forms an insoluble precipitate.
- The precipitate remains suspended in floccules, rxn known as flocculation.
Types:
1) Ring test
- 1ml of serum poured out into a narrow test tube.
- Then 1ml of Ag slowly layered on the wall with a pipette.
- +ve result: a cloudy precipitation ring appears on the borders of serum & Ag.
2) Immunodiffusion/ precipitation in gel ( Ringel)
- Antiserum is incorporated in agar gel poured on slide.
- The Ag added to the wells cut on the surface of the gel.
- The Ag diffuses radially and forms ring shaped bands of precipitation (halos) concentrically
around the well.
- Diameter of halo gives an estimation of the concentration of the Ag.
COMPLEMENT FIXATION TEST (CFT)
Def: the ability of Ag-Ab complexes to <fix> complement.
Consist of 2 steps and 5 reagents (Ag, Ab, complement, sheep erythrocytes and amboceptor- rabbit Ab).
Scheme
Test tubes for the ingredients
Ag
Inactivated immune serum 1:5
Complement (guinea pig serum)
Physiological solution
*Incubation at temp 37C, 45mins
Steps:
I.
II.

Specific system
Trial
0.25
0.25
0.25
-

Control of Ag
0.25
0.25
0.25

- Inactivated serum incubated with specific Ag and fixed amount of complement.

- If serum contains the specific Ab, complement should be used during Ag-Ab rxn.
- If no Ab, no Ag-AB rxn, so complement is left intact.
- adding sensitized cells (sheep erythrocytes coated with 4 MHD hemolysin) into complement &
incubated to indicate whether the serum had Ab or not.
- Lysis of RBC indicates that complement was not fixed in Ist step , therefore serum didnt have
Ab (-ve CFT).
- Absence of RBC lysis indicates that complement as used up in Ist step, therefore serum contain
the Ab (+ve CFT)

Examples :

Wassermann reaction for syphilis.

Bordet- Gengou reaction for gonorrhea.

IMMUNOFLUORESCENT TESTS
Fluorescent dyes can be conjugated to Ab and show up brightly under ultraviolet light as they convert
ultraviolet into visible light.
Types:
I.
Direct
- Used for identification of bacteria, viruses & other Ag using specific antiserum labelled with
immunofluorescent dye (eg fluorescein isothiocyanate).
- Test is used as a sensitive method of diagnosing rabies.
- DisAdv: separate fluorescent conjugates have to be prepared against each Ag to be tested.
II.
Indirect
- Using an antiglobulin conjugate.
- A drop of antimicrobial serum (eg from rabbit) palced on fixated smear.
- After incubation, slide is washed to remove all free serum, leaving only Ab globulin, if present,
coated on the surface of microbes.
- Smear then treated with fluorescent-labelled antiserum to rabbit -globulin.
- Slide examined under ultraviolet illumination, the detected microbes seen as bright objects
against dark background (+ve).
- Only dark phone revealed in view (-ve)

Q.17 ACQUIRED IMMUNITY. IMMUNOBIOLOGIC DRUGS. VACCINES:

IMMUNE SERA, IMMUNE GLOBULINS, IMMUNOMODULATORS.
ACQUIRED IMMUNITY
Def: the resistance that an individual acquires during life.

Types: active natural & artificial

Passive natural & artificial
Active Immunity
Def:
- Resistance developed by an individual as a result of an antigenic stimulus.
- leads to synthesis of antibodies/ immunocompetent cells.
- sets in only after a latent period.
- have a negative phase when level of Ab lower than before antigenic stimulus. This is due to Ag combine
with any preexisting Ab.
- Once developed, it is long lasting.
- immune response occurs more quickly than first encounter. [secondary response]
I. Natural
-

Results after exposure to infectious agent.

Long lasting but varies with pathogen:
Mumps, measles = life long
Influenza, common cold = short lived
Syphilis = premunition (immunity to reinfection lasts only as original infection remains
active, once cured patient become susceptible again).

II.Artificial
-

Resistance induced by vaccines.

Passive Immunity
Def:
- the resistance that is transmitted to a recipient in a readymade form.
- immune system plays no active role.
- no antigenic stimulus, instead prefomed Ab are administered.
- no latent period.
- no negative phase.
- immunity is transient, lasts for several days or weeks.
- no secondary response occurs.
- when foreign Ab administeres 2nd time, it is eliminated more rapidly.
- less effective.
Main Adv : immediate in action.
I.Natural
-

Resistance passively transferred from mother to baby.

Mother Ab (IgG) transmitted to infant thru placenta.
Human fetus able to synthesize Ab (IgM) from 12th week.

Infant acquired satisfactory level of immunological independence age of 3 month.

II.Artificial
-

Resistance passively transferred to a recipient by administration of AB.

Agents used are hyperimmune sera of human origin (homologous sera), convalescent sera & pooled
human -globulin.
Used for prophylaxis and therapy.

IMMUNOBIOLOGIC DRUG
VACCINES: preparation of live or killed microorganism or their product or extracts used for immunization.
Classification:1) Live Vaccines
a) Bacterial against TB (Bacille Calmette-Guerin - BCG), plaque, tularemia, antrax.
b) Viral against rubella, mumps, measles, yellow fever, influenza.
c) Rickettsial against Q fever.
- Initiates infection without causing injury or diseases, bcoz they are prepared with organism limited in
their ability to cause diseases (avirulent).
- Administered by natural routes of entry & induce local immunity.
- Following immunity :
As caused by natural infection.
Elicit both cellular & humoral.
Lasts several years but booster doses maybe necessary.
- 2 problems with Live Vaccine :
Still dangerous for immunosuppressed people and pregnant women.
May revert to virulent form (rare).

2) Killed Vaccines(Inactivated)
A. Corpuscular
a) Bacterial vaccines
-for prophylaxis of cholera, leptospirosis, whooping cough.
- For treatment of staphylococcal, dysentery, gonorrhea, brucellosis.
b) Viral vaccines
-for prophylaxis of rabies, tickborne spring summer encephalitic, hepatitis A
influenza.
B. Subunits/ fractions
-against hepatitis B, meningococcal & pneumococcal infection.
- contain segment of bacterial cell/ viral.
C. Toxoids, or anatoxins
-against diphtheria, tetanus, staphylococcal infection, gas gangrene, botulism.

Corpuscular
-

Confer protection against most bacteria and viruses that maybe too virulent or can cause recurrent
infection or are oncogenic.

Produced by :
Chemically (formalin, formaldehyde)
Heat inactivation of bacteria, bacterial toxins or viruses.
Purification of components/ subunits of infection agents.
Administered with an adjuvant (absorbant) eg alum, which boost their immunogenicity.
Safe except in people allergic to vaccines component eg eggs.
Following immunity:
Usually not lifelong
Limited to humoral
Does not elicit local IgA response
Require booster shoots.
Require larger dose.

Subunits
-

Developed after identification of bacterial/ viral components that elicit protective immune response.
The immunogenic component:
Isolated from bacteria, virus or virus-infected cell biochemically.
Prepared thru genetic engineering, involving expression of cloned vial genes in bacteria or
eukaryotic cells.
Eg: hepatitis B subunit vaccine initially prepared from surface Ag obtained from human sea
of chronic carriers of the virus but the vaccine is no purified from yeast with gene for Ag.

Toxoids
-

Inactivated by 0.3 0.4% formalin, 28 30 days, 37-40C.

Bacterial toxins that have lost their toxicity but able to induce antibody.
Eg: diphtheria and tetanus toxins are adsorbed with aluminium salts to produce their toxoids.

IMMUNE SERA (IS) & IMMUNE GLOBULIN (IG)

-

Produce artificial passive immunity = resistance passively transferred to a recipient by administration

of Ab.
Agents used: (for prophylaxis & treatment.)
Hyperimmune sera & human origin (homologous sera).
Convalescent sera
Pooled human Gamma globulin.

a) Hyperimmune sera
- Humans sera gives longer lasting protection.
- Animals not recommended: cause allegic complication. But usually horses sera is used.
b) Convalescent sera
- Contain high level of specific antibody.
- Specific antibody produced by fractioning large pools of blood pools selected for high antibody
titer to a specific microorganism, microbial toxins or virus.
- Classified as:
i)
Antibacterial Ig = to treat whooping cough, leptopiosis, antrax, plaque.
ii)
Antitoxic Ig
= as prophylaxis & to treat diphtheria, tetanus, gas gangrene, botulism.
iii)
Antiviral Ig = as prophylaxis of hepatitis A & B, rabies, varicella, rubella, tickborne spring
summer encephalitis.
c) Pooled human Gamma globulin
- Contain Ab against all common pathogens prevalent in the region.
- To treat patients with immunodeficiency.

Tend to aggregate and when injected IV, may cause anaphylactic rxn due to complement activation.
Given IM only.
Preparation must be ensured free from risk of infection with Hep. B & C, HIV.

IS + IG (Combined immunization = passive + active immunization)

- Eg :
Protection of nonimmune individual with tetanus prone wound.
- inject tetanus Ig in one arm and 1st dose of tetanus toxoid in the other.
- tetanus Ig provide protection necessary till active immunity can take effect.
Patient exposed by bites/ contamination of open wound and animal infected with rabies.
- immunize patient: inactivated rabies vaccine + human rabies Ig
- human rabies Ig provide initial Ab until patient produces own Ab.
IMMUNOMODULATORS
-

Drug administered for correction of immune system during diseases.

Immunocorrection is aimed for rehabilitation of adequate immune response.
Immunomodulators = subs of different chemical & biological nature, which can influence
immunocompetent cells and their cooperation.
Classified into:
Immunostumulors;
Immunosuppressors;
Drugs for immune substitutional therapy.

