PHARMACEUTICAL MICROBIOLOGY

Identification of Microorganisms:

MICROBES IDENTIFICATION CHARACTERISTIC

Cocci Spherical in shape
Bacilli Rod like in shape
Spirillium Coil shaped
Vibrios Coma shaped
Mycoplasma Lack cell wall, septate and filamentous, shapeless
Actinomycetes Chains of rod-shaped cells, rigid cell wall

IDENTIFICATION:

Types of Staining
1) Simple staining-
- Basic stain: Methylene Blue
- Heat fixed
- Elucidate morphology and arrangement of cells

2) Negative staining-
- Acidic stain: Eosin or Nigrosin
- No heat fixing
- Elucidate Bacterial Capsule

3) Gram staining/Differential staining-

Smear Heat fixed Crystal violet Gram’s iodine (mordant black)

(Primary stain)
Red (Negative)
Safranin Decolorising agent
Blue (Positive) (Counter stain) (95% alcohol/acetone)

4) Acid fast staining-

Smear Heat fixed Carbol fuschin Gram’s iodine

(Primary stain)
Red (Acid fast)
Methylene Blue Decolorising agent
Blue (Non-acid fast) (Counter stain) (95% alcohol/acetone)

Note: Waxy substance, mycolic acid gives acid fast staining, e.g. Mycobacteria
5) Spore staining-

Smear Heat fixed Malachite Green Gram’s iodine

(Primary stain)

Safranin Decolorising agent

(Counter stain) (95% alcohol/acetone)
6) Capsule staining- India Ink method

Note: Crystal Violet + Iodine = Gram stain

Other staining techniques:

 Giesma’s stain: Ricketssia & Protozoa staining
 Fontana’s stain: Spirochetes staining

Types of Bacteria:
1) Gram-positive bacteria: 40-90 % peptidoglycan + Teichoic acid
2) Gram-negative bacteria: 5-10 % peptidoglycan + Lipoproteins

Nutritional requirement:

Nutritional Type Energy Source Carbon Source Examples

Photoautotroph Light Carbon dioxide Photosynthetic
bacteria,
cyanobacteria, algae
Photoheterotroph Light Organic compound Purple and Green
non-sulphur bacteria
Chemo-autotroph Usually electrons Carbon dioxide H, S, Fe and
from inorganic Nitrifying bacteria
compounds
Chemo-heterotroph Usually electrons Organic compound Most bacteria, fungi,
from organic protozoans
compounds

Temperature requirement:

Type of Microorganisms Meaning Temperature

Psychrophiles Cold loving Below 25oC
Mesophiles Moderate temperature 25-45oC
Thermophiles Heat loving 55-65oC (NLT 45oC)

Oxygen requirement:

Type of Microorganisms Meaning Examples

Obligate aerobes Require O2 for growth Pseudomonas sp.
 Obligate anaerobes Do not require O2 Clostridium sp.
Facultative anaerobes Aerobic, but can grow E.coli,
in absence of air Clostridium sp.
Microaerophiles Require very low conc. -
of O2

pH requirement:

 Microbes can grow well in the pH range 5-8, but optimum is 6.5-7.5

Type of microbes pH Examples

Acidophilic 3.5 Lactobacilli
Basophilic 9.0 Vibrio cholera

ISOLATION OF BACTERIA:

Methods Points To Remember

Streak Plate Simple and most widely used
Pour Plate Unsuitable for Psychrophiles
(a) Loop dilution
(b) Serial dilution
Spread Plate Surface colonies formed and counting microorganisms
Micromanipulator Hanging drop preparation
Roll tube Isolation of Stringent anaerobes

Other Microorganisms-

Actinomycetes Fungi
Bacteria with fungal morphology Aerobic or facultative anaerobic
Gram-positive Source of antibiotics
Cycling of organic matter Study of fungus- Mycology
Heterotrophs Chemoheterotrophs

MICROSCOPY:

 Dye used in fluorescence microscopy – Auramine.

IMMUNOLOGY:

Antigens:

- Any substance introduced into host produce antibodies.

- They are proteins, carbohydrates, lipids or nucleic acids.
- They have immunogenicity and reactivity.

Hapten:
 - Also known as “Incomplete Antigen”.
- Has Reactivity but not Immunogenicity.

Hapten + Carrier molecule = Complete Antigen

(Protein)
- Antigen binding site: Paratope
- Antigen determinant site: Epitope

TYPES OF IMMUNITY:

Types Mediated Examples

Humoral Antibody mediated B-cells, antibodies
Cellular Cell mediated T-cells, macrophages

Note: Immune Tolerance is due to the suppressor T-cells

Antigen-Antibody (Ag-Ab) reactions, in vitro, are called as Serological reaction.

