Basic Principle of Microbiology

-Different Between Eukaryotes & Prokaryotes :-

1-Eukaryotes ] Nucleus [ :-
This Cells Form (( Animals –Protozoa- Plants – Fungi –Algea-Helminths )) .
- most eukaryotic cells would lyse at temperature extremes (both hot and cold), with
dryness, and with very dilute and diverse energy sources.

2- Prokaryotes ] No Nucleus [ :- This Cells Form (( Bacteria – Blue green Algea-

Mycoplasm-Chlamydia-Rickettsia ))
- Use smaller Ribosome ( 70S ribosome ) .
- Bacteria can survive and, grow in hostile environments in which the osmotic
pressure outside the cell is so low.
- Bacteria have evolved their structures and functions to adapt to these conditions.
-Differences Among Prokaryotes :-
Bacteria can be distinguished from one another by their morphology (size, shape, and
staining characteristics) and metabolic, antigenic, and genetic characteristics.
Although bacteria are difficult to differentiate by size, they do have different shapes.
A spherical bacterium, such as Staphylococcus, is a coccus; a rod-shaped bacterium,
such as Escherichia coli, is a bacillus; and the snakelike treponeme is a spirillum. In
addition, Nocardia and Actinomyces species have branched filamentous appearances
similar to those of fungi. Some bacteria form aggregates such as the grapelike clusters
of Staphylococcus aureus or the diplococcus (two cells together) observed in
Streptococcus or Neisseria species.

- Bacterial Morphology Shapes :-

- Gram Stain :-

is a powerful, easy test that allows clinicians to distinguish between the two major
classes of bacteria. Bacteria that are heat fixed or otherwise dried onto a slide are
stained with crystal violet a stain that is precipitated with iodine, and then the
removed by washing with the acetone-based decolorizer and water. A red
counterstain, safranin, is added. This process takes less than 10 minutes.
-Gram Stain Procedures :-

-Result Of Gram Stain :-

1- Gram-positive bacteria :, Color is purple, Because They Have Thick

Peptidoglycan layers .
2- Gram-negative bacteria:- Color is Red, Because They Have Thin Peptidoglycan
layers .

- There are Bacteria cannot be classified by Gram stain Like mycobacteria, which
are distinguished with the acid-fast stain, and mycoplasmas, which have no
peptidoglycan.

Gram Positive Bacteria Gram-negative bacteria

Cell Wall :- Thick Peptidoglycan Cell Wall :- Thin Peptidoglycan Layers

Layers + Tichoic acid + Lipotichoic + Lipopolysacchraide ( LPS ) +
acid . Phospholipid + Protein

- Function Of Cell Wall :-

1- Protect the internal parts of the cell
2- Give The shape of bacteria .
3- Prevent bacteria cell from lysis .

- functions of plasma membrane :-

1- selective barrier which material enter and exit the cell (selective permeability ).
2- important in break down of nutrient and production of energy .
-Spores :- Found in some gram ve+ bacteria (( e.g bacillus anthracis , clostridium ))
are spore forming , but never gram ve- bacteria .
- spore be within a cell , and is a characteristic of the bacteria and can help in
identification of the bacterium.
- bacterial spores are so resistant to environmental factors .

-Endospore : when essential nutrient or water is unavailable , some gram positive

bacteria like clostridium and bacillus form endospore .

Function of endospore :
1-resistance dehydration and heat .

-Function of ribosome: is synthesis of protein .

- External structures of bacteria :-

1- Capsules :- Loose polysaccharide or protein layers surround the bacteria .
- it is major virulence factor .
- vi , k antigen found in capsule .
*function of capsule: protect bacteria from antibodies and phagocytosis

2- Flagella :- Provides motility for bacteria (( Motor Protein )) .

