Bacteriology of Water Milk Air

1. WATER MILK AIR
- Dr. ANKUR KUMAR

2. • Wholesome water
water that is fit to use for drinking, cooking, food
preparation or washing without any danger to human
health.
• Drinking water should have the following properties-
Biological quality: should be free from potentially
pathogenic microorganisms
Chemical quality: should be free from harmful chemical
substances such as metals, solvents, pesticides and
hydrocarbons
Physical quality: should have pleasant in taste, color &
odor.

3. Bacterial flora in water

4. Factors determining the Number of Bacteria in Water
 Salinity: - more is the salinity, lesser is the number of bacteria.
 Acidity: - Acidity of water has a deleterious effect on most of
the bacteria.
 Temperature: - Body temperature usually favours survival of
the bacteria.
 Light: - Sunlight with the wavelength of 300–400 nm is highly
bactericidal.
 Storage: - decreases bacterial count in stored water due to
sedimentation .
 Organic matter: - When organic matter is plenty, the
microorganisms tend to multiply and are present in large
numbers whereas when it is less, the organisms are few.
 Type of water: - Surface water is more likely to be
contaminated than the deep water.

5. Water-borne pathogens

6. Transmission of water related pathogens:
Ingestion
Inhalation &
aspiration
Skin, mucous
membrane
wounds, eye
Contact
(bathing)
Gastrointestinal Respiratory
Bacteria
Campylobacter spp.
E. coli
Salmonella spp.
Shigella spp.
V. cholerae
Y. enterocolitica
Viruses
Adenovirus
Astrovirus
Enterovieus
Hepatitis A & E
Rotavirus
Norovirus
Protozoal &
Helminthes
C. parvum
E. histolytica
G. intestinalis.
D. medinensis
L. pneumophila
Atypical
mycobacteria
N. fowleri etc
Acanthamoeba
spp.
Aerpmonas spp.
B. pseudomallei
Leptospira spp.
p. aeruginosa
S. mansoni

7. Bacteriological Examination of water
 It is impractical to detect the presence of all different kinds of
water borne pathogens, any of which may be present
intermittently.
 Instead, reliance is placed on testing the supply for
microorgsnisms which indicate that fecal pollution has taken
place.

8. Indicator organisms
• Indicator organisms themselves are not pathogens.
• Their presence in water supplies- indicates
contamination of sewage and the water supplies
needs to be disinfected.
• These organisms satisfy 2 properties----
1. present in excess number than any pathogen & should
not be able to proliferate in water to any extent
2. more resistant than the pathogens

9. Indicator organisms

10. Indicator organisms
1. Coliforms (presumptive
coliforms)
2. Faecal (Thermotolerant)
coliforms
3. E. coli or ‘fecal E. coli’
4. Faecal streptococci
5. Sulphite – reducing clostridia
6. Pseudomonas aeruginosa
7. Bacteriophage that infect
Escherichia coli.

11. Methods of Analysis
 Membrane filtration method
Multiple tube method. Membrane Filtration Method

12. Methods of Analysis
• Presumptive coliform count (Multiple Tube Method)
– for estimation of presumptive coliform count, which is
expressed as most probable number (MPN) of coliform
organisms in100 mLof water.
– Medium: MacConkey broth (double strength and single strength) in
bottles / tubes, Durham's tube- used to detect gas
production.
• Differential Coliform Count (Eijkman Test)
– to confirm that coliform bacilli detected in the presumptive test are
fecal E. coli.
– Method- Sub culturing the positive tubes for detection of lactose
fermentation with production of acid and gas at 440C
– Demonstrating positive indole test at 440C.

14. McCrady's probability table
for calculating most probable
number (MPN)/100 ml of water

15. Membrane Filtration Method
• Method-
measured volume of the water sample is filtered through a
membrane of pore size small enough to retain the
indicator bacteria to be counted on its surface.
– The membrane is then placed on a suitable selective
indicator medium and incubated- indicator bacteria grow
– bacterial content of the water is calculated by counting the
total number of colonies grown.