TYPES OF HYPERSENSITIVITY:

Types Name of the reaction Examples

(acronym: “ACIDS”)
I Anaphylactic/Immediate IgE Anaphylaxis
II Cytotoxic/Cell stimulating Lupus erythematosus
III Immunocomplex/Toxic complex Arthus reaction, Steven Johnson
syndrome
IV Delayed/T-cell mediated Phototoxicity
V Stimulatory/Antireceptor -

CLASSES OF IMMUNOGLOBULIN:

Class Features

(acronym: “GAMED”)
IgG - Pass through placenta
- Highest half life (21 Days)
- Protect body fluid (☻GF BF)
- Present in highest concentration
- Present since birth
- Long-acting thyroid stimulator (LATS)

IgA - Protect body surfaces (☻ A=Angrakshak)

- Passes through milk
 IgM - Protect blood stream (☻M=Mother = Blood
relation)
- Pentamer

IgE - Shortest half life (2 Days)

- Type-I hypersensitivity

IgD - B-cell receptor function

DISINFECTANTS:

- Schedule O: Standards for Disinfectant Fluids.

- Evaluation of Antiseptic, Antimicrobial action.

Methods:

1) Tube dilution and Agar plate method.

2) Filter paper and Cup plate method.
3) Ditch plate method.
4) Phenol Coefficient method.
a) Rideal Walker Test
b) Chick Martin Test
c) US- FDA test
d) AOAC test (Association of official Agricultural Chemist)
5) Kelsey Sykes method (Concentration of bacteria should be 109/ml)

 Rideal-Walker (R-W) Phenol Coefficient Test-

Used to check:

- Antiseptic,
- Bactericidal,
- Disinfectant activity.
 Salmonella typhi species used in Phenol-coefficient test.

Grades of Disinfectant R-W Phenol Coefficient

1 18
2 10
3 5

Chromosomes:

Autoploidy Multiplication of chromosomes of single species.

Alloploidy Multiplication of chromosomes following/between two

species.
Polyploidy Multiplication of chromosomes between 3,4 species.

Aneuploidy No. of somatic chromosomes varies irregularly/widely.

STERILIZATION:

Sterility: It is a complete freedom from viable microorganisms within a probability limit of

10-6 .

D-value or Decimal reduction time – Time, in minutes, at any defined temperature, which
destroys 90% of viable organisms.

Z-value or Thermal destruction value – The number of degrees of temperature change to

produce a ten-fold change in D-value.

Q-value or Temperature coefficient – Q = Time to kill at XoC

Time to kill at X + 10oC

STERILIZATION PROCESSES:

Sterilization Mechanism Application Biological Chemical

indicators indicators
Dry Heat Oxidation of Glass Bacillus subtilis Browne’s
Proteins syringes, var. niger tubes/Bowie
Needles, Dry Dick heat
powders, sensitive
Metal tapes
instruments,
Oils and Fats
Moist Heat Denaturation Aqueous Bacillus sterothermophilus/ Stripe
and solution, Clostridium sporogenes papers
coagulation of Suspension,
proteins Emulsion,
Surgical
Dressings,
Rubber
Gaseous Alkylation of Disposable Bacillus subtilis Royce
Proteins syringes, var. niger sachet
Fragile rubber,
Bottles,
Dressing,
Rooms
(Formaldehyd
e)
Radiation Nucleoprotein Plastic Bacillus pumilus Dosimeters
damage by UV syringe,
of λ 265 Rubber
nm/Ionisation/ gloves,
 Free radical Disposable
formation instruments
Filtration Retention of Thermolabile Bubble point Pseudomon
bacteria liquids and test as diminuta/
solutions Serratia
marcescens

DRUGS ASSAYED:

Microorganisms Drugs Assayed

Staphylococcus aureus Amikacin
Kanamycin
Doxycycline
Tetracycline

Staphylococcus cerevisiae Amphotericin B

Nystatin

Staphylococcus epidermidis Gentamycin

Neomycin
Novobiocin

Micrococcus luteus Bacitracin

Erythromycin

Bacillus subtilis Framycetin

Rifampicin (☻
TB drugs)
Streptomycin

Bacillus pumilus Kanamycin

Framycetin

Bacillus cereus vermicoides Oxytetracycline

Mycobacterium smegmatis Bleomycin

Pseudomonas aeruginosa Carbenicillin

Bordetella bronchiseptica Polymyxin-B

SEROLOGICAL TESTS:

Sr. Test Disease

No.
1. Dick test Scarlet fever
2. Schick test Diphtheria
3. Schultz Charlatan test Scarlet fever
4. Frei test Lymphogranuloma venereum
5. Tuberculin test Tuberculosis
6. Mantoux test Tuberculosis
7. Ducrey test Haemophilia
8. Widal test Typhoid
9. Cold hemagglutination Pneumonia
test
10. Paul Bunnel test Mononucleosis
11. Weil Felix test Typhus fever
12. VDRL test Syphilis
13. Kahn test Syphilis
14. Radial Influenza virus
Immunodiffusion
15. Ouchterlony Small-pox
16. Elek test Diphtheria bacilli
17. Tube agglutination Typhoid, Brucellosis, Typhus
18. Rose water test Rheumatoid arthritis
19. Streptococcus MG Atypical Pneumonia caused
agglutination test by mycoplasma
20. Lepromine Leprosy
21. Wassermann test Syphilis
22. Radio Immuno Assay To detect human chronic
(RIA) gonadotropin in the serum of
women as a test for pregnancy
23. Enzyme Linked AIDS
Immunosorbant Assay
(ELISA)
24. Coombs test Brucellosis
25. Karpus test AIDS
26. Fuji Korai test AIDS

