Module 5

Important Scientists and Discoveries in Microbiology

Basic Laboratory Techniques

1. Aseptic technique – Method used to prevent contamination of cultures and media.
2. Sterilization – Process of killing or removing all forms of life including spores.
3. Disinfection – Killing of pathogens only (not spores).
4. Sanitization – Reduction of microbial load to a safe level.
5. Autoclave – Used for sterilization by moist heat at 121°C, 15 psi for 15–20 min.
6. Hot air oven – Used for dry heat sterilization at 160°C for 2 hours.
7. Pasteurization – 72°C for 15 sec (HTST) or 63°C for 30 min (LTLT) to kill pathogens in milk.
8. Filtration – Used for sterilizing heat-sensitive liquids (e.g., antibiotics, vitamins).
9. Membrane filters – 0.22 µm pore size commonly used.
10. UV sterilization – Used for surface sterilization of laminar airflow chambers.
11. Laminar air flow chamber – Provides sterile air through HEPA filters.
12. HEPA filter – Removes 99.97% of particles ≥ 0.3 µm.
13. Inoculation – Transfer of microbes into nutrient medium under aseptic conditions.
14. Incubation – Keeping inoculated media under optimum conditions for growth.
15. Colony – Visible mass of microorganisms arising from a single cell.
16. Pure culture – Population of cells derived from a single organism.
17. Mixed culture – Contains more than one species of microorganisms.
18. Streak plate method – Used for isolation of pure cultures.
19. Pour plate method – For counting viable microbial colonies.
20. Spread plate method – For surface colony growth observation.

Staining Techniques

1. Staining – Enhances visibility of microorganisms under microscope.
2. Simple staining – Uses single dye (e.g., methylene blue).
3. Differential staining – Uses multiple dyes to differentiate organisms.
4. Gram staining – Developed by Christian Gram in 1884.
5. Gram-positive bacteria – Retain crystal violet; appear purple.
6. Gram-negative bacteria – Lose crystal violet, take safranin; appear pink/red.
7. Acid-fast staining – For Mycobacterium (Ziehl–Neelsen stain).
8. Spore staining – Detects endospores using malachite green.
9. Capsule staining – Uses India ink or nigrosin (negative stain).
10. Flagella staining – Used to visualize bacterial flagella.
11. Lactophenol cotton blue – Common stain for fungi.

Preservation of Microbial Cultures

1. Sub-culturing – Transferring microbes periodically to fresh medium.
2. Refrigeration – Short-term preservation (4°C).
3. Deep freezing – Long-term preservation (-20°C to -80°C).
4. Lyophilization (Freeze drying) – Best method for long-term storage.
5. Cryopreservation – Preservation in liquid nitrogen (-196°C).
6. Glycerol stock (10–20%) – Common method for bacterial preservation.
7. Silica gel preservation – For fungal cultures.
8. Soil culture method – For actinomycetes.
9. Storage in mineral oil – Common for fungi like Aspergillus.
10. Periodic revival – Checking viability of preserved strains.

Microbial Assays and Enzyme Tests

1. Catalase test – Detects oxygen bubble formation (positive in aerobes).
2. Oxidase test – Detects cytochrome c oxidase enzyme.
3. Indole test – For tryptophanase enzyme.
4. Methyl red test – Identifies acid production from glucose.
5. Voges-Proskauer (VP) test – Detects acetoin production.
6. Citrate utilization test – Detects use of citrate as sole carbon source.
7. Starch hydrolysis test – Uses iodine to detect starch degradation.
8. Gelatin liquefaction test – Detects proteolytic activity.
9. Urease test – Detects ammonia production from urea.
10. Nitrate reduction test – Checks for reduction of nitrate to nitrite.
11. H₂S production test – Detects hydrogen sulfide formation.
12. Carbohydrate fermentation test – Acid and gas production detected by pH indicator.
13. Amylase activity – Clear zone on starch agar.
14. Protease activity – Clear zone on skim milk agar.
15. Lipase activity – Clear zone on tributyrin agar.
16. Cellulase test – CMC agar + Congo red zone clearing.