microbiology

1. Bacteriology of Water and Air

2. • Wholesome water-
Fit to use for drinking,cooking or washing
without any potential danger to human
health.

3. • Drinking Water (3 properties)
-Biological Quality
-Chemical Quality
-Physical Quality

4. WATER SURVEILLANCE
Need for water surveillance
• To know water is fit for consumption
• To rule out water borne infections

5. Water borne pathogens
Bacterial Vibrio cholerae, Salmonella Typhi, S. Paratyphi A, B and C,
Shigella species, Yersinia enterocolitica, Campylobacter
jejuni ,Diarrheagenic Escherichia coli
Viral Hepatitis A virus ,Hepatitis E virus ,Polio virus, ,Rota virus
Protozoal Entamoeba histolytica, Giardia lamblia, Balantidium coli,
Cryptosporidium, Isospora
Helminthic Ascaris lumbricoides, Enterobius vermicularis
Trichuris trichiura
Helminths
transmitted through
aquatic hosts
Dracunculus medinensis Diphyllobothrium latum
Schistosomes

6. When to do??
• Outbreaks
• Dialysis units
• Routinely: optional

7. Criteria for indicator organisms:
• Present in feces in abundant number
• Should be a member of the intestinal micro-
flora of humans
• Longer survival time
• Should have some direct relationship to the
degree of faecal pollution

8. Indicator organisms
• Fecal (thermotolerant) Escherichia coli
– Confirms recent fecal contamination of water
– Most sensitive indicator
• Other indicators:
– Coliform other than fecal E.coli
– Fecal Streptococci
– Clostridium perfringens
– Pseudomonas aeruginosa
– Bacteriophages

9. Indicator organisms of fecal pollution
of water
Indicator organisms Interpretation-
(Presence in water indicates-)
Coliform (other than
E.coli)
Remote contamination- either fecal
(presumptive) or soil and vegetation
Fecal (thermotolerant)
Escherichia coli
Confirms recent fecal contamination of
water
Most sensitive indicator
Fecal streptococci Confirms remote fecal pollution
Clostridium perfringens Remote contamination
Pseudomonas aeruginosa Least reliable indicator
Useful in hospitals and food
establishments
Bacteriophages Phage specific for E.coli- indicate fecal
pollution of water Indirectly indicates
viral pollution

10. Methods of analysis
• Standard tests usually employed for
bacteriological analysis of water are-
 Presumptive coliform count ( Multiple tube
method)
 Differential coliform count (Eijkman test)
 Membrane Filtration method

11. Multiple tube method
• Used for the estimation of presumptive coliform count
expressed as the most probable number (MPN) of
coliform organisms in 100 ml water.
• Medium- MacConkey purple broth (double strength and
single strength) in bottles or tubes is the standard
medium of choice.
• Durham's tube is used to detect gas production
• Bromocresol purple is used as indicator

12. Multiple tube method (cont..)
• Procedure - Measured amounts of water samples are added
to tubes containing MacConkey purple broth by sterile
graduated pipettes as under:
o 50 ml of water to one bottle of 50 ml double strength medium
o 10 ml of water each to five tubes of 10 ml double strength
medium
o 1 ml of water each to five tubes of 5 ml single strength medium
• Result:
o Inoculated tubes are incubated at 37° C for 48 hours
o Positive test is indicated by a color change in the medium to
yellow from purple (due to lactose fermentation) and gas
collected in the Durham’s tube.

13. • Interpretation- The interpretation of presumptive coliform
count is as follows:
o Presumptive Coliform count (Most probable number)- An
estimate of coliform count per 100 ml is calculated from
the tubes showing acid and gas production using the
McCradey’s probability table.
Multiple tube method (cont..)

14. • Quality of water supply is determined by the
presumptive coliform count. The of most probable
numbers of 0, 1-3, 4-9 and ≥10 per 100 ml are
interpreted as:
o Excellent
o Satisfactory
o Intermediate
o Unsatisfactory respectively.
• Detection of coliform bacteria does not always indicate
fecal contamination as some of them may be found in
environment.
• Hence, it is further tested by differential coliform count
to detect the fecal E.coli.
Multiple tube method (cont..)

15. Presumptive coliform count
Most probable number (MPN)/100mL
McCrady’s table
1 tube of
50 ml
5 tube of
10 ml each
5 tube of
1 ml each
MPN/
100ml
0 0 0 <1
0 0 1 1
0 0 2 2
0 1 0 1
0 1 1 2
0 1 2 3
0 2 0 2
0 2 1 3
0 2 2 4
0 3 0 3
0 3 1 5
0 4 0 5

16. Presumptive coliform count
Most probable number (MPN)/100mL
1 tube of
50 ml
5 tube of
10 ml each
5 tube of
1 ml each
MPN/
100ml
1 0 0 1
1 0 1 3
1 2 2 10
1 0 3 6
1 1 0 3
1 1 1 5
1 1 2 7
1 1 3 9
1 2 0 5
1 2 1 7
1 2 2 10
1 2 3 12
1 3 0 8
1 3 1 11
1 3 2 14
1 3 3 18
1 3 4 21

17. Presumptive coliform count
Most probable number (MPN)/100mL
1 tube of
50 ml
5 tube of
10 ml each
5 tube of
1 ml each
MPN/
100ml
1 4 0 13
1 4 1 17
1 4 2 22
1 4 3 28
1 4 4 35
1 4 5 43
1 5 0 24
1 5 1 35
1 5 2 54
1 5 3 92
1 5 4 161
1 5 5 >180