TYPES OF REACTION:

Reaction Test
Neutralisation reaction Schick and Dick test
Agglutination test Widal test
Precipitation reaction Ouchterlony test
Complexation fixation test Wassermann test

SOURCE OF ANTIBIOTICS:

Antibiotic Microorganism
Amphotericin B Streptomyces nodosus
Bacitracin Bacillus subtilis
Bleomycin Streptomyces verticillus
 Chloramphenicol Streptomyces venezuelae
Chlortetracycline Streptomyces aureofaciens
Colistin Bacillus calistinus
Dactinomycin or Streptomyces parvulus
Actinomycin D
Demeclocycline Streptomyces aureofaciens
Framycetin Streptomyces lavendulae
Gentamicin Micromonospora purpurea
Mithromycin Streptomyces argillaceous
Streptomyces tanashiensis
Mitomycin C Streptomyces verticillatus
Neomycin Streptomyces fradiae
Nystatin Streptomyces noursei
Oxytetracycline Streptomyces rimosus
Paramomycin Streptomyces rimosus
Penicillin Penicillium notatum (Original)
Penicillium chrysogenum (Present)
Sisomicin Micromonospora inoyensis
Streptomycin Streptomyces griseus
Streptozotocin Streptomyces achromogenes
Tetracycline Streptomyces aureofaciens
Tobramycin Streptomyces tenebrarus

SOURCE OF DISEASES:

Disease Causative Agent

Amoebiasis Entamoeba histolytica
Candidasis Candida alicans
Cholera Vibrio cholera
Conjuctivitis Moraxella lacunata
Diarrhoea Helicobacter pylori
Diphtheria Corynebacterium diphtheria
Dysentery Shigella dysenteriae
Gas gangrene Clostridium perfringens
Gastritis Helicobacter pylori
Giardiasis Giardia lamblia
Gonorrhoeae Neisseria gonorrhoeae
Influenza Haemphilus inflenzae
Keratitis Acanthamoeba species
Leprosy Mycobacterium leprae
Leshmaniasis (Kala- Leishmania donovani
Azar)
Local inflammation Escherichia coli
Malaria: Benign Plasmodium vivax
tertian
Malaria: Malignant Plasmodium falciparum
tertian
Malaria: Quartan Plasmodium malariae
Meningitis Neisseria meningitides
Paratyphoid Salmonella paratyphi
Pneumonia Diplococcus pneumonia
Pseudomembranous Clostridium difficile
colitis
Q-fever Coxiella burnetii
Syphilis Trepenoma palladium
Tetanus Clostridium tetani
Toxoplasmosis Toxoplasma gondii
Tuberculosis Mycobacterium tuberculosis
Typhoid Salmonella typhi
Typhus-Rocky Rickettsia rickettsia
Mountain spotted
fever
Urinary tract Proteus species
infection
Vaginitis/Urethritis Trichomonas vaginitis
Whooping cough Bordetella bronchiseptica

VIRUSES:

Groups Infection Associated

Adenoviruses Respiratory tract infection/Conjuctivitis
Arboviruses Yellow fever/Dengue fever/Leishmaniasis
Arenaviruses Meningitis
Coronoviruses Severe Acute Respiratory Syndrome
(SARS)
Herpes viruses Chickenpox/Infectious mononucleosis
(Viral infections)
Myxoviruses (Ortho) Influenza
Myxoviruses (Para) Mumps fever
Picornaviruses Poliomyelitis
Reoviruses Diarrhoea
Retroviruses AIDS
Rhabdoviruses Rabies
Togaviruses Rubella
Variola virus Small pox

VACCINES:

Live Attenuated Killed (inactivated)

BACTERIAL
Bacillus Calmette Guerin (BCG) Cholera
Typhoid-Ty 21a Paratyphoid (TAB)
Plague
Whooping cough
Meningococcal
 Haemophilus inflenzae
Type B
VIRAL
Poliomyelitis oral live (OPV, Poliomyelitis
Sabin) inactivated (IPV, Salk)
Mumps Rabies
Measles Influenza
Mumps Hepatitis A
Rubella Hepatitis B
COMBINED VACCINES
Double Antigen (DT-DA): Tetanus + Diphtheria
Triple Antigen (DPT): Diphtheria + Pertussis + Tetanus
TOXOIDS
Tetanus (fluid / adsorbed)
Diphtheria (adsorbed)