- it is allow cell to swim ((chemotaxis ))
- chemotaxis is :- toward food and away from poisons .
- H antigen is found in flagella .
The 4 arrangement of flagella :-
1- monotrichous :- single polar flagella .
2- amphitrichous :- single flagellum at both ends
3- lophotrichous :- two or more flagella at one or both ends .
4- peritrichous :- flagella distributed all over the cell .
5- Atrichous :- without flagella

3- Fimbriae (( Pili )) :- it is promote adherence (( Attachment )) to other bacteria or

the host .
- pili smaller than flagella in diameter . but shorter and thiner than Flagella.
- adherence factor (( Adhesin )) is pili important virulence factor for E.coli .
- O antigen is found in Pili .

-Different between capsule and slime layer :

-capsule : it is organized and firmly attached to cell wall .
- slime layer or(glycocalyx) : it is unorganized and loosely attached to cell wall .

- Biochemical Metabolism and Growth :-

obligate anaerobes :- bacteria cannot grow in the presence of oxygen.
obligate aerobes :- bacteria require the presence of oxygen for growth .
facultative anaerobes.:- bacteria grow in either the presence or the absence of
oxygen. .
autotrophs (lithotrophs) :- can rely entirely on inorganic chemicals for their energy
and source of carbon (CO2) .
heterotrophs (organotrophs):- bacteria and animal cells that require organic carbon
sources .
Photosynthetic bacteria :- bacteria which contain Chlorophyll .

- Population dynamics :-

Lag phase :- adapt with environment in media without growing

Log phase :- the bacteria will grow and divide with doubling time ( multiply ) .
Stationary phase :- stop growing .
Decline :- death increased and stop growing.

Summary of various types of microscopes :-

1- Brightfield (( light )) microscope :-this microscope stained by Gram stain.

the basic components of this microscope consist of :-
a- light source (( to illuminate the specimens on the stage )) .
b- condenser (( used to focus the light )) .
c- two lenses to magnify image (( objectives + ocular )) .
- there are 3 different objective lenses are commonly used .:-
1- low power(( 10 fold magnification )), used to scan specimens .
2- high dry (( 40 fold )), used to look large microbes(( parasites – fungi ))
3- oil immersion (( 100 fold )) . used to observed(( bacteria + yeast )) and
morphologic details of larger organisms.
- the best brightfield microscope have resolving power approximately 0.2um .

2- darkfield microscope :- the same lenses used in brightfield microscope are used in
darkfield microscope .however special condenser is used in this microscope.
- resolving power is 0.02um . this allow to see extremely thin bacteria such as
(( Treponema pallidum : etiologic agent of syphilis ))

3- phase – constrast microscope :- this microscope enables the internal details of

microbe to be examined .
The annular rings use in condenser and the objective lenses .
This microscope give three –dimensional image ((3D)) .
4- Fluorescence Microscope :- this microscope use fluorescent dyes ((stain)).
The microscope use high pressure mercury halogen or xenon vapor .

5- Electron Microscope :- unlike other forms of microscope , magnetic coils ((rather

than lenses )) this microscope used beam of electron instead((‫ )) بدل من‬of light .
Samples are usually stained by metal ions to create contrast .
There are two types of electron microscope:
a- transmission electron microscope : in which electrons pass directly through the
specimen , magnification of it is ((10000 – 100000 X)) and image in this type
not give ((3D)) picture
b- scanning electron microscope : magnify ((1000-10000X)) , the image Is ((3D))

General Information On Microbiology :-

* Microorganism are divided into three groups on the basis of temperature :-

1- psycrophiles (cold loving microbes (15-20 C) ) .
2- mesophiles (moderate temperature loving microbes (25-40 C) ).
3- thermophiles (heat loving microbes( 50-60 C) ).

* Microorganism are divided into three groups on the basis of PH :-

*-Most bacteria grow Best on PH 6.5-7.5 .
1-acidophilic (grow in PH below 7 and can grow on PH 1 ) ‫ بكتيريا حامضية‬..
2- Neutrophilic ( grow in PH 7.2-7.4 ) ‫بكتيريا معتدلة‬..
3-basophilic (grow in PH above 7) ‫ بكتيريا قاعدة‬..