Acc. to action:

Specific
Non specific

Acc. to origin:
I)

Homologic@ endogenous.
Natural subs produced by host (proteins, oligopeptides).
Play role in natural process immune response & their regulation.
Have strict target of action.
They regulate(activate, suppress or normalize) the separate or all stages of immune response.
Eg: IL, IFN, myelopeptides, hormone of thymus, TNF.
Produced by : genetic engineering

II)
Heterologic
- Subs from different origin, source of production & principle action on immune system.
- It is artificial bcoz not natural for human.
- Activate/ suppress immunocompetent cells (T, B-cells and macrophages) & modulate fx for whole
immune system.
- Eg:
Antibodies ( cyclosporine A)
Chemical subs (levamisol, levakadin, LPS from microbial origin eg pirogenal)
Antilymphocytic serum (adrenocorticosteroids)
Produced by:
i)
Biotechnological methods from plants, humans ( ie antilympholytic seum, hormones)
ii)
Biosynthesis after cultivation of bacteria & fungi(ie antibiotics, polysaccharides)
iii)
Chemical synthesis (ie myramyldipeptide).

19. Immunological memory and immunological tolerance

Immunological memory

Is the capacity of bodys immune system to remember on encounter with an antigen due to activation B/T
cells specificity for that antigen by means of these activated cells in a later encounter

Immunological tolerance
-

process which the immune system does not attack an antigen

it can either be
natural/self-tolerance (the body does not mount an immune response to self-antigen)
induced (tolerance to external antigen created by manipulating the immune system)
it occurs in 3 forms
1. central tolerance
during lymphocyte development
operates in thymus and bone marrow
T and B cells that recognize self-antigen are deleted before they develop into fully
immunocompetent cells, preventing autoimmunity
most active in fetal life, but continuous throughout life
2. peripheral tolerance
developed as T and B cells mature and enter the periphery
T cells that leave thymus are relatively but not completely safe. some will have
receptors that can respond to self-antigen that are present in such high concentrations
that can bind to weak receptors and that T cells did not encounter in thymus
these self-reactive T cells can inflict cell injury unless they are deleted
3. acquired tolerance
refers to immune system adaptation to external antigens
characterized by specific non reactivity of lymphoid tissue to given antigen
most important natural acquired tolerance is immune tolerance in pregnancy (placenta
must be tolerated by maternal immune system)
in adults tolerance may be induced by repeated administration of very large doses of
antigen/small doses required for stimulation of an immune response
in clinical practice tolerance is important in organ transplantation (when body must be
forced to accept organ from another individual)

20. General characteristics of Viruses. Structure. Classification. Viral multiplication.

Cultivation.
General characteristics

obligate intracellular parasites (dependent on hosts)

unicellular
contain either DNA or RNA
do not possess cellular organization
lack enzymes for protein and nucleic acid synthesis
cannot produce energy on its own
multiply by replication not by binary fission
unaffected by antibiotics
sensitive to interferon

Structure of viruses

Basic particle of virus is known as VIRION that contains either DNA or RNA but never both
Capsid
-protein coat surrounding the nucleic acid core
-protects nucleic acid from inactivation
-helps to introduce viral genome into host cell
Capsomers
-repeating protein subunits that make up capsid
-arranged around coiled nucleic acid = helical arrangement (eg:coronavirus)
-as cubes around spheroidal nucleic acid=icosahedral arrangement (eg:adenovirus)
Capsid + Capsomers = Nucleocapsid
Protomers polypeptide chains which make up capsomers
Virions may be enveloped or non-enveloped (naked)
Envelope
lipoprotein in nature
Protein subunits may be seen as projecting spikes on surface of envelop (peplomer)
may have more than one type of peplomer (eg: influenza virus)
confers chemical, antigenic and biological properties
susceptible to lipid solvents
Naked viruses
stabile in hostile environment
not damaged by drying, acid, detergent and heat
released by lysis
can infect GI tract and survive acid and bile
spread easily via hands, dust, fomites etc
neutralizing mucosal and systemic antibodies are needed to control the establishment of infection
Small viruses of diameter about 20nm = Parvovirus ; large of diameter up to 400nm = Poxviruses

Classification of viruses
They can be classified based on;
1. absence or presence of envelope
2. type of nucleic acid (DNA or RNA) and number of strands of nucleic acid
enveloped viruses
- single-stranded RNA eg Togaviridae, Coronaviridae
- double-stranded RNA eg Retroviridae
- double-stranded DNA eg Poxviridae, Herpesviridae

- mixed strand eg Hepadnaviridae

non-enveloped viruses
- single-stranded RNA eg Caliciviridae, Filoviridae
- single-stranded DNA eg Parvoviridae
- double-stranded RNA eg Reoviridae
- double-stranded DNA eg Papaviridae, Adenoviridae
3. symmetry of nucleocapsid
- helical symmetry
- icosahedral symmetry
4. size
- parvoviruses
- poxviruses
5. shape
- spherical
- rod-shaped
- brick-shaped
- tadpole-shaped
- bullet-shaped
- filament

Viral multiplication

depends on synthetic machinery of host cell because it lacks enzymes

5 stages

1. Adsorption
-occurs at specific receptor on host cell only when there is affinity between virions and cells
2. Penetration
- virus particles engulfed by mechanism called viropexia (like phagocytosis)
- for enveloped, fuse with plasma membrane and release nucleocapsid into cytoplasm

3. Uncoating
- in some cases affected by lysosomal enzyme of host cell
- in poxvirus, first outercoating removed by enzyme then internal core of virus released into
cytoplasm and is effected by viral uncoating enzyme thus releasing DNA
4. Biosynthesis
- DNA viruses synthesize in host cell nucleus except poxviruses
DNA enters host cell nucleus
uses host cell enzymes for transcription
for partially double stranded or single stranded, DNA changed into duplex form by viral DNA
polymerase in host cytoplasm before moving into nucleus for transcription
- RNA viruses synthesize in cytoplasm except orthomyxoviruses, paramyxoviruses and
retroviruses
transcription of messenger RNA from viral nucleic acid
translation of mRNA into early proteins. they initiate and maintain the synthesis of virus
component and shut down the host protein and nucleic acid synthesis. this is the most critical
step
replication of viral nucleic acid
synthesis of late proteins, which are components of daughter virion capsid

depending on method of mRNA transcription, single stranded RNA is classified into positive and
negative strand
positive RNA strand itself acts as mRNA and translated directly into viral proteins in the host
cytoplasm (it itself is infectious)
negative RNA strand possessed their own RNA polymerases for transcription (they are not
infectious)
oncogenic RNA ciruses (retroviridae) have unique replication as their RNA strand is converted into
RNA:DNA hybrid by viral reverse transcriptase enzyme. double stranded DNA then synthesized by
the hybrid and is intergrated into chromosome and development of neoplasia occurs

5. Maturation
- assembly of daughter virion
- make take place in nucleus (herpes, adeno) or in cytoplasm (picorna, pox)
- enveloped viruses gets enveloped from the cell membrane of host during process of budding
- non-enveloped are present intracellularly as fully developed virion
6. Release
- takes place by lysis of infected bacterium
- in animal viruses, release occur without lysis (myxoviruses)
- viruses like polio cause cell lysis during their release
cycle of replication in animal virus takes about 15-30 hours while in bacterial phage only about 1530 minutes
eclipse phase is the time from stage of penetration of viruses until appearance of mature daughter
viruses. In this phase virus cannot be demonstrated in host cell
Cultivation of viruses

cannot grow on inanimate culture medium

3 methods of cultivation ;

1.
-

Animal inoculation
growth of animals in inoculated animals may be indicated by their death, disease or visible lesions
serial blind passages may be necessary before evidence of virus growth are obtained
disadvantage is that immunity may interfere with viral growth so latent viruses may harbored
also used for study of pathogenesis, immune response, epidemiology and oncogenesis

2.
-

Chick Embryo
better because they are clean and bacteriologically sterile
do not have immune mechanism and do not need feeding and caging
have several sites for cultivation (chorioallantoic membrane, allantoic cavity, amniotic sac and yolk
sac)
several vaccines are produced from chick embryo
disadvantage is susceptibility of chick embryo is limited to a few viruses only and some bacteria may
kill the embryo

3.
-

Cell culture
very popular and useful technique
from tissue fragments, cells are dispersed by proteolytic enzymes
cells suspended in growth medium and distributed in Petri dishes
3 types of cell cultures ; primary cell cultures (normal cells freshly taken from body grown for the
first time), diploid cell strains (capable of 100 divisions in culture) and continuous cell lines (single
type cells mainly derived from cancer cells)

Detection of virus growth

1. Cytopathic effect
Many undergoes morphological changes when they grow; degeneration of entire cell sheet,
syncytium formation, formation of inclusion bodies, transformation. These can be observed under
microscope.
2. Plaque assay
Is a quantitative method when dilution of viral suspension is added to a monolayer of cultured cells
in a flask. After incubation, localized focus of infected cells can be seen with naked eyes. They are
known as plaques and each indicates an infectious virus.
3. Metabolic inhibition, or colour probe
Normally medium turns acid due to cellular metabolism. When viruses grow in cell cultures,
metabolism is inhibited and no acid produced. This can be indicated by adding phenol red
incorporated in the medium.
4. Hemagglutination
Occurs due to hemagglutinin receptors on surface of virus.
5. Hemadsorption
When hemagglutinating viruses grow in cell culture, their presence can be indicated by addition of
erythrocytes of defined animal or fowl species to culture. If viruses multiplies, erythrocytes will
adsorb on surface of cells.
Identification of viruses can be carried out by neutralization of cytopathic effect, plaque assay and metabolic
inhibition and also by hemagglutination and hemadsorption inhibition by specific immune serum.

