1. Bacteriological Examination of water, milk and air

2. Examination of water, milk and air

3. Importance of water examination for pathogens
Water intended for human consumption should not contain any pathogenic organisms.
Water is used for many applications either at home for cooking ,washing or drinking or in industries such as food and pharmaceuticals.

4. It is also important for hospitals for example haemodialysis unit
Testing of water samples are done regularly to make sure of its safety

5. Supplies of drinking water contaminated with sewage may cause diseases such as: typhoid fever and cholera.
All sources of water should be tested regularly.
Microorganisms which indicate the fecal pollution in water are usually common intestinal commensal bacteria.

6. Most important indicators of fecal pollution of water
Escherichia coli:
The essential indicator of fecal pollution of human /animal origin.
It is an important member of the coliform bacteria.
Coliforms are members of the enterobacteriaceae family and they
grow in the presence of bile salts.
produce acid and gas from fermentation of lactose at 37°C.
It is the commonly-used bacterial indicator of sanitary quality of food and water.

7. Enterococcus faecalis:
less numerous than E.coli in human feces, but more resistant to chlorination.
Clostridium perfringens:
Less numerous in human feces
Its spores can survive in the environment
Resist treatment processes than most of the indicators.

8. Media used in bacteriological examination of water
1. For coliforms:
MacConkey’s broth
Containing bromocresol purple as the pH indicator.
To confirm the presence of E.coli :
EMB agar + IMVC

9. Enterococcus faecalis:
Glucose azide broth.
Clostridium perfringes:
Differential reinforced clostridial medium.

10. Methods Used in Bacteriological Examination of Water
Membrane Filtration Method
Determination of Most Probable Number (MPN) by dilution method
Pour plate technique

11. Membrane Filtration Method
Using Millipore Filter Apparatus
MacConkey’s agar

12. Determination of MPN of Coliforms by Dilution Method
50 ml water
sample
10 ml water
sample
1 ml water
sample
Water Sample
50 ml
DSMB
5 x 10 ml
DSMB
5 x 5 ml
SSMB

13. Results: Positive tubes: showing production of acid or gas.
Acid production: change color of tube from purple to yellow
Gas production: detected in the Durham’s tube.

14. Gas
Yellow
Purple
Determine no. of coliforms per 100 ml water sample (MPN) using the standard probability table.

15. i.e: No. of coliform bacilli per 100 ml water sample is 14 cells.3 2
MPN = 14
i.e: No. of coliform bacilli per 100 ml water sample is 14 cells.

16. Most probable number of coliforms by McCrady’s table

17. Viable Bacterial Count
Using 10 fold serial dilution method
1 ml water
1 ml
1 ml 1 2 3
9 ml Saline
Water sample
1/10 x 1/10
1/100
1/100 x 1/10
1/1000
1/10
Melted NA
1 ml
1 ml
1 ml 1 2 3

18. Results: No. of colonies per plate Dilution factor 1 2 3 X X . y 10 x1
102 x2
X2.y2
103 x3
X3.y3
No. of cells per 1 ml =
X1.y1 + X2.y2 + X3.y3 3

19. Examination of water, milk and air19

20. Introduction: Human infections may be caused by
theingestion of animal milk which
contains microorganisms derived from:
Animal e.g. by contamination with its feces
The environment
Milk handlers such as dairy workers

21. Importance of milk examination for pathogens
It is important to examine milk for pathogens to ensure that it is safe to be consumed by man.
Milk is further used for obtaining many milk products like cheese ,cream , butter and ice cream

22. Pathogenic bacteria present in milk
E.coli
Streptoccus pyogenes
Mycobacterium bovis
Bacillus anthracis
Salmonella sp.
Brucella sp.

23. Determination of viable bacterial count:
Using the pour plate method after preparation of 10 fold serial dilution from the milk sample with ringer solution.
Permissible number of bacterial flora in pasteurized milk is 5 x 104 cfu/ml
Permissible number of bacterial flora in long life milk is 10 cfu/ml

24. Methylene Blue Reduction Test To determine quality of the milk
Increasing the number of bacterial flora will reduce
the color of methylene blue more rapidly due to
increasing consumption of oxygen.
i.e.: The speed of reduction of methylene blue color is
directly proportional to the number of bacteria present
in milk sample.

25. Methylene Blue Reduction Test
Results:
The shorter the decolorization time, the higher
the number of bacterial flora present in milk,
and the poor quality of milk
Decolorization time
Result
30 min – 2 hrs
Poor quality
2 – 6 hrs
fair quality
6 – 8 hrs
good quality
Over 8 hrs
excellent quality

26. Test for coliforms
Done by inoculation of MacConkey’s broth with 0.1 ml of milk sample.
Examine for the production of acid detected by changing the color of the medium from purple to yellow.
-ve result
+ve result with gas production

27. Examination of water, milk and air27

28. Importance of keeping the micro- organisms count low in air
Surgical theaters
Food preparations
Drug materials
Cross infection and out breaks in hospitals

29. Number on bacteria in air depends on
Number of persons
Body movement
Disturbance of clothing

30. Methods of examination of air
a. Settle plate:
Petri dishes containing an agar medium are left open for a measured period of time.
Large bacteria-carrying dust particles settle on the medium.
The plates are incubated and a count of the colonies is formed

31. Blood agar is suitable for over all count
For detection of a particular microorganism suitable media is used .
Disadvantage of this method :
Despite its simplicity it measures only the
rate of deposition of large particles from
the air

32. b. Slit sampler
It draws in air from the environment at a fixed rate and causes the suspended particles to fall on the surface of the agar plate.

33. c. Air centrifuge
Centrifuging particles from the air on to a culture medium.
The sampled air passed along a tube lined with nutrient agar which was rotated on its long axis.
After sampling the strip is removed from the instrument and incubated then colonies can be counted.

34. Notice:
No level of contamination however low can be regarded as certainly safe.
Infection can be initiated by deposition of a single infected particle at a favorable site.
The probability of S. aureus initiated infection is low in comparison with Mycobacterium tuberculosis

35. Demonstration: Air examination Settle plate Water examination
Determination of MPN
Milk examination
Methylene blue reduction test

36. Thank you