18. Classification of quality of drinking water supply according
to bacteriological tests
Quality of
drinking water
supply
Most probable number
(MPN)/100 ml of water
Coliform
counts / 100
ml
E.coli count /
100 ml
1. Excellent 0 0
2. Satisfactory 1 – 3 0
3. Intermediate 4 – 9 0
4.
Unsatisfactory
≥10 ≥1

19. Differential coliform count
(Eijkman test)
• Done to confirm that the coliform bacilli detected
in the presumptive test are fecal E.coli.
• This is done by:
o Sub culturing the positive tubes on lactose containing
medium such as brilliant green bile broth for detection
of lactose fermentation with production of acid and
gas at 440C and
o Demonstrating positive indole test at 440C

20. Fecal streptococci detection
• When presumptive coliforms are present but
fecal E.coli are absent, detection of fecal
streptococci would confirm the fecal origin of
coliform bacilli.
• Subcultures are made from positive tubes - tubes
containing 5 ml of glucose azide broth and
incubated at 45°C for 48 hours.
• Presence of acid - indicates fecal streptococci
• Further confirmation can be done by plating onto
bile esculin azide agar (black colonies formed).

21. Clostridium perfringens detection
• By first heating the water sample (which kills all vegetative
bacteria retaining the spores)
• Then performing multiple tube method by sub culturing it
in differential reinforced clostridial medium (DRCM).

22. Enzyme methods
• Detection of specific enzymes for the detection and
confirmation of coliform bacilli and fecal E.coli is rapid and
novel method described recently.
o β galactosidase – is a coliform bacilli specific enzyme
o β glucuronidase- is a fecal E.coli specific enzyme

23. Membrane filtration 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.
• Membrane is then placed on a suitable selective indicator
medium and incubated
• Indicator bacteria grow into colonies on its upper surface

24. CDC recommends targeted air
surveillance
• Investigation of an outbreak
• For research purpose
• After reconstruction or newly constructed buildings
• After fumigation (fumigation itself is not routinely
recommended)
• For short term evaluation of a change in infection-control
practice.

25. Evaluation of the quality of air in OT
Microbiological parameters
1. Microbiological sampling method
2. Particle count method

26. Particle count method
• Airborne particle concentrations in OT is measured by means of
a laser light scattering instrument (particle counter)
• The particle count of an OT is considered acceptable only when
it falls in the acceptable range according to the international
standards system ISO 14644-1.

27. Settle plate Method
• Petri dishes containing an agar medium of
known surface area are left open for 30
minutes to 1 hour.
• Plates are incubated at 37°C for 24 hours
• Colony count: Large bacteria carrying
dust particles settle onto the medium.

28. Slit Sampler Method
• Most efficient and convenient method
• Number of bacteria carrying particles
suspended in a unit volume of air.
• Air is sucked through the equipment at a
rate of one cubic foot (28.3 liter) per
minute for 10 minutes
• Directed onto a plate containing culture
medium through the slit
• The plate is rotated mechanically so as
to allow the organisms to spread out
evenly on the medium

29. Non-microbiological parameters
Properties Recommendations
Air changes per
hour
Minimum 20 numbers of air changes per hour;
out of which four should be fresh air.
Air velocity 25-35 FPM (feet per minute)
It is checked by anemometer.
Positive pressure
(PP)
 Difference in positive pressure between OT
and adjoining areas is required to prevent
outside air entry into OT.
 PP should be maintained in OT at all times
(operational & non-operational hours)
 Minimum PP of 2.5 Pascal is recommended.
Temperature 21 +/- 30C
(ortho 180C +/- 20C)
Relative humidity 20 to 60% (ideal 55%)

30. Non-microbiological parameters
Properties Recommendations
Air handling in the OT There should be dedicated AHU (air handling unit) for each
OT and should not be linked to air conditioning of any other
area/OT.
 Window & split AC should never be used in OT as they are
pure re circulating units and have pockets for microbial
growth.
 Air is supplied through Terminal HEPA filters in the ceiling
 The minimum size of the filtration area should extend one
feet on each side of the OT table to cover the entire OT
table and surgical team.
 Validation of HEPA Filters is done biannually by DOP
(Dispersed Oil Particulate) test.
Wall, floor and ceiling  Should be anti-static and made up of non-porous, smooth,
seamless materials
 Paints used should have antibacterial, anti-fungal

31. SURFACE SURVEILLANCE
• Environmental surface sampling has been used to determine
a) potential environmental reservoirs of pathogens
b) survival of microorganisms on surfaces
c) the sources of the environmental contamination.

32. SURFACE SURVEILLANCE
• Locations: It is required for high risk locations such as
operation theatres and ICU settings.
• Sites for sampling (high touch areas)
• Indications (CDC recommendation)- Indicated for research,
as a part of an epidemiologic investigation, or during an
outbreak investigation.
o Routine periodic surface surveillance is not recommended.
• Method: Moistened sterile swabs (soaked in sterile saline)
are used to take the samples from high risk areas as
mentioned above and then inoculated on to non-selective,
nutrient-rich agar (e.g. blood agar) for the recovery of
aerobic bacteria.

33. SURFACE SURVEILLANCE
• Reporting: Only pathogenic organisms isolated are reported.
• Newer technique such as luminometer (expresses bacterial
contamination as CFU/ml) and glow gel techniques are
available which are easy to perform though expensive.