21. Main methods of laboratory diagnosis of infectious diseases.

The main methods are
1. microscopic method (refer to Q4)
2. bacteriological method (refer to Q7)
3. biological method / animal models of investigations
Importance; can study:

pathogenicity
virulence
clinical symptoms
effect of treatment/vaccination
factors of pathogenicity/toxins

Biological method
-

consist of infecting animals for purpose of isolating the culture of causative agent and their
subsequent examination for pathogenicity and virulence
the choice of animals depends on aim of study
most frequently used are rabbit, guinea pigs, mice, rats, dogs and monkeys
sometimes gnotobiotic animals are used (without microflora)

stages;
Selection of animals
Only on healthy animals that are feed vigorously. Before inoculation, details of animals
(weight, age, sex, growth) must be recorded in protocol marking on animals and fixation of
animals.
Infecting the animals
Inoculation by subcutaneous, intramuscular, intraperitoneal, intravenous, or per os performed
with observation aseptic and antiseptic.
Observation of clinical disease
Observe change in body temperature, behavior, clinical symptoms, diarrhea, vomiting,
common cold, coughing, necrosis, formation of toxin etc.
Autopsy of animals
To observe changes in internal organs and to prepare smears stained with gram method and
are then fixed in slides.
For introduction of pure culture, agent have to do test in part of infected organs in nutritive
medium to study the properties of microbes
Prior to autopsy, body is soaked in disinfectant to prevent inoculation of operators and
contamination of surrounding objects. Instruments are washed in disinfectants and sterilized.

Aim of biological method;

reproduction of inflectional processes, to study pathogenicity and effective treatment

introduction of pure culture in microbes if growth in nutritive medium is impossible
to determine pathogenicity and virulence

virulence can be determined in doses of DL min, DL 50 and DCL

Dosis letalis minima ; is the minimum number of microbes cause death of 85% animals

 Dosis letalis 50 ; minimum number of microbes cause death of 50% animals

Dosis cerma letalis ; minimum number of microbes that cause 99% animal deaths

22. General characteristics of Enterobacteria. Escherichia coli. Salmonella. Methods of

laboratory diagnosis. Treatment and prevention.
Enterobacteria
largest, most heterogeneous Gram negative rods
present in large intestine
non-sporing
aerobic or facultative anaerobes
motile by peritrichate flagella or non-motile
grow on ordinary media: MEA or MEB
also on specific media : Endos, MacConkeys, Levins, Ploskirevs, Vi-Sagas
ferments glucose with acid and gas or only acid
reduce nitrates to nitrites
catalase positive and oxidase negative
Based on lactose fermentation they can be classified into:
1. Lactose fermenters eg Escherichia, Klebsiella
2. Late lactose fermenters eg Shigella sonnei
3. Non lactose fermenters eg Salmonella, Shigella
Commensal intestinal bacteria = LF (red on MacConkeys)
Intestinal pathogens = NLF (colourless on MacConkeys)

Based on modern taxonomy group together bacteria that possess:

1. common morphological and biochemical properties
2. similar DNA base composition
3. family/tribe and group/genera
Tribe 1 Escherichiae (Escherichia, Edwardsella, Citrobacter, Salmonella, Shigella)
Tribe 2 Klebsielleae (Klebsiella, Enterobacter, Hafmia, Serratia)
Tribe 3 Protecae (Proteus, Morgarella, Provideneia)
Tribe 4 Envinieae (Envinia)
Escherichia coli
Domain: Bacteria
Order: Enterobacteriales
Family: Enterobacteriaceae
Genus: Escherichia
Species: E. coli

Morphology

Gram negative

facultative anaerobes
nonsporing rods
usually motile with peritrichate flagella
some produce polysaccharide capsules
capsules and fimbriae may found in some strains
ferments lactose

Cultural and biochemical properties

grows well on ordinary media

produces colonies which are pink on Endos and MacConkeys, blue on Levins
smooth form (S) : colonies are large, thick, greyish white, moist, smooth opaque or partially
translucent
rough form (R) : colonies are flat, irregular, granular surface and often autoagglutinable in saline
ferments glucose, lactose, mannitol, maltose but not sucrose
positive indole production and methyl red test
does not occur in KCN media

Antigenic structure

serotyping is based on 3 antigens : somatic O, capsular K and flagellar H antigen

more than 170 O type, 100 K type and 75 H type have been recognized

Diseases caused by E. coli

Urinary tract infections (UTI)

Neonatal meningitis
Intestinal diseases (Gastroenteritis)

Epidemiology

occur by fecal-oral mechanism

can be transmitted through infected food, water or contact
Man is the only reservoir

Pathogenesis
5 classes of E.coli that causes diarrheal diseases;

Enterotoxigenic E.coli (ETEC)

Enteroinvasive E.coli (EIEC)
Enterohaemorrhagic E.coli (EHEC)
Enteropathogenic (EPEC)
Enteroaggregative E.coli (EAggEC)

Enterotoxigenic E.coli (ETEC)

cause diarrhea in infants and travelers
pathogenesis involves intestinal colonization by fimbrial adhesion (noninvasive) and diarrheagenic
enterotoxins : Heat Labile (LT) or Heat Stable (ST)
LT is similar to vibrio cholera enterotoxin. they are of 2 types: A = activated by cleavage of peptide
bond and internalized and B = binds toxin to target cells via specific receptor

 activated A then catalyzes ADP-ribosylation which bound adenylate cyclase which produces cAMP
leading to hypersecretion of water and elecrolytes into bowel lumen
ST causes increase in GMP in host cell cytoplasm. they are of 2 types: A = stimulates guanylate
cyclase increasing cGMP which inhibits intestinal fluid uptake and B = other mechanism
controlled by preventing transmission and by stressing importance of breast feeding of infants
best treatment is oral fluid and electrolyte replacement
antibiotics are not recommended

Enteroinvasive E.coli (EIEC)

shigella-like strain
dysentery-like diarrhea (mucous and bloody), severe inflammation, fever
penetrate and multiply within epithelial cells
does not produce shiga toxins
Sereny test cause rapid keraoconjunctivitis when placed on guinea pigs eye
virulent carries usually 140 megadalton plasmids

Enteropathogenic E.coli (EPEC)

nonfimbrial adhesion (intimin)

moderately invasive
does not produce LT or ST but may shiga-like toxins
usually infantile diarrhea, watery diarrhea, some inflammation, no fever

Enterohaemorrhagic E.coli (EHEC)

single strain
fimbriae adhesion
moderately invasive
produces shiga toxins but not LT and ST
copious bloody discharge, intense inflammation and may be complicated by hemolytic uremia
pediatric diarrhea may be fatal due to acute kidney failure (hemolytic uremic syndrome HUS)

Enteroaggregative E.coli (EAggEC)

nonadhesion
noninvasive
produces St-like plasmid encoded toxin (EAST) and hemolysin
persistent diarrhea in young children without inflammation, no fever

Laboratory Diagnosis
Bacteriological method is the main method
1. Inoculation into different diagnostic medium (Endos, Levins) and simultaneously into Ploskirevs
and bismuth-sulphite agar. Pink on Endos and blue on Levins.

2. Isolated culture identified by its morphological, cultural and other properties. Smears are made
followed by Gram stain.
3. Biochemical identification
4. Serological identification using slide agglutination with polyvalent OK-antisera, O-monovalent
antisera and also with diagnostic antisera for detection of O-, H- and K- antigens. Bacterial
suspension is warmed up to 100C beforehand for destruction of K antigen. This test is important to
distinguish ETEC and EPEC from other strains.

Treatment

based on symptomatology
fluid replacement is primary treatment
antibiotics not used unless in severe cases that has progressed to a systemic stage (resistant to
penicillin but sensitive to ampicillin, streptomycin, tetracycline)
bacteriophage therapy

Prevention

no specific prevention : active and passive immunization

Salmonella
Domain: Bacteria
Order: Enterobacteriales
Family: Enterobacteriaceae
Genus: Salmonella
Species: Salmonella typhi, parathypi A, B, C, enteritidis, choleraesuis, thyphimurium

Morphology

Gram negative
motile with peritrichate flagella
noncapsulated and nonsporing rods
may possess fimbriae

Cultural properties

facultative anaerobes
grows on ordinary media at optimum temperature of 37C
colonies are large, circular low convex, smooth and translucent
on MacConkeys, Endos and Levins, colonies are colorless (lactose fermentation negative)

Biochemical properties

ferment glucose, mannitol and maltose forming acid and gas

does not ferment lactose, sucrose and salicin

indole negative
methyl red positive
Voges-Proskauer negative
citrate positive
H2S produced, urea not hydrolyzed
enteric fever group may be separated biochemically

Antigenic composition

can be serotyped according to somatic O, surface (envelope) Vi, phase 1 flagellar and phase 2
flagellar H antigens
according to these antigens salmonella are divided into serogroup A until T
classification is based on Kauffman-White scheme which depends on identification by agglutination
on structural formula of O and H antigens of the strain
initially they are based on O antigenic factors and designated 1,2,3 etc
phase 1 antigens are either specific for species or shared by a few species only [hence specific phase]
and are designated a,b,c,d etc
phase 2 are widely spread [nonspecific/group phase] and are designated 1,2,3 etc
within each group differentiation is by identification of phase 1 and phase 2 flagellar antigens
Kauffman-White scheme gives species status to each serotype

Resistance

Salmonella destroyed at 55C in one hour or at 60C in 15 minutes

boiling/chlorination/pasteurization
in polluted water, soil, they survive for weeks and in ice for months
may viable for years if prevented from drying
killed within 5 minutes by mercuric chloride (1:500) or 5% phenol

Factors of pathogenicity

invades and replicates in lumen of small intestine

stimulates rearrangement of actin with subsequent engulfment of bacteria within endocytic vesicles
chromosomal invasion genes mediate attachment/penetration
acid tolerance response genes protects from stomach acid/acid pH
catalase and superoxide dismutase protect from intracellular killing
inflammation mediates release of prostaglandins thus active fluid secretion

Epidemiology, Pathogenesis, Clinical syndromes

found in virtually all animals including poultry, livestock, rodents, domestic animals, birds and
humans
cause diseases in human and nonhuman hosts

