2. Bacteriology of water
• Drinking water should have the following properties
1. Biological quality
2. Chemical quality
3. Physical quality
3. Bacterial flora in water
1.Naturally occurring bacteria e.g. Micrococcus,
pseudomonas
2.Soil bacteria e.g. Bacillus subtilis, Bacillus meganterium
3.Sewage bacteria e.g. E.coli, Enterococcus faecalis,
C. perfengens, salmonella , vibrio
4. WATER BORNE PATHOGEN
BACTERIAL Vibrio cholera, E. coli, salmonella typhi, salmonella
paratyphi A,B & C, Shigella spp. , Yersinia enterocolitica
Viral Hepatitis A virus, Hepatitis E virus, Rotavirus
Protozoal E. Histolytica, Giardia lambliya, Balantidium
coli.,cryptosporium, Isosporium
Helmenthic Ascaris lumbricoides, Trichuris trichura.
5. Bacteriological Examination of Water
• INDICATOR ORGAMISM- Which satisfy two properties
1.Should be present in excess number , should not be able to
proliferate in water.
2.Should be more resistant than the pathogen.
Indicator organisms themselves are not pathogen , but their presence
in water supply indicate that there is a contamination of sewage &
the water supply needs to be disinfected.
7. Total coliforms
• Bacteria that occur in large number in faeces & sewage but also found
in environment in the absence of fecal contamination.
• Their presence in water does not necessarily signify faecal
contamination
• Member of Enterobacteriaceae
• Only E.coli is a reliable indicator as it is not found in other source.
8. Thermotolerant (faecal) Coliforms
• Term “fecal coliforms” has been used in water microbiology to denote
coliform organism which grow at 44 & 44.5 ˚ c & ferment lactose to
produce acid and gas
• Presence of thermotolerant coliforms nearly always indicates faecal
contamination.
• In thermotolerant coliforms E. coli more than 95% present.
• E. coli does not survive in water for long time & therefore is the best
indicator of recent human or animal fecal pollution of water
9. Faecal streptococci
• Faecal streptococci is evidence of faecal contamination
• Faecal streptococci tend to persist longer in the environment than
thermotolerant or total coliforms & are highly resistant to drying
• Faecal streptococci grow at 37 & 44˚c
10. Pseudomonas aeruginosa
• It can survive in the environment for the long time
• It can multiply in various aquatic habitats hence its presence in water
is not necessarily good indicator for fecal contamination
11. Bacteriological examination of water
• Collection and transport of water sample
1.Use heat sterile bottles containing sufficient volume of sodium
thiosulphate to neutralize the bactericidal effect of any chlorine
2.Each bottle of 100ml capacity should contain 0.1 ml of fresh 1.8%
aqueous solution of sodium thiosulphate.
3.When collecting the sample from taps, allow water to run to water
for 2-3 min before running it to bottle
4.Water from steam or lakes- the bottle should be opened only after
immersed at a depth of 30 cm with its mouth facing the current
5.Transport- bottle should be properly labeled and sent to lab as
quickly as possible at least within 6 hours
12. Presumptive coliform count (multiple tube method)
• Standard test employed for bacteriological analysis of water.
• Only procedure that can be used if water sample are very turbid or if
semi solids such as sediments are to be analyzed.
• In multiple tube method, the presumptive coliform count is
determined, which is expressed as the most probable number (MPN)
of coliform organism in 100 ml of water.
Medium – MacConkey purple broth ( double strength and single
strength) in bottles or tubes.
Inverted Durham’s tube is used to detect gas production
Bromocresol purple is used as indicator
13. Requirement
• One bottle with loose fitting caps calibrated at 50 and 100 ml for 50
ml of sample + 50 ml of culture media
• 10 test tubes for 10 ml of sample + 10 ml of culture medium
• Test tube rack
• Measuring cylinder of different capacity
• Durham's tubes
• Double and single strength MacConkey broth
14. MacConkey broth, double strength
Peptone 40g
Sodium taurocholate 10g
Lactose 20g
Sodium chloride 10g
Bromocresol purple , 1% (w/v) in ethenol 2ml
Distilled water 1liter
Dilute the double strength medium with an equal volume of D.W. to make
single strength
15. procedure
• Place 50 ml and 10 ml volume of double strength concentration and 5
ml volume of single strength into suitable size of bottle or tube
containing an inverted Durham's tube
• Aseptically pipette one 50 ml volume and five 10 ml volume of the
water in the vessel containing corresponding volume of double
strength medium.
