Microbiological Analysis: Water, Disinfectants, Air, and Fungi

Posted on May 7, 2026 in Microbiology

Experiment 4: Detection of Coliform Bacteria in Water

Coliform Groups

  • Total coliforms: Citrobacter, Enterobacter, Klebsiella, Escherichia (Environmental and fecal).
  • Thermotolerant (fecal) coliforms: Grow at 44–44.5°C; ferment lactose to produce acid and gas.
  • E. coli: The most specific fecal indicator; comprises 80–95% of human fecal bacteria.

Hierarchy

Total coliforms → Thermotolerant coliforms → E. coli.

Indicator Organism Criteria

  • Not normally present in water or soil.
  • Easy to detect.
  • Survive slightly longer than pathogens.
  • Associated with feces.
  • Testing is simpler than direct pathogen detection.

Why Detect Coliforms?

  • Suggests fecal contamination.
  • Indicates possible pathogens (e.g., Shigella, Salmonella).
  • Prevents waterborne diseases like cholera, typhoid, and diarrhea.

Methods

1. Multiple Tube Fermentation (MTF / MPN)

Presumptive Test Method
  1. Prepare lactose broth tubes with inverted Durham tubes.
  2. Inoculate with 1 mL and 0.1 mL water samples.
  3. Incubate: 35–37°C for total coliforms; 44°C for thermotolerant coliforms.
  4. After 18–24 hours, check for turbidity, acid, and gas.
  5. Gas production >10% indicates unsafe water.
Confirmatory Test (EMB Agar) Method
  1. Streak from positive lactose broth onto EMB agar.
  2. Incubate for 24 hours.
  3. Interpretation: E. coli (metallic green sheen), lactose fermenters (dark colonies), non-fermenters (pale pink).

Key Principles

  • Triclosan (soap): Inhibits fatty acid synthesis.
  • Alcohol gels: Denature proteins.
  • Soap: Removes microbes via emulsification rather than killing.
  • Efficiency: Higher microbial loads require longer kill times; clumped cells are harder to kill than dispersed cells.

2. Membrane Filtration (MF)

  1. Filter 100 mL of water through a membrane.
  2. Place membrane (grid side up) on lactose agar.
  3. Allow 2 hours of acclimatization if incubating at 44°C.
  4. Incubate: 35–37°C for total coliforms; 44°C for thermotolerant coliforms.
  5. A yellow halo under the membrane indicates coliforms.

Advanced Methods

  • Enzymatic: β-galactosidase (coliforms), β-glucuronidase (E. coli).
  • Solid phase cytometry: Laser scanning of stained cells.
  • PCR: lacZ (coliforms), uidA (E. coli).
  • FISH: Probes for Enterobacteriaceae 16S rRNA.

Experiment 5: Testing Soaps and Disinfectants

Microbial Resistance (Most to Least Resistant)

  1. Bacterial spores
  2. Coccidia
  3. Mycobacteria
  4. Non-lipid viruses
  5. Fungi
  6. Vegetative bacteria
  7. Lipid viruses

Biofilms

  • Microbial communities attached to surfaces.
  • Up to 1000x more resistant than planktonic cells.
  • Mechanisms: Thick matrix, enzymes, gradients, and genetic variation.
  • Found in pipes, catheters, lenses, and endoscopes.
  • Chlorine and monochloramines can inactivate biofilm bacteria.

Methods

1. Handwashing Effectiveness

  1. Make a pre-wash fingerprint on a TSA plate.
  2. Wash hands using assigned soap for 10 sec, 30 sec, or 3 min.
  3. Use proper technique: hot water, vigorous scrubbing, brush, rinse, and dry.
  4. Make a post-wash fingerprint on a new TSA plate.
  5. Incubate at 35°C for 24–48 hours and compare colony counts.

2. Environmental Sampling (RODAC Plates)

  1. Press a RODAC plate onto a surface before disinfection.
  2. Apply disinfectant (5–10 mL), allow contact time, and wipe.
  3. Press a second RODAC plate onto the surface after disinfection.
  4. Incubate at 35°C for 24–48 hours and count colonies.

Interpretation

  • 0–5: Excellent | 6–15: Good | 16–30: Moderate | 31–50: Poor | >50 or TNTC: Unacceptable.

Experiment 6: Air Sampling

Why Sample Air?

  • Airborne transmission of pathogens.
  • Critical in hospitals, pharmaceutical, and food industries.
  • Prevents contamination and nosocomial infections.
  • HEPA filters reduce airborne Aspergillus.

Methods

1. Passive Method (Settle Plates)

  1. Place open agar plates in various lab locations.
  2. Expose for 5, 10, or 15 minutes.
  3. Close, label, and incubate at 28°C for up to 1 week.
  4. Pros: Simple, cheap. Cons: Only large particles settle; no air volume measurement; desiccation risk.

2. Active Method (Air Sampler / Impaction)

  1. Place an agar plate inside the air sampler.
  2. Draw 250 L of air from each location.
  3. Incubate at 28°C for up to 1 week and count colonies.
  4. Pros: Quantitative, collects all suspended particles. Cons: Expensive, noisy, potential oversampling.

Experiment 7: Examination of Fruit Fungi

Why Fruits Spoil

High sugar, low pH, and rich nutrients support fungal growth.

Common Spoilage Fungi

Aspergillus, Rhizopus, Alternaria, Penicillium, and Fusarium. Risks include spoilage, allergies, and mycotoxins.

Fungal Basics

  • Eukaryotic; cell wall contains chitin.
  • Yeasts are unicellular; molds form hyphae (mycelium).
  • Reproduce via conidia.

Identification Methods

  • Morphological: Colony color, texture, hyphae structure, and conidia shape.
  • Molecular (PCR): Targets the ITS region between 18S and 28S rRNA.

Lactophenol Cotton Blue (LPCB) Staining

Purpose: Stains fungal structures blue, preserves morphology, and prevents lysis.

Components

  • Phenol (kills cells), Lactic acid (preserves structure), Glycerol (prevents drying), Cotton blue (stains chitin).

Methods

  1. Tease Mount: Place LPCB on a slide, add specimen, tease with needles, add coverslip.
  2. Scotch Tape Method: Press tape to colony, place on LPCB drop, add coverslip.