1. Gastroenteritis (food-poisoning)
- Transmission by fecal-oral route through contaminated food or water or by contact from
patients or carrier, infected animals such as cattle, domestic cats, hamsters, birds
- symptoms usually begin 6-48 hours after ingestion of contaminated food or water

coordinal manifestation is diarrhea

nausea, vomiting, abdominal cramps, myalgia, headache, fever and chills are common
duration of fever or diarrhea normally 2-7 days
eg S. cholerasuis, S. thypimurium and S. enteritidis

2. Septicemia
- commonly found in children or adult with low immunity
- an intermediate stage of infection
- no internal symptoms and bacteria cannot be isolated from fecal specimens
- may remain localized in intestine or disseminate to the bloodstream
- eg S. hirshfeldii, S. thypimurium, S. cholerasuis

3. Enteric fever
- S. thypi, S. parathypi A, B and C
- transmitted by fecal-oral route through contaminated food or water or by contact with
patients and carriers
- stages:
i.
Ingestion
Salmonella ingested orally. Passes stomach barrier and enters small intestine.
ii.
Invasion
Penetrates mucus layer and enter mononuclear phagocytes of ileal Peyers patches and
mesenteric lymph nodes. Proliferate. Incubation period 7-21 days.
iii.
Bacteremia
1st week of disease. Spread to blood.
iv.
Bacterial dissemination
2nd week of disease. Enters spleen, liver and bone marrow (reticuloendothelial system).
From gallbladder many bacteria enters intestine again.
v.
Hyperergia and excretion
3rd week. Ulceration of mucous in region of Peyers patches of small intestine causing
hemorrhage and perforation. Excretion through feces and urine.
vi.
Final
4th week. 3 results: recovery, forming carrier or fatal case.
-

incubation period is accompanied by fever which is the main presenting symptom

also accompanied by general malaise, headache, sweating, diarrhea, abdominal pain and
vomiting
if not treated, intestinal infection may be severe and cause CNS involvement as delirium and
coma
in fair skin people, some skin rash may appear called rose spots
organisms live intracellularly in macrophages of reticuloendothelial system eg liver, spleen,
and lymph nodes

Laboratory diagnosis

depends on isolation and identification of Salmonella from sample of patients blood or feces by
bacteriological method

1st day; inoculation of 5-10ml of blood in 50-100ml of 10% bile broth or Rappaports medium 1:10 at
37C for 24hours
2nd day; investigation of morphological and tinctorial properties by Grams Method. Investigation of
mobility under dark-field microscope. Subculture on Endos, Ploskirevs and Levins medium.
3rd day; investigation of cultural properties. Isolation of pure culture on Ressels media (green colour)
at 37C for 24 hours or on Kliglers media (red colour)
4th day ; identification of pure culture by morphological and tinctorial properties, biochemical
properties on Ressels, antigenic properties using slide agglutination reaction with monoreceptor
serum, bacteriophage typing and antibiotic sensitivity test.
use Widal Test in diagnosis of enteric fever to measure O and H agglutinins
- adding suspension of dead typhoid bacterial cells to series of tubes containing patients serum
which has been diluted to various concentrations
- after incubation for 30 minutes at 37C, they are centrifuged and examined to note the
amount of agglutination occurred
- reciprocal of highest dilution at which agglutination is seen designated as antibody titer of
patients serum
- naturally the higher the titer the higher the antibody response to disease
- in typhoid cases, titer of specific O antibodies is 1:80 or titer of specific H antibodies is
1:160
- in paratyphoid cases, titer of H antibodies is 1:80

Treatment

antibiotics such as chloramphenicol (best drug), ampicillin, cotimoxazole etc

replacement fluid if there is dehydration

Prevention

administration of vaccines through injections or oral route

- Oral: live oral vaccine (typhoral) given before food with a glass of water or milk on 1st, 3rd
and 5th day (one capsule). No antibiotics should be taken during this vaccination.
- Injection: typhim-Vi contains purified Vi polysaccharide antigen derived from S. typhi.
Single dose. Given only to 5 years old and above.
- TABTe vaccine: effective in prevention of paratyphoid fevers and tetanus.

23. Causative agent of bacterial dysentery and cholera. Methods of laboratory

diagnosis. Treatment and prevention.
Causative agent of bacterial dysentery: Shigella
Kingdom: Bacteria
Order: Enterobacteriales
Family: Enterobacteriaceae
Genus: Shigella
Species: S. boydii, S. dysenteriae, S. flexneri, S. sonnei

Morphology

Gram negative
non motile
facultative anaerobe
nonsporing rods
failure to ferment lactose or decarboxylate lysine
closely related to E.coli

Cultural properties

Grows well on ordinary media at 37C and pH 7.4

colonies are small, round, convex, smooth and translucent
on Endos and MacConkeys they are colorless due to absence of lactose fermentation

Biochemical properties

catalase positive
citrate not produced
H2S not formed
nitrates reduced to nitrites
biochemically inactive
glucose and mannitol fermented forming acid

lactose and sacrose not fermented except by S. sonnei after 24 hours

Classification
Based on fermentation
1. Non-mannitol fermenters Shigella dysenteria
2. Mannitol fermenters Shigella flexneri, boydii and sonnei
Based on serotypes
1.
2.
3.
4.

A [S. dysenteriae , 12 serotypes]

B [S. flexneri, 6 serotypes]
C [S. boydii, 18 serotypes]
D [S. sonnei, 1 serotype]

Antigenic properties

somatic O antigen
some strains have K antigen in cell wall
fimbriae may present

Virulence
1. Invasin
- encoded by large extra chromosomal elements (plasmids)
- induces the endocytic uptake of Shigella by M cells, epithelial cells and macrophages
- deform the plasma membrane of contiguous cells
- IcsB plasmid-encoded protein lyses the plasma membranes, resulting in intracellular bacterial
spread
2. Endotoxin
- cause fever, shock, bloody and mucous stool and abdominal pain (cramps and tenesmus)
3. Exotoxin
- Shiga toxin (verotoxin)
- chromosomally encoded
- toxin inhibits protein synthesis
- makes disease appears as diarrhea

Epidemiology and pathogenesis

source: patients, carrier, recovered people

infected from contaminated food, water, hands
bacilli infect epithelial cells of large intestine and multiply inside them
inflammatory reaction follows and leads to necrosis of patches in epithelium
bacteremia in children
short incubational period about few hours
symptoms: diarrhea with blood and mucus, high temperature, hemolytic syndrome
disease lasts for 1 week

Laboratory diagnosis of dysentery

1. Bacteriological method
1. 1st day
- inoculation f investigated material on Ploskirevs medium using portions of stool
containing pus and mucus obtained directly from rectum
- incubation at 37C for 24 hours
2. 2nd day
- investigation of cultural, morphological and tinctorial properties
- investigation of mobility under dark-field microscope
- isolation of pure culture on Ressels media
- temperature at 37C for 24 hours
3. 3rd day
- identification of pure culture by morphological and tinctorial properties by Grams
Method
- investigation of biochemical properties on Ressels medium
- investigation of antigenic properties of antigenic properties using antiserum of
shigella dysenteria, flexneri, boydii, sonnei using slde agglutination reaction (positive
with dysenteria)
- determination of sensitivity to bacteriophages (lysed)
- antibiotic sensitivity test (high to tetracycline, middle to ampicillin and low to
penicillin)
2. Agglutination reaction in test tubes similar to the Widal reaction (retrospective diagnosis)
3. Reaction of passive hemagglutination for chronic dysentery
4. The fluorescent method is used for direct identification of Shigella in smears from feces by means of
specific sera treated with fluorochromes
5. An allergic test consisting in intracutaneous injections of 0.1ml dysenterin is applied in diagnosis of
dysentery in adults and children. Hyperemia and papule from 2 to 3.5cm in diameter developed at the
site of injection in 24 hours in a person who has dysentery. The test is strictly specific
6. Biological method by injecting into guinea pigs conjunctiva resulting in keratoconjunctivitis

Treatment

mild cases are treated without antibiotic therapy

sever cases treated by IV replacement of fluid and electrolytes and by using antibiotics
inactivated vaccine consisting of S. flexneri and S. sonnei used for chronic cases

Prophylaxis

hospitalization or isolation at home with observance of the required regimen

treatment with a phage of all persons who have been in contact with sick individuals
observance of sanitary and hygienic regimens in childrens institutions

Causative agent for cholera: Vibrio cholera

Domain: Bacteria
Order: Vibrionales
Family: Vibrionaceae
Genus: Vibrio
Species: V. Cholerae, Eltor, and Bengal

Morphology

Gram negative
curved shaped with rounded ends (short)
are seen arranged in parallel described by R. Koch as fish stream
motile with monotrichate flagella
nonacid fast bacteria
noncapsulated and nonsporing

Cultural properties

facultative anaerobic
grows better in alkaline nutrient agar (optimum pH 8.2)
colonies are small, moist, translucent, round disks with bluish tinge in transmitted light
in peptone water, growth occurs in about 6 hours as a fine surface pellicle which on shaking breaks
up into membranous pieces
on MacConkeys colonies are colorless
on blood agar, colonies are surrounded by hemolysis
on TCBS (thiosulphate, citrate, bile salt, sucrose), colonies are large, convex, yellow colour

Biochemical properties

ferments glucose, maltose, mannitol, mannose, sucrose forming acid but no gas
does not ferment inositol, arabinose and lactose
indole and H2S negative
nitrates reduced to nitrites
catalase and oxidase positive
gelatin is liquefied
methyl red and urease test positive

Resistance

susceptible to heat, drying and acids

destroyed at 55C in 15 minutes
dried on linen or thread, it survives for 1-3 days
susceptible to common disinfectants
killed in a few minutes in gastric juice of normal acidity but may survive for 24 hours in achlorhydric
gastric juice

able to survive and replicate in contaminated water with increased salinity and at temperature of 1030C

Antigenic structures

has all somatic O thermostable antigen

belong to 01 (V. cholera and V. eltor) and 0139 (V. Bengal) subgroup
all can occur in epidermic and pandemics
01 can be divided into 3 fractions A, B, C
1. Ogawa (A,B)
2. Inawa (A,C)
3. Hikojima (A,B,C)
each H-flagella thermostable antigen is common for genus vibrio