16. BACTERIOLOGY OF MILK
• Microorganisms in raw milk are derived from:
1. Animals (fecal contamination or through infected
udder, teat canals and skin)
2. Hands of the milk handlers
3. Environment

17. Milk borne diseases
(By Joint FAO/WHO Expert committee on Milk Hygiene 1970)

18. Methods used for Disinfecting/Sterilizing Milk
• Thermized milk- raw milk heated at 57- 68°C for 15
seconds. Efficacy is tested by methylene blue
reduction test
• Pasteurization- Milk is heated to high temperature
for short time (720C for 15 seconds). Efficacy is tested
by phosphatase test.
• Ultra heat treated milk- milk is heated to very high
temperature of 1350C for 1 second
• Sterilized milk- milk is heated at 1000c for long
periods such that it can pass the turbidity test

19. Methods for Bacteriological Examination of Milk
• Colony count
Viable count
Coliform count
• Chemical tests
Methylene blue reduction test
Phosphatase test
Turbidity test
• Detection of specific pathogens
Tubercle bacillus
Brucella

20. BACTERIOLOGICAL EXAMINATION
OF MILK
Viable count
Test for
coliform bacilli
Methylene blue
reduction test
Phosphatase
test
Turbidity test
Examination for specific pathogens
• Tubercle bacillus
• Brucella

21. Viable Count
• by plate counts with serial dilutions of the milk sample.
• rough and indirect assessment of the visible bacteria in the
milk.
Test for coliform bacilli
• by inoculating varying dilutions of milk into MacConkey’s
broth.
• Production of acid and gas is noted after incubation.

22. Methylene blue reduction test
• Depends on the reduction of methylene blue by bacteria
in milk when incubated at 37°C in complete darkness.
• Raw milk is considered satisfactory if it fails to reduce the
dye in 30 minutes under standard conditions.

23. Phosphatase test
• Check on the pasteurization of milk.
• Enzyme phosphatase is inactivated if pasteurization
has been carried out properly.
• Phosphatase test if positive after proper
pasteurization of milk indicates contamination after
pasteurization.

24. Turbidity test
• All heat coagulable proteins precipitates if the milk is
boiled or heated to the temperature prescribed for
‘sterilization’.
• No turbidity results if ammonium sulphate is then
added to the milk.

25. Examination for specific pathogens
Tubercle bacillus:
• Microscopic examination for tubercle bacilli is
unsatisfactory.
• Tubercle bacilli may be isolated in culture- LJ media,
BACT.
• Milk is centrifuged at 3000 rpm for 30 minutes and
the sediment inoculated into two guinea pigs.
• Animals are observed for a period of three months
for tuberculosis.

27. Organisms implicated –
• Mycobacteria
• Staphylococcus
• Streptococcus
• Bacillus
• Nocardia
• Actinomycetes
• Pseudomonas
• Burkholderia
• Klebsiella
• Escherichia
• Proteus
Airborne Agents
Innumerable microorganisms are present in air.
Apart from bacteria, moulds and viruses are also present and can be transmitted
from person to person in the form of aerosols.

28. • The proportion of the dust particles or aerosols
reaching the lung depends on their size.
Air born infection :
 produced by respiratory droplets less than 5µm in size.
 Reach upto the lung and are retained in the alveoli.
Droplet infection :
 produced by respiratory droplets more than 5µm in size
 retained in the nose

30. Purpose - measure microbial contamination in air
Types:
Passive air sampling -
• Settle plate method
Active air sampling -
• Slit-type Impactors
• Sieve-type Impactors
• Centrifugal air samplers
• Filtration samplers
• Cascade impaction
• Electrostatic precipitation
Air Sampling

31. • Open plates of culture media exposed to room air -
 Nutrient media (Surface area 65cm2)–
•BA
•TSA(Trypticase soy agar)
 1m height above floor
 1m away from the wall
 for 1 hour.
• Plates incubated at 37°C × 24 hrs
→ number of colonies counted.
•Large bacteria-carrying dust particles settle on the
medium.
Settle Plate Method

32. Settle Plate Method
• Gives an idea of relative no. & type of organisms-
• Used for testing surgical theater and hospital ward air
• The result is expressed as-
 the number of bacteria-carrying particles settling on a given area in a
given period of time.
• The method has the advantage of simplicity, but measures
only the rate of deposition of large particles from the air.

33. Slit sampling machine
Known vol. of air is directed onto a plate containing culture medium through
a slit 0.25 mm wide
• Plate being mechanically rotated
organism is evenly distributed
• Valuable in areas of low microbial contamination
•The efficiency of collection, even for the smallest bacterial particles, is very high.
•Disadvantages of the slit sampler: noisy and relatively cumbersome.
Slit Sampler

34. THANK
YOU