*Fungi grow between PH 5-6 .

halophiles (grow at high salt , 30% salt )

Acid Fast Stain Procedures :- (used to identify organisms that have waxy
material in their cell wall.)
1- carbol fuchsin.
2- acid-alcohol decolorizing.
3- Counterstain with methylene blue.
Staining using in microbiology :-
1- Gram stain :- (( most common ))
2- Ziehl-neelsen stain (( acid fast stain ))
3- Wright – giemsa stain
4- India ink
5- Iron hematoxylen stain
 6- Auramin – rhodamine stain
-There are different method for staining in microbiology showin in this table:
Staining Method
1- Direct Examination :-
A wet mount method
A- 10% KOH ( potassium hydroxide )
B- India Ink
C- Iodine
2- Differential Stains :-
A- Gram stain
B- Iron Hematoxyline Stain
C- Methenamine silver stain
D- Trichrome stain
E- Wright-Giemsa stain
3- Acid fast Stain :-
A- ziehl-Neelsen stain
B- Kinyoun stain
C- Auramine-rhodamine stain
4- Fluorescent Stains :-
A- Acridine orange stain
B- Auramine-rhodamine stain
C- Calcofluor white stain
Use of stain
This method doing by adding water or
saline ( wet mount ) mix with KOH or
lactophenol cotton blue.
Facilitate detection of fungi when added
to lactophenol cotton blue. and dissolve
bacteria.
Used to detect capsule ssorrounding the
organisms shuch as yeast, Cryptococcus
neoformans.(fungi) .
Used to different between ameba and
WBC.
Most commonly used stain in
microbiology used to different between
gram + from gram -
Used for detection fecal protozoa.
Used in histology lab .
Used for detection fecal protozoa.
Used to detect blood parasites
( Chlamydia).
-It is the oldest method of acid fast stain .
- used to stain mycobacteria and other
acid fast bacteria ( Heat acid fast stain )
Cold acid fast stain ( Not require heating)
Same principle as other acid-fast stains,
except that fluorescent dyes are used for
primary stain .
-Used for detection of bacteria and fungi.
-Same as acid-fast stains.
- Used to detect fungi , some lab replaced
 KOH stain with this stain.

*Mycoplasm: is microorganisms (bacteria) that lack cell wall.

*Mycoplasm can not be identified by Gram stain.

*Bacteria cells divided by binary fission.

*Bacteria exchange genetic information carried by plasmid.
* substanse use in catalyse reaction is H2O2 .

Citrate test :- detects the ability of an organism to use citrate as source of energy.
If the organism has the ability to use citrate, the medium changes its color from green
to blue.
Examples:
 Escherichia coli: Negative
 Klebsiella pneumoniae: Positive
 Frateuria Aurantia: Positive

Indole test:-

Indole-Positive Bacteria :- E.coli , Haemophilus influenzae, Proteus sp ,

Plesiomonas shigelloides, Streptococcus faecalis, and Vibrio sp.

Indole-Negative Bacteria :- Actinobacillus spp ,most Haemophilus sp., most

Klebsiella sp., Neisseria sp Proteus mirabilis, Pseudomonas sp., Salmonella sp
Yersinia sp. most Bacillus sp., Bordtella sp., Enterobacter sp.,
 CULTURE MEDIA :-

- types of media :-

1- Nutrient agar : Use for growth all species of bacteria is not have
special environment .

Type of nutrient media :

a-simple (basic ) media :- this media allow growth of all species of bacteria .
because this media contain most bacterial requirement for growth . this media non-
selective media .

b- Enriched ( complex ) media : Some bacteria are fastidious and their growth
requires the presence of highly nutritive .