Factors of pathogenicity

V. cholera has adhesins and produces enzymes and toxins

adhesion to epithelial surface and colonization may be facilitated by special fimbria such as toxin
conjugated pilus
vibrio elaborate several enzymes collagenase, elastase, chitinase, decarboxylase, lipase, mucinase
and neuraminidase
enzymes enable the causative agent to overcome the protective mucosal barrier and reach the
enterocytes. Throughout the course of infection, vibrio remain attached to epithelium but do not
damage or invade cells
produce exotoxin called cholera toxin which is very similar to LT but they are more potent
also possesses endotoxin

Epidemiology, pathogenesis and clinical syndromes

exclusively human disease
sources: patients, carriers, recovered people
transmission: food, water and hands
stages:
i.
after ingestion passing through acid barrier of stomach
ii.
multiplication in alkaline environment of small intestine where choler toxins produced
cholera toxin characterized by abrupt onset of watery diarrhea and vomiting
clinical forms of cholera:
i.
incubation period 2-3 days
ii.
distinguished 3 stages in development of disease
- cholera enteritis
lasts 1 or 2 days
diarrhea
in some cases, the infectious processes terminate in this period and patient
recovers
- cholera gastroenteritis
characterized by profuse diarrhea (rice water stools) and continuous vomiting
symptom may lead to degradation of patients body
- cholera algid
skin becomes pale due to loss of water

cyanosis appear
body temp fall to 34C
characterized by dyspnea, uremia, cholera coma and death

Laboratory diagnosis
1. microscopical method
- under light microscopy, morphological, tinctorial and immobilization examined
- can be viewed directly in stool samples, particularly by dark-field microscopy
2. immunofluorescent test
- identifying observed cells as V. cholera

3. bacteriological method
Day 1
Results
1. Inoculation of investigated material on
1. On alkaline agar, colonies are circular,
alkaline agar and 1% peptone water
smooth, moist, transparent, colorless
for 6-10 hours at 37C
with bluish shiny
2. Investigation of cultural properties
2. On 1% peptone, there is delicate
(after 6-10 hours)
pellicle on surface
3. Investigation of morphological and
3. Gram negative, coma shaped rods
tinctorial properties by Grams
Method. Investigation of mobility
under dark-field microscopy
4. Slide agglutination with O-antiserum
4. very mobile
5. Isolation of pure culture on slant
5. positive agglutination reaction
alkaline agar at 37C for 10-12 hours

Day 2
1. Identification of pure culture:
morphological, tinctorial, biochemical
properties
2. Investigation of antigenic properties
by agglutination test with O-antiserum
and antiserum Inaba & Ogawa
3. Identification cholera and eltor
i.
hemolysis of sheep erythrocyte
ii.
hemagglutination with chicken
embryo
iii.
sensitivity to polymexin B
iv. susceptibility to bacteriophage

1. Gram negative, comma shaped rods,

mannose and sucrose positive forming
acid. Arabinose negative
2. Positive with Ogawa antiserum
3. cholera
+

eltor
-

+
+

+
+

Difference of Biovars of Cholera Vibrio

Different properties
V. Cholerae biovar Cholerae
Sheep erythrocyte hemolysis
Sensitivity to polymyxin B
+

V. Cholerae biovar Eltor

+
-

Lysis by bacteriophages
-cholera phage C
-phage eltor
Hemagglutination with
chicken erythroctes
Voges- Proskauer reaction

+
-

+
+

4. serological method
detection of antibody in patients serum in tube agglutination test
passive hemagglutination
ELISA test
5. genetic method DNA mapping
6. rapid method
immobilization test of vibrio, cultivated in alkaline peptone water by V. cholera 01 antiserum
in dark-field microscopy
Ermolrevas method

Treatment

rehydration and replacement fluid and electrolyte with commercial or hand-mixed sugar-salt solution
or massive injections of liquid given IV in advanced cases
antibiotics usually tetracycline or doxyclicline
cholera bacteriophage
syndrome therapy

Prophylaxis

isolation and hospitalization of source of infection

avoid contaminated food or water
appropriate hygiene
chemoprophylaxis with tetracycline or doxycycline for 2 days
killed cholera vaccine enriched with cholera toxoid
live vaccines administered orally

29. CAUSATIVE AGENTS OF ZPPNOTIC DISEASES ; TULAREMIA,

BRUCELLOSIS ANTHRAX AND PLAQUE
Anthrax
Causative agent : bacillus Anthracis
Morphology non acid fast

Rod shape (largest ), gram +, in tissue it is found singy in pairs or in short chain, surrounded by
colourless in organism
In miropreparation, the bacilli are arranged in long chain, look like bamboo stick
Spores are formed and arranged in center of baciliflagella are absent

Physiology

Facultative anaerobe- wirh temp 12 45 but optimum temp 36 37

Good growth on ordinary media (MEA, colony are raised and opaque, greyish white, under low
power microscope the edge of colony is composed of long chain of bacilli (medusa head)
On blood agar, colony are non haemolytic, in MEB, bacillus anthracis are growth as flocular deposit
with little turbidity
Selective media called MLET consist of polymyxin, lysoxin, ethylene, diamine, tetra acetic acid
colony are greyish white, opaque, raise, large in size
B, anthracis may grow in medium with penincilin. After 6 hours, if we prepeare micropreparation,
we cant see rod but oval- string of pearls

Biochemistry

Glucose,maltose, sucrose are fermented and forming acid, nitrate reduced to nitrite, catalase +, no
gas

Resistant

Vegetative bacilli are not particularly resistant, destroyed at temp 60 celc in 30 mins
In organ or animal, which died due to anthrax, the bacilli remain variable in bone marrow for 1 week,
and in skin for 2 weeks

Spores
o Increase resistant to physical and chemical agent
o Example, dry heat spores may survive for 1 3 hours, and boiling for 10 mins
o Mey present in soil for many years

Pathogenicity

2 virulent factor have been identified

Capule polypeptide
Inhibit phagocytosis and major virulent factor in early stages of diseases,
Anthrax toxin
Complex of 3 factors edema fator protective antigen factor lethal factor
They are not toxic individually, but the whole complex cause local anemia and
generelized shock

Pathogenesis and disease in man

Anthrax is zoonotic disease. Animal are infected by injection of spores present in soil and water
Direct spread from animal to animal is rare
Human anthrax is contacted from animal directly or indirectly
Disease can be in 3 clinical form
Cutaneous
Pulmonary
Intestinal
All types lead to lethal septicaemia
Cutaneous anthrax
Following entry if infection through skin. Clinical syndrome : face, hand, neck are3 ususal
size. The lesion starts as populla 1-3 day after infection and become vesicular containing fluid
which may be clear of blood stained. The name anthrax which mean coalcome from the
balck color of eschar ( dead tissue).
Pulmonary anthrax
Occur as result of inhalation of spores. This is haemorhagical pneumonia with high fatality
rate
Intestinal anthrax
Rare disease. Main clinical syndrome : vomiting with blood
Septicaemia form ; cause death

Laboratory diagnosis
Material : exudate from vesicular, papula, sputum, feces and urine, blood if patient have septicaemis
1. Microscopical examinations
a. Prepare micropreparataion stained with gram method
b. See gram + large stepto bacilli may surrounded with capsule
2. Bacteriological
a. 1st day inoculate in MEA 37 c 24 hours
b. 2nd day in MEA, colony are regular : large opaque : greyish white , irregular edges ( medusa
head)
- in MEB, we can see cotton wool which sink to bottom of tube
c. From colony, prepare micropreparation stained with gram method and find large gram + rod,
arranged in chains resemble bamboo stick
d. Then isolate of pure culture on slant mear agar
e. 3rd day : identify of pure culture

i. Prepare mpreparation stained with gram method. See large gram + and rod
ii. Biochemical properties, glucose + forming acid, sucrose + forming acid , maltose +,
HS +, milk is coagulated and peptonized formed acid but no gas
iii. Exam factor of pathogenicity
-hemolysin absent, lechitinaeactivity present in yolk salt agar, coagulase +, cattalose +
-prepare penicillin test; b anthrax growsn and after 6 hours may see oval shaped
-prepare bacteriophage typing and immunoflurosent method on blood agar; hemolysin
-, yolk salt agar : lechitinase +

3. Serological examination
a. Ascolis test place anthrax antiserum in to test tube with extract from tissue/organ - +
result, ring of precipitation is formed
4. Biolpgical
a. Infected animal, see clinical syndrome, after animal death, exam changes in internal organ,
prepare micropreparation and inoculate on culture medium
Treatment and prophylaxis
1. Anti anthrax globulin IM and antibiotic (penincilin)
2. To prevent : use 2 vaccine; living vaccine, prepare capsulated anthrax bacilli and consisiting of
suspension of life spores of vaccine strain
3. Chemical anthrax vaccine: consist of protective antigen, noncapsulated anthrax
4. Penincilin test
o Culture of microbe is inoculated on MPA contain penincilin temp 37c 3-6 hours
o Result- cells become large, spherical and occur in chain in the form of pearls
5. Allergy test
o Inject SC anthracin and see the reaction
o Healthy; no changes
6. Express diagnosis /immunodiagnose test
Plaque
Causative agent: yersenia pestis
Morphology

Short, ovoid bacillus with rounded end convex sides

Arranged singly in shot chain
Gram chain in smear stain with methylene blue it shows bipolar staining
Non motile, non sporing, capsule may be present

Physiology

Facultative anaerobes and aerobes. Optimum temp 25c - 30c

Can also grown 0 - 45 , pH 6.9 7.0
In MEA, colony are small delicate look like transparent disc
In MPB, cultures formed pellicle at surface with thread like growth thread
Sodium suphate, fresh haemolytic blood are used as growth stimulator

