FOOD AND WATER ANALYSIS

1. M
ICROBIOLOGYOF WATER,
AIR, FOOD, SURFACES

2. SLO
MI 8.8 Describe the methods used and significance of assessing the microbial
contamination of food, water and air.
1 List foodborne, waterborne and airborne infections.
2 Enumerate bacterial organisms indicative of faecal contamination of water.
3 Describe collection & transportation of water & food for bacteriological
analysis.
4 Mention various methods of bacteriological analysis of water w.r.t community
and hospital settings (dialysis water, RO) and interpretation of results.
5 Discuss different microbiological parameters involved in evaluation of quality
of air in OT with a brief note on surveillance methods
6 List out different methods for examination of milk and food and their clinical
importance

3. Importance
• Outbreak investigations
• Evaluation of infection control practices
• Research purpose

4. M
ICROBIOLOGYOF WATER

5. Wholesome water
• Biological quality
• Chemical quality
• Physical quality
Bacterial flora in water
Naturally occurring water bacteria: Micrococcus, Pseudomonas, Flavobacterium
Soil bacteria: Bacillus subtilis, Enterobacter spp
Sewage bacteria: E.coli, Enterococcus faecalis, Clostridium perfringens, Proteus spp

6. Water borne pathogens
Bacteria Viruses Protozoa Helminths
Diarrheagenic
E.coli
Enterovirus Entamoeba
histolytica
Trichuris trichiura
Salmonella spp Hepatitis A, E Giardia lamblia Strongyloides
stercoralis
Shigella spp Norwalk virus Cryptosporidium Ascaris lumbricoides
V. cholerae Rotavirus
Campylobacter
jejuni
Common hospital pathogens: E. coli, Klebsiella, Pseudomonas, Acinetobacter, NTM,
Legionella pneumophila

7. Need for water analysis
• Detecting fecal contamination of water
• Investigation of outbreak
• Usage of new water source
• Assessing efficacy of chlorination
• Checking the potability of water

8. Indicator organisms
Commensals in intestine
Indicates contamination of water with sewage, need for disinfection
2 properties:
Presence in large numbers
Highly resistant to environmental stress, disinfection
• Faecal coliforms (Klebsiella, Citrobacter) – presumptive evidence
• Faecal E.coli (thermotolerant) – most sensitive
• Faecal Streptococci – remote fecal pollution
• Clostridium perfringens – remote contamination
• Pseudomonas aeruginosa – least reliable indicator
• Bacteriophages

9. Collection and Transport of water
sample
• Screw-capped wide sterile containers
• At least 150-200 ml water to be collected
• Avoid contaminations
• Collected from taps, lakes, well
• Hospitals – endoscopy rinse water, dialysis water, etc.
• Transported within 6 hours of collection

10. Routine tests used in bacteriological
examination of water
A. Multiple tube method: Presumptive coliform test
B. Eijkman test: Differential coliform test
C. Membrane filtration method
D. Plate count method
E. Presence-Absence method

11. MULTIPLE TUBE METHOD – Presumptive coliform count
Principle – most probable number of coliforms in 100 ml
1. Water sample added to series of tubes containing indicator broth.
2.Tubes showing color change and gas production is taken positive.
3.The most probable number (MPN) of indicator organisms/100 ml is estimated by
reference statistical table.

12. • 50 ml water in 1 bottle of 50 ml double strength medium
• 10 ml water each in 5 tubes of 10 ml double strength medium
• 1 ml water each in 5 tubes of 5 ml single strength medium
• 0.1 ml water each in 5 tubes of 5 ml single strength medium
• Compared with McCrady’s probability table
• 0 is excellent
• 1-3 is satisfactory
• 4-9 is intermediate
• ≥10 is unsatisfactory
Negative Positive

13. McCrady’s probability table
Double strength medium

14. EIJKMAN TEST – Differential coliform count
Detection of Fecal E.coli
Positive tubes of presumptive coliform test sub-cultured on lactose
containing medium (brilliant green bile broth)
24 hours incubation at 44 °C
Tubes exhibiting gas production are deemed to contain E.coli.
Further confirmed by biochemical tests (citrate & indole).