-Type of enriched media :

blood agar : Use for identify bacteria by their hemolytic action -1

chocolate agar : It is heated blood agar the temp raised to 100 ºC . and it selective -2
. media use for growth Neisseria , Haemophilus group. And inhibit other bacteria

. Loffler serum : use for growth clostridium diphtheriaa((diphtheriaa bacilli )) -3

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selective media : This media contain substance that inhibit all -2
. bacteria but allow growth a few type of bacteria

-: Types of selective media

,A- MacConkey : the most common selective media
That support the growth of most gram –ve rods espically (Enterobacteriaceae spp) but
.inhibits growth gram +ve organisms and some fastidious gram –ve bacteria

.B- - Lowenstein Jensen (L-J): Use for growth mycobacterium tuberculosis (TB)

C- Blood tellurite : Use for growth diphtheria , inhibits all normal flora of upper
respiratory tract
D- Thayer –Mattin :many special growth factors by N. gonorrhoea

E - Desoxycholate Citrate agar (DCA): use for growth Shigella and Salmonella . and
.Inhibits growth intestinal flora
this media also differential and selective media for shigella & salmonella -
.((enterbacteriaces))

.F- TCBS : use for Vibrio cholerae (yellow colonies)

G-shigella - salmonella agar ( SS AGAR ) :Selective media use for growth
salmonella and shigella , and inhibt other types of bacteria
a- salmonella (colorless with black center due to produce H2S )
b- Shigella (colorless with no change in center )

.
H- mannitol salt agar ( MSA ) :- selective media use for growth staph spp , and
inhibit growth all other types of bacteria . because contain high concentration of salt
( Nacl 7.5% )

I- saburod dextrose agar ( SDA) :- selective media for growth of fungi .(( General
media in mycology ))such as (( Aspergillus flavus, Candida albicans)) , and inibit growth
of all bacteria spp . because their PH acidic ( ph Below 7 )
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3- Differential Media :- media distinguish one microorganism

type from another growing on the same media .

:Types of differential media

-: MacConkey : different media to differentiate between -1
a- lactose fermenter ( red to pink ) e.g : E.coli , Klebsilla , Enterobacter
. b- Non lactose fermenter ( colorless ) e.g proteus , salmonella , shigella
this media contain bile salts + crystal violet that make it allow growth of gram
. negative bacteria and inhibit growth of gram positive bacteria

2-Blood agar plate (BA) :- different media to differentiate between :-

Streptococcus pneumoniae : Good growth, Alpha - hemolysis
Streptococcus pyogenes : Good growth, Beta-hemolysis

Cystine lactose electrolyte deficient ( CLED): different media to differentiate -3

-: between
. a- lactoe fermenter ( yellow ) e.g E.coli
. b- non lactose fermenter ( colorless ) e.g acintobacter
.this media Use for urine culture

:Triple sugar iron (TSI) agar -4

-: different media to differentiate between
pseudomonas, E.coli, proteus mirabilia ,

-: Xylose lysine desoxycholate (XLD) : different media to differentiate between -5

a- salmonella ( pink color with black center due to produce H2S )
b- Shigella ( pink color with no change in center )
this media Use for stool culture
 shigella – salmonella agar ( SS AGAR ) : different media to differentiate between -6
a- salmonella (colorless with black center due to produce H2S )
b- Shigella (colorless with no change in center )

-: mannitol salt agar ( MSA ) :- different media to differentiate between -7

. Staph spp ( not ferment mannitol )
Staph aureus ( ferment mannitol with golden color )

-: bile esculine : different media to differentiate between-8

Strepto spp
Streptococcus gamma enterococcus (( E.faecalis )), that produce enzyme form black
color
This media for stool sample . this media contain Iron (( ferrous )) .

9-Eosin Methylene Blue Agar (EMB)

-: different media to differentiate between
Gram-negative enteric bacteria , Bacteria that ferment lactose, especially the coliform bacterium
Escherichia coli from non lctose fermentive
. Escherichia coli : Good growth, green metallic sheen
.Klebsiella pneumoniae : Good growth, purple colonies, no sheen
Shigella flexneri : Good growth, transparent colonies (lactose negative)
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:Other type of media*

-: Mueller – Hinton agar *
Less nutrient media
the best use of this media is for Sensitivity tests .. because it not contain any chemical
. substances
bordet gangou medium is the best isolation of brodetella retusis-

Hektoen agar: for isolation Shigella -

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-: General information about media
*:Blood agar contain
agar (1
mineral salts(2
tryptone(3
meat extract(4
-: Media to become nutritive must contain the *
. source of carbon -1
source of nitrogen -2
water -3
minerals -4