Biochemistry

Methyl red +
Citrase Glucose, maltose, galactose, arabinose, manitole ferment forming acid
Not ferment lactose, HS +, indole -, catalase + oxidase and urea test -, gelatine not liquefied, reduce
nitrate nitrite

Antigenic

Contain several antigents (D,F,T,W)

Virulent Y. pestis contain thermostable somatic antigen, increase toxic for mice and rats. It may
change to anatoxin under effect of formalin

Toxin production

Microbes is very vitulent. It produces thermolabile toxin in 2 forms : A and B and mouse toxin. It
cause hemolysis of RBC and dissolve fibrin

Resistant

Y. Pestis can withstand low temperature 0c for 6 months aand survive on clothe or 6 months
In water, 1 month
In bubo pus, 20days
Sputum 10days
Fruit and vegetable 1-2 weeks
In bread 4 days
Very sensitive and dying in increase temperature
Boiling kill microbes in 1 min in 5% phenol sol 5 mins

Pathogenesis and disease in men

Plaque is zoonotic disease, plaque basili is naturally paracytic

In rodents, infection is transmitted among them by rat fleas. The fleas acquired the infection by
feeding in infected rodents
In fleas, the bacilli multiply in stomach to such an extend that they block the proventriculus. When
such fleas bite on other rodents, it cant suck in blood because the bacteria mass blocked the passage
mechanically.
The blood mix with bacteria is regurgitated into bile and transmit the infection
Infection alsso transmitted by contamination of bile wound with the feces of infected fleas
Y, pestis enter huma body through abrasion of mucous membrane in pneumonic form, the plaque
bacilli are spread in air with sputum
Several formed of human plaque
o Cutaneous
o Bubonic
o Cutaneous bubonic
o Pulmonary
o Intestinal
o Sceptical
Plaque devide quicly without prodromal stage, its characterized by severe headache, face is pale and
bluish facies pestica
Each form of plaque show characteristic clinical syndrome, phagocytosis is inhibited , there is
marked disintegration and necrosis of bulb
, small haemorrhages formed in skin, liver enlarged showing necrosis + haemorrhage
Spleen is enlarged too and dark red in colour
After recovery, stable immunity is acquired

Laboratory diagnosis
1. Material from lymph node, skin lesion, bubo, mucous from pharynx, sputum , blood
2. Exam is carried in special lab and in anti-plaque protective clothes
3. Microscipical exam
o Mpreparation is fix in nikiforov mixture, DONT under flame , because yerseria may change
form
o Stained with gram method or methylene blue. Gram oval shape, rod with bipolar staining
4. Bacterial method
o Inoculate on MPA and MPB
o MPA colony with turbid white center
o MPB : form pellicle SURFACE WITH STALACTIC

o PREPARE micropreparation and exam morphology properties and subcultured on slant meat
agar
o Identify pure culture of microbe
Micropreparation stained in gram method
Biochemical, glucose,a rabinose, maltose + produce acid
Pathogenic factor; vibrinolysin +, plamna coagulase + on heminagar, colony are
brown
Prepare slide agglutination test with plaque anti serum and we can see clumps
o Serological exams
Prepare immunoflorosent test : see green light
o Biochemical exams
Infected guinea pig with plaque of after death (5-7 days) we see changes in internal
organ and isolate microbes from these organs
Treatment and prophylaxis
For treatment : anti laque serum, anti plaque gamma globulin, specific bacteriophage and
antibiotic (streptomyxin, gentamyxcin, teramyxcin)
For prevention : live vaccine and non specific prophylaxis : early diagnosis immediate
isolation and hospitalization, disinfection and extermination of rats and systematic
observation carried out by plaque control library.
Francisella Tularensis
Causative agent for tularemia
Morphology

Small , capsulated, non motile gram rods

In culture tends to be pleomorphic and filamentious
Capsule have lipid nature
In infected animal, act as intracellular parasite, found in mass inside the liver and spleen cell

Cultural properties and biochemical properties

Strict anaerobes
Fastidious growth requirements and special media Francis dextrose blood agar
Minute droplike colonies appear after incubation for 3-5 days
Weakly catalyse + and oxidase
Slow catabolism of carbohydrates is slow, produce acid, no gas
H2S is produced

Resistance

Killed by moist heat at 55c after 10 mins

May remain for many years in 10c
Many days in moist soil and infected water by animal

Factors of pathogenicity

Intracellular parasite, can survive in prolonged periods in macrophages of the reticuloendothelial

systemposses antiphagocytic capsule , loss of capsule is associated with decrease virulence
Have endotoxin but less active than others

Epidemiology, pathogenesis and clynical synfromes

Found in many wild mammals, domestic animals, fish, and blood sucking anthoropods as well as
contaminated water
Human tularaemia is acquired by the bites if infected arthropod, contact with infected animal,
consumption of contaminated meat and water
Subgroups
o Ulceroglandular
o Oculoglandular
o Glandular
o Typhoidal
o Oropharyngeal
o Pneumonic
o Gastrointestinal

Lab diagnostic

Specimen ; ulcer scraping, lymph node biopsy specimen, gastrinc washings, sputum and blood
Can be established by immunofluorescence, bacteriological, biological serological method and skin
test
Indirect immunofluorescence used for smears obtain from skin lesion
F. Tularensis can grow on chocolate blood agar, cysteine blood agar or glucose cysteine agar
Identification is made by cultural properties a, biochemical properties and alide agglutination
Guinea pigs and mice is used for inoculation of clinical specimen, liver and spleen are studied post
mortem
Skin test with tularin is aimedfor revealing of delayed hypersensitivityand resistance to tularemia

Treatment

Streptomycin is the antibiotic of choice for treatment of all forms of tularemia,

Gentamicin is an acceptable alternative
F.Tularensis strains produce Beta-lactamase, which renders penicilins and cephalosporins ineffective

Prophylaxis

Live attenuated vaccine is abailable for people at high risk for contacting disease.

33) Causative agent of hospital infection. Principle of laboratory diagnosis &

prophylaxis
- Infection developing in hospitalized patient, not present or incubate at the time of their admission, infection
evident during their stay or after discharge.
- Can be exogenous or endogenous origin
- Exogenous = from environment & medical personnel
- Endogenous = patient own skin, GIT / resp flora
- Infection caused by microorganism which present in general population. Patient with higher risk of dying
have higher risk infection
- Transmission from indivual to other, hosp staff to patient. Between individual may be direct( hand contact)
& indirect ( Inhalation, ingestion, puncture through integument). Viral may spread through air. Blood for
transfusion may contaminated with Hep A, B C or HIV. Food in hospital maybe contaminated.
- Exogenous microorganism acquired from improper sterilse instrument or contaminated disinfectant sol.
- Patient acquire infection as result of break down of their own immune system.
Causative agent of hosp infection

Gram + cocci (S.Aureus, S.epidermis)

Enteric gram ve rod (E.coli, Enterobacter,proteus)
Candida spp
Fungi ( aspergillus, mucor group)
Virus (Hep b,c rubella, influenza virus)
Protozoan parasite (toxoplasma gondi & entamoeba histylitica)

-Most pathogen ass. With nosocomial infection has insignificant multiple antibiotic resistance by R-plasmid
- Most common type of hosp infection : wound infection, urinary tract infection, resp.infection, bacteremia,
septicaemia
- May occur as a sporadically or as outbreak
- Preventive medicine apply to patient & medical personel. Vaccination. Hand wash. Use of antiseptic
technique during surgery.

34. POXVIRUSES. SMALLPOX VIRUSES

BIOLOGICAL PROPERTIES
Taxonomy
Family

Genus

POXVIRIDAE

ORTOPOXVIRUS

Members
Variola Virus (SMALLPOX)
*
Vaccinia Virus

Morphology

brick shaped
seen under microscope when stained by silver impregnation enclosed by outer membrane &
envelope containing many enzymes and other proteins
genome: large double-stranded, linear DNA (which is fused at both ends)

Replication

Entire multiplication cycle take places within the host cell cytoplasm. As a result, the poxviruses
must encode the enzymes required for mRNA and DNA synthesis as well as the other DBA viruses
normally obtain from the host cell
Viral penetration occurs within phagocytic vacuoles. After this, the outer membrane is removed in
the vacuole and early gene transcription is initiated.
the uncoating proteins (which are produced in the virion core among others
proteins,enzyme,activator etc.) will removes the core membrane and liberating the Viral DNA into
cell cytoplasm
Next, the viral DNA replicates in Guanieris inclusion bodies = the factories
Late mRNA is translated into structural and virion proteins
*unlike other viruses, poxviruses assemble around the core factories. The viral particles are produced
and are released on cell lysis

Resistance

poxviruses are stable, if protected from sunlight can remain viable for a month at room temperature
survived for years in the cold or freeze dried
susceptible to UV light and other irradiations
resistant to 50% glycerol and 1% phenol but are readily inactivated by formalin and oxidizing
disinfectant

Cultivation

cultivated in chick embryo, tissue culture and lab animals

cytopathic effect on tissue cultures; formation of eosinophilic Guanieris inclusion bodies
*vaccinia virus can infect a wide range on animals (monkeys,calves,rabbits) experimentally but
variola virus produces small lesions on monkeys

Antigenic structure

(NP) nucleoprotein antigen common antigen for poxviruses

20 different antigens have been identified ,these includes;

-LS antigen (complex of heat labile L & heat stable S antigens)
-agglutinogen
-hemagglutinin (responsible for the agglutination of erythrocyte)

SMALLPOX VIRUS
Epidemiology, pathogenesis & clinical syndromes

exclusive to human infection, with no animal reservoir , & no carriers because the virus is totally
eliminated completely from patient on recovery
source of infection was a patient in the early phase of disease
infected host through inhalation and the virus replicated in upper resp. tract
dissemination occurred via lymphatic and cell-associated viremic spread
internal and dermal tissues were inoculate after a second, more intense viremia
the skin lesions eventually became vesicular and pustular
forms of smallpox- 1) variola major (more virulent) *2)variola minor/alastrium (less virulent)