15. • Testing dialysis water
• Testing clean water
• Measured volume of water
• Cellulose membrane pore size 0.2 or 0.45 µm
Bacterial growth
Result: CFU/100 mL
Membrane filter

16. Water testing in Dialysis Settings
• Membrane filtration method-microbial count
• Endotoxin detection
• Methods: gel clot assay, turbidometric method, chromogenic method
• Total microbial count <100 CFU/mL
• Endotoxin unit <0.25 EU/mL

17. AIR SURVEILLANCE

18. • Airborne infection: respiratory droplets <5µm
• Suspended in air
• M.tuberculosis, Varicella-Zoster virus, Influenza viruses
• Droplet infection: respiratory droplets >5µm
• Close contact
• N.meningitidis, C.diphtheriae, B.pertussis, Mumps virus
Indications
• Outbreak investigation
• New constructions / Reconstructions
• Post fumigation
• Evaluation of infection control practices

19. Evaluating air quality in OT
Non-microbiological parameters:
• Total Air changes/hr-20
• Air velocity: unidirectional downwards on the OT table (25-35 FPM)
• Positive pressure: 2.5pascal
• Air filtration: HEPA filters
•Temperature & humidity: 210c+20c, 20-60%
Microbiological parameters: 2 methods:
• Passive Monitoring - settle plate method
• Active monitoring - slit sampler method

20. Settle plate method
• Open plates with culture media are exposed for specific periods
• 1, 1, 1 method
• Incubation
• Colonies counted
• Maximum acceptable levels
• ≤5 CFU/9 cm diameter plate/h at rest
• ≤25 CFU/9cm diameter/h when operational
• Relative number and type of microorganisms
• Quality of air in Operation theatres and wards

21. Slit sampler method
• Known volume of air is directed to a plate through a slit
• Vacuum pump, perforated lid
• Known volume of air
• Quantity measured in CFU/m3 of air

22. M
ICROBIOLOGYOF MILK

23. Milk borne diseases
• Classification of milk borne diseases according to Joint FAO/WHO expert committee on
Milk Hygiene, 1970:
A.Infections of animals that can be transmitted to man by milk.
eg: Tuberculosis, Brucellosis, Salmonellosis
B. Infections primary to man that can be transmitted through milk.
eg: Typhoid

24. Methods to sterilize Milk
• Thermized milk: raw milk heated for 15 secs at 57-68°C
• Pasteurization: 72°C for 15 secs
• Ultra heat treated milk: 135°C for 1 sec
• Sterilized milk: 100°C for long period

25. Bacteriological examination of milk
• Colony count tests:
1. viable count test.
2. coliform count test.
• Chemical test:
1. Methylene blue reduction test.
2. Phosphatase test.
3. Turbidity test.
Detection of specific pathogens

26. VIABLE COUNT TEST
• Plate dilution method
• Serial dilution of milk in yeast extract milk agar
Significance
Rough and direct estimate of viable bacteria in milk

27. METHYLENE BLUE TEST
• Reduction of methylene blue by bacteria
• 37°C in complete darkness
• 1 ml methylene blue in 10 ml milk
• No decolourization in 30 mins
Satisfactory – if milk fails to reduce dye in 30 minutes

28. PHOSPHATASE TEST
• Evaluates effectiveness of pasteurization
• Phosphatase is inactivated on pasteurization
TURBIDITYTEST
• To evaluate sterilization of milk
Heat coagulable proteins are precipitated

29. FOODM
ICROBIOLOGY

30. Food-borne pathogens
• Staphylococcus aureus (poultry, mayonnaise)
• Bacillus cereus (fried rice)
• Clostridium perfringens (beef, poultry, legumes)
• Salmonella spp (Beef, poultry, eggs, dairy products)
• Campylobacter jejuni (poultry, raw milk)
• Vibrio parahaemolyticus (mollusks)

31. VIABLE PLATE COUNT
Food sampling
• 10 g food homogenized in 90 mL of sterile diluent
Food processing
• Serial dilutions of sample
• Cultured on suitable medium

32. SURFACE SURVEILLANCE

33. SURFACE SURVEILLANCE
• Evaluate environmental contaminations and detect reservoirs of pathogens
• High risk locations – OTs, ICUs
• High-touch areas are sampled
• Samples collected using moistened sterile swabs
• Inoculation into culture media
• Growth of pathogens with growth rate provided