Immunity

cell-mediated immunity is essential to resolve the poxviruses infection

however the poxviruses encode protein can help the virus to evade the immune control
(These include protein that impede the interferon, complement, and inflammatory responses)
Lab. Diagnosis

smallpox can be diagnosed by microscopical, virological and serological methods

rapid diagnosis on distinctive morphology was possible by electron microscopy
isolation of virus can be achieved by cultivation in tissue cultures of blood in early phase in severe
cases or from eruptive lesion in all cases

Prophylaxis

vaccination against smallpox is highly effective, involves the intradermal admin of live viruses
preparation with an antigenic composition identical to that of variola
however, it can occasionally causes generalized infection that can be fatal depending on the immune
system status

35. ADENOVIRUSES
Taxonomy
Family ; Adenoviridae
Genus ; Mastadenovirus
Morphology

non-enveloped, double-stranded DNA viruses

Icosahedral in shape, comprising 10 structural proteins
capsid comprises capsomers which consist of hexons & peptons
the 12 pentons, which are located at each of the vertices, contain a penton base a fibre
the fibre contains the viral attachment proteins and can act as hemagglutinin. The penton base abd
fiber are toxic to cells. They also contain type-specific antigens
the core complex within the capsid contain viral DNA and at least 2 major proteins

Replication

viral fiber protein interact with cell surface receptors, and virions enter the cell by endocytosis
the virus lyses the endosomal vesicle and the capsid delivers the DNA genome to the nucleus
the penton and fiber proteins of the capsid are toxic to the cell. They inhibit cellular macromolecular
synthesis
the first mRNAs code for non-structural early proteins which shut off most of host cell activities,
while switch on the host cell DNA-dependent DNA-polymerase which required for replication of
viral DNA
the remaining genes are transcribed from it to form late proteins which are produces in cytoplasm
They are back to the nucleus where new virus particles are assembled.
Empty capsids first assembled and then the viral DNA and core proteins enter the capsid through an
opening at one of the vertices
Cleavage of several of the capsid proteins and of the terminal protein attached to the DNA causes the
particle to mature into a stable and infectious virions
The effect of shutting down the host cell metabolism and the accumulation of thousands of new
virions result in rupture (lysis) an death of infected cell with release of the particles

Resistance

Adenoviruses are relatively stable, remaining viable for about a week at 37C
Readily inactivated at 56C and by UV radiation
Resist ether, chloroform and bile salts

Cultivation

Adenoviruses are host specific and so lab animals are not susceptible to adenoviruses infecting
human
Human adenoviruses grow only in tissue cultures of human origin, such as human embryonic kidney,
HeLa or HEP-2
Cytopathic changes; cell rounding and aggregation into grape-like cluster
Infected cells swell and become ballooned.
Intranuclear inclusion may be seen in stained preparations

Antigenic structure

More than 40 serotype have been found which belong to one of 6 serogroups (A-F)

Epidemiology, pathogenesis, and clinical syndromes

Spreads exclusively by human-human transmission, mainly by respiratory of fecal-oral contact, with

no apparent animal reservoirs for the virus
Capable of causing lytic, latent and transforming infections
These viruses infect epithelial cells lining resp. and enteric organ
Viremia may occur after local replication of the virus
Dissemination is more likely to occur in immunocompromised patients than in immunocompetent
people
The viral fiber proteins determine the cell target specificity among adenovirus serotypes
The penton base proteins inhibit cellular mRNA transport and protein synthesis, cell rounding and
tissue damage
The virus can be latent in lymphoid tissue
Causes infection of resp. tract, eye, bladder, and intestine.
Main clinical syndrome; pharyngitis, pneumonia, acute resp disease, pharyngoconjunctival fever,
epidemic keratoconjunctivitis, acute follicular conjunctivitis, acute hemorrhagic cystitis, diarrhea,
genital infections and hepatic disorders.

Immunity

Cell mediated immunity is important in limiting virus outgrowth, as borne out by the fact that
immunosuppressed people suffer recurrent disease

Lab diagnosis

By miscroscopical, virological, serological, immunefluorescent methods and genetic molecular

analysis
-Immune electron microscopy is used to detect enteric adenoviruses
-virological; isolation of adenovirus types in cell cultures derived from epithelial cells. The virus
causes lytic infection with characteristic intranuclear inclusions
-serological essays (e.g complement fixation, hemagglutination inhibition, ELISA, and neutralization
techniques have been used to detect type-specific antibodies after adenovirus infection.
-immunofluorescent tests are significant for detection of virus inside infected tissues
-PCR method and DNA probe analysis can be done for detection of group and type of virus in
clinical specimens and tissue cultures
Treatment and prophylaxis

No known treatment
Live oral vaccines to prevent infection (only in military recruits in US but not for civilians)

36. RABDOVIRUSES (rabies)

Family: Rabdoviridae
Genus: Vesiculoviruses, contains vesicular stomatitis virus and etc.
Lyssavirus, contains rabies virus etc.
Morphology

Bullet shaped
Lipid bilayer envelope derived from host cell plasma membrane and G-protein encoded by the virus
The surface G-protein bind specifically to cellular receptors and to confer the neurotropism in
infected animals
On the inner surface of membrane, is the M-protein which play a role in virus-budding
The internal,nucleocapsid core is a helix of single stranded, negative-sense RNA associated with a
phosphorylated nucleoprotein (N protein), a nucleocapsid-asscociated phosphoprotein (NS protein)
and a RNA-dependent RNA polymerase (L protein)

Replication

Virus attaches to host cell via G protein.

Entry by endocytosis; transcription of 5 mRNA species is catalyzed by the virion RNA polymerase.
Viral RNA is replicated on a positive-strand template by viral polymerase. Only negative strands are
enclosed new virions
The M protein appears to be important in packing the RNA and protein N and linking it to the
envelope.
Virions are formed by budding in association with the endoplasmic reticulum of the cell

Cultivation

Cultivation in warm-bloooded animals (rabbits,g. pigs, rats, mice)

Growth also occurs in chick or duck embryo and in range of cell cultures e.g. baby hamster kidney,
mouse neuroblastoma cells, etc.)

Resistance

The virus is sensitive to ethanol, iodine preparations, quaternary ammonium compounds, and lipid
solvent, such as ether, chloroform and acetone
It is activated by phenol, formalin, beta-propiolactone, proteolytic enzymes, UV irradiation and
sunlight
Thermal inactivation occurs in one hour at 50C and 5mins at 60C. It survives at 4C for weeks

Antigenic structure

Rabies viruses from all sources appear to be a sign immunologic type

Epidemiology, pathogenesis, and clinical syndromes

Rabies is classic zoonotic infection, spread from animals(cats, dogs,bats)to humans

Rabies usually result from the bites of a rabid animal
Rabies infection of the animal causes secretion of the virus in the animals saliva and promotes
transmission of the virus ,which in turn promotes transmission of virus

The virus can also be transmitted through the inhalation of aerosolized virus, in transplanted infected
tissue (cornea), and by inoculation through intact mucosal membranes
Virus may directly infect nerve ending or muscle before progressing to CNS
Once the virus reach the spinal cord, the brain becomes rapidly infected
The virus then disseminates from the CNS via afferent neurons to highly innervated sites (e.g skin of
head& neck, salivary glands, retina, nasal mucosa etc.)
After the brain & spinal cord are infected, encephalitis develops and neurons degenerate
The incubation period is 3-8 weeks but can be as short as 6 days
Symptoms are first noted in the prodromal period and include irritability and abnormal sensations at
the wound site.
Clinical diseases becomes apparent with a change in muscle tone leading to problems such as
swallowing
Neurological phase leads to coma and death resulting from neurological and pulmonary
complications

Immunity

Cell mediated immunity plays a little or no role in protection against rabies

Antibody can block the spread of virus to the CNS and to the brain if administered or generated
during incubation period

Lab Diagnosis

The lab tests are usually done to confirm the diagnosis (postmortem)
Diagnosis is made by microscopical, virological and serological methods
Detection of of intracytoplasmic inclusions consisting of aggregates of viral nucleocapsids (Negri
bodies) in affected neurons
Immunofluorescent is most widely used method for diagnosing rabies by antigen detection
Rabies can be grown in cell culture or intracerebrally inoculated infant mice.
ELISA method to establish rabies antibody titers in serum & CSFluid

Treatment

Clinical rabies is invariably fatal unless treated. Once symptoms have appeared, little other than
supportive care can be given

Prophylaxis

Postexposure prophylaxis for preventing overt clinical illness in the affected person
Prophylaxis should be initiated for anyone exposed by bite or by contamination of an open wound or
mucous membrane to the saliva or brain tissue of an animal suspected to be infected with virus
Local treatment of wound as first protective measure.
The wound should be washed with soap&water, detergent or any substances that inactivates the virus
Immunization with vaccine in vaccine in combination with administration of human rabies immune
globin (HRYG) is recommended
Preexposure vaccination on animal workers, lab workers who handle potentially infected tissue, and
people travelling to the rabies endemic area
Rabies vaccine is a killed-virus vaccine prepared by chemical inactivation of rabies-infected tissue
culture human diploid cells

37. RETROVIRUS. (HIV* & HTLV)

Family;
subfamily;

Retroviridae
Lentivirinae (HIV-1, HIV-2)
Oncornavirinae (HTLV-2, HTLV-5)
Spumavirinae

Morphology

Roughly spherical, enveloped RNA viruses

The envelope surrounds a capsid that contain 2 identical copies of positive strand RNA genome
inside the electron-dense core
The virion also contains 10-50 copies of reverse transcriptase and integrase enzyme and 2 tRNAS
which are base-paired to each copies of the genome to be used as a primer for the reverse
transcriptase
The HIV virions core resembles a truncated cone
3 major genes that encode polyproteins of the virus;
-gag (group-specific antigen, capsid protein)
-pol (polumerase,protease and integrase)
-env (envelope polyproteins)
At each end of genome are long-terminal repeat( LTR) sequences containing promoters, enhances
and other gene sequences use for diff cellular transcription factors
Oncogenic viruses may also contain a growth-regulating oncogene that operates at the expense of
other sequences
HTLV and HIV also encode several regulatory proteins that require more complex transcriptional
processing (splicing) than the simple retroviruses
The viral glycoproteins are produced by proteolytic cleavage of the protein encode by the env gene.
(e.g. the gp160 of HIV is cleaved into gp41 and gp120)
The larger of the glycoproteins is responsible for producing the tissue tropism.
The smaller subunit forms the lollipop stick and promotes cell-cell fusion. The gp 120 of HIV is
glycosylated, and its antigenicity can drift during the course of chronic HIV infection

Replication

Initiated by the binding of the viral glycoprotein spikes to specific cell surface receptor proteins
The gp120 of HIV binds with the CD4 surface molecule expressed on T-helper cells and cells of
macrophage lineage. A 2nd receptor, a 7-transmembrane G-protein-coupled chemokines receptor is
also required
HIV enters the cell by fusion of the envelope with the cellular plasma membrane.
The reverse transcriptase uses the virion tRNA as a primer and synthesizes a complementary,
negative-strand DNA, and then to double stranded DNA (provirus) which is integrated into host cell
chromosome
in response to viral promoters, the provirus initiates viral replication by directing synthesis of viral
RNA and other components

during viral replication, naked virus buds through the host cell surface membrane, it acquires a
lipoprotein envelope, which consists of lipid derived from cell membrane and glycoproteins which
are virus coded
HIV can also spread from cell to cell through the production of multinucleated giant cells, or
syncytia. Syncytia are fragile, and their formation enhances the cytolytic activity of the virus
HIV (HUMAN IMMUNODEFICIENCY VIRUS)
Causes acquired immunodeficiency syndrome (AIDS)

Cultivation

Difficult to culture HIV. The continuous cultures for isolation of HIV had been obtained from
lymphomas, consisting a CD4 positive T-lymphocytes

Resistance

Relatively stable in dried or lyophilized state, but the virus is very sensitive to amny detergents,
including soap, and can be eliminated rapidly by bleach.
Alcohol and acetone-alcohol mixtures can also inactivate virus at room temperature
HIV strains are also sensitive to iodophores.
For inactivation of virus in serum prior to serological testing, specimens should be routinely heated
at 56C for 30mins

Antigenic structure

HIV is highly mutable virus

It exhibits frequent antigenic variations as well as differences in other features such as nucleocapsid
sequences, cell tropism, growth characteristics and cytopathology.
Not only are these differences between isolates of HIV from different places or persons but also
between sequential isolates from the same person and even between those obtained from different
sites of the same person at the same time.
This great variability of HIV is believed to be due to the error prone nature of reverse transcription.
Antigenic variation is most frequent in respect of the envelope proteins but is also seen less often
with other antigens. Based on antigenic differences, 2 types of HIV have been recognized
(HIV1&2) . HIV-1 strains have been classified into at least 9 subtypes based on sequence analysis of
their gag and env genes (from A- I)

Epidemiology, pathogenesis, and clinical syndromes.

The presence of HIV in the blood, semen and vaginal secretions of infected people and the long
incubation period are factors that have promoted the spread of the disease through sexual contact and
exposure to contaminated blood and blood products. The virus can be transmitted perinatally to
newborns
Initially, infection occur in macrophages with a macrophage tropic virus directed toward the CD4
molecule and CC CKR5 chemokine receptor T-cell tropic strains, which recognize CD4 and fusin
chemokine receptor, evolve later in the infection
The course of the disease parallels the CD4 T-cell numbers and the amount of virus in the blood. The
large burst of virus production and viremia corresponds to the occurrence of a mononucleosis- like
syndrome
Virus level in blood decreases during a clinically latent period, but viral replication continues in
lymph nodes
Late in the disease, CD4 levels are significantly decreased, the structure of lymph nodes is destroyed
and the patient becomes immunosuppressed. Then macrophage-lineage cells from lymph nodes
become infected.
Monocytes and macrophages are probably the major reservoirs and mean of distribution of HIV.

HIV induces several cytopathological effects that may kill the cell. These include an accumulation of
nonintegrated circular DNA copies of the genome, increased permeability of the plasma membrane,
syncytial formation, and the induction of apoptosis.
The increases release of virus into the blood and decrease level of CD4 correlates directly with the
development of the symptoms of AIDS.
HIV can also cause neurological abnormalities
Symptoms; resemble influenza or infectious mononucleosis, with an aseptic meningitis
the onset of symptoms correlates with a reduction of T-cells to less than 450/microliter and increase
level of virus and protein p24 in blood
In aids, CD4 T-cells count are less than 200/microliter and involves the onset of more significant
diseases including HIV wasting syndrome, and the occurrence of indicator diseases such a Kaposis
sarcoma or specific opportunistic diseases , etc.
AIDS may manifest in one of several different ways, including lymphoadenopathy and fever,
opportunistic infections, malignancies, and AIDS-related dementia

Immunity

The ability of HIV to incapacitate the immune system, persist in macrophages and lymphocytes, and
alter its antigenicity allows the virus to escape immune clearance and prevents resolution of the
disease

Lab diagnosis

The main method of diagnosis is serological.

ELISA is used for routine screening
More specific procedure Western blot analysis is subsequently used to confirm seropositive results
Viral RNA in blood can be detected by the reverse transcriptase-PCR method

Treatment & Prophylaxis

Anti-viral drugs against HIV are classified into 3 groups

1) nucleoside analogue reverse transcriptase e,g; azidothymine (AZT), dideoxyinosine (ddl),
didooxycytidine (ddC), stavudine (d4t), lamivudine (3TC)
2)nonnucleoside reverse transcriptase inhibitors- nevirapine, delavirdine
3) protease inhibitors- saquinavir, ritonavir, indinavir,nelfinavir
No vaccines against HIV available

38. SLOW VIRAL INFECTION

The term slow viral diseases is applied to a group of infection, characterized by incubation period
ranging for months to years, course of illness lasting for months or years with involvement of central
nervous system and invariable fatal termination.
These diseases are spongiform encephalopathies, which are slow neurogenerative diseases
Slow viral infection may classified into 2 groups;
1) Group A
-Includes human diseases kuru, Creutzfeldt-lakob disease (CID), Gestmann-StraulerScheinker (GSS) disease, and fatal familial insomnia (FFI) and the animal disease scrapie, bovine
spongioform encephalopathy (mad cow disease), chronic wasting disease (in mule, deer, an elk)
and transmissible mink encephalopathy
2) Group B
-Includes measles, rubella virus, herpes simplex virus and HIV

*only Group A are going to be explained since group B have been described in their own questions
before
Group A

The slow virus agents are filterable and can transmit disease but otherwise do not conform to the
standard definition of a virus
Unlike conventional viruses, no virion structure or genome has been detected, no immune response is
elicited, and the agents are extremely resistant to inactivation by heat, disinfectants, and radiation.
The slow virus agent appear to be a modified host protein known as a prion (a small proteinaceous
infectious particle) which can transmit the disease

Structure

The prion lacks detectable nucleic acids and consists of aggregates of proteaseresistance,hydrophobic glycoprotein, which for scrapie is termed PrPSc .
Humans and animals encode a protein PrPC (cellular prion protein) that is closely related to PrPSc in
its protein sequence but differs in many of its other properties. Differences in posttranslational
modifications cause the 2 proteins to behave very differently. PrPSc is protease resistant, aggregates
into amyloid rods (fibrils), is found in cytoplasmic vesicles in the cell, and is secreted. The normal
PrPC on the other hand is protease sensitive and appear on the cell surface.

Physiology

PrPSc binds to the normal PrPc on the cell surface, then cause it to be processed into PrPSc released
from the cell, and aggregated as amyloid-like plaques in the brain
Next, the cell replenished the PrPC , and the cycle continues.
The fact that these plaques consist of host protein may explain the lack of immune response to these
agents in patients with the spongioform encephalopathies.
The similar aberrant protein are found to cause CID and kuru

Resistance

The agents are resistant to a wide range of chemical and physical treatments, such as formaldehyde,
detergents, UV and ionizing radiation, and heat to 80oC
Autoclaving at 15 psi for 1 h instead of 20 mins or treatment with 5% hypochlorite solution or 1.0 M
sodium hydroxide can be used for decontamination

Epidemiology, pathogenesis, and clinical syndromes

CID transmitted predominantly by injection, the transplantation of contaminated tissue (corneas),

contact with contaminated medical devices (brain electrodes). CID, FFI,and GSS disease are aslo
inherited, and families with genetic histories have been identified.
This diseases are rare but occur worldwide
Transmission of kuru is clearly a result of handling and consuming infected brain tissue. Cessation of
ritualistic cannibalism in new Guinea has stopped the spread of kuru
The term Spongioform encephalopathy is derived from the characteristic degeneration of neurons
and axons of the gray matter that occurs in affected patients.
Vacuolation of the neurons and the formation of amyloid-containing plaques and fibrils, a
proliferation and hypertrophy of astrocyte, and the fusion of neurons and adjacent glial cells are
observed. Prions can also be isolated from tissue other than the brain, but only the brain shows any
pathology
The slow virus agents cause a progressive, degenerative neurological disease with a long incubation
period (in some cases more than 30 years) but with rapid progression to death after the onset of
symptoms.
The spongioform encephalopathies are characterized by a loss of muscle control, leading to
shivering, myoclonic jerks, tremors, loss of coordination, and rapidly progressive dementia

Immunity

No inflammation or immune response is induced to the agent, which distinguished this disease from
classic viral encephalitis

Lab. Diagnosis

The disease is diagnosed clinical manifestations. There are no methods for directly detecting virus in
tissue using electron microscopy, antigen detection, or nucleic acid probes. No serological tests can
detect viral antibody

Treatment

No treatment exists for kuru or CID