• Pipette five 1 ml volume into vessel containing single strength
medium.
• Incubate the media aerobically at 37˚C
16. Cont…..
• After 24 h and 48h of incubation inspect the media and note the
culture of each volume of water that show the production of acid
(color change) and gas (a bubble at the top of Durham's tube)
• In respect of the combination of positive and negative result read off
the MOST PROBABLE NUMBER (MPN) of presumptive coliform
bacteria present in 100 ml of the sampled water.
17.
18.
19. Eijkman test
• It is done to confirm that the coliforms bacilli detected in the
presumptive test are fecal E.coli. This is done by
Sub culturing the positive tubes on lactose containing medium such
as brilliant green bile broth for detection of lactose fermentation of
acid and gas at 44˚c
Demonstrating positive indole test at 44˚c.
20. Other methods of water analysis
Membrane filtration method
• Principle –
• this method is based on the filtration of known volume of water
through a cellulose membrane with pore diameter of 0.45 -0.2µm.
• the bacteria are retain on the surface of membrane filter , such
membrane is transferred on to a petri dish containing selective
differential culture medium at an appropriate temperature ( 37 and
44˚c) and incubate; charrecterstics colonies of coliforms developes
which can be count didectly.
21. Air surveillance
• Air is important vehicle of transmission of many pathogenic
organisms
• The examination of air to detect the number of bacteria carrying
particle is important particularl in critical areas such as OTs. ,bone
marrow transplant units etc.
• Routine air sampling is not recommended because-
• There is no standard guideline mentioning for permissible level of
microbial contamination of environmental surface or air.
• HAI rates are not related with levels of general microbial
contamination of air or environmental surface .
22. CDC recommends targeted air surveillance , which should be carried out for-
• Investigation of an outbreak.
• For research purpose.
• After reconstruction or newly constructed buildings.
• After fumigation or fogging ( to monitor the quality)
23. EVALUATION OF QUALITY OF AIR IN OT
Microbiological parameters
• Can be performed routinely by two broad methods
1. Microbiological sampling method
2. Particle count method
24. Microbiological sampling method
• Can be done in two ways
1.Method that can measure bacteria carrying particle settle down by
gravity from air e.g. settle plate method
2.Method that can count number of bacteria carrying particle in a given
volume of air e.g. The slit sampler and air centrifugation method.
25. Particle count method
• The airborne particle concentrations in OT is measure by means of
laser light scattering instrument ( particle counter)
26. Settle plate method
• Petri dishes containing an agar medium of known surface area are left
open for 30 min. to 1 hour. Then , the plates are incubated at 37 c for
24 hours
• Colony count : large bacteria carrying dust particle settle onto the
medium. The number of colony formed onto the plate indicates the
number of settle particle containing bacteria.
• Blood agar is the preferred medium for an overall count of
pathogenic, saprophytic and commensal bacteria.
• Malt extract agar may be used for molds
27.
28. Slit sampler method
• Most efficient and convenient method for counting the number of
bacteria carrying particle suspended in a unit of volume of air
• Procedure – A special equipment called “ slit sampler” has three part
1.An area to hold a petri dish.
2.Suction pump.
3.Outer surface has a slit of 0.33 mm width and 27.5 mm length and
3mm depth.
29.
30.
31. Milk surveillance
• Milk can be occasionally contain bacteria which are derived from
three sources
1.From animals
2.From hand of the milk handler
3.From the environment
32. Methods used for disinfecting/Sterilizing milk
1.Thermized milk- it is raw milk that had been heated for 15 sec at 57-
68˚c
2.Pasteurization- milk is heated to high temperature for short time (
72˚c for 15 sec.)
3.Ultra heat treated milk- milk is expose to very high temperature of
135˚c for 1 sec so that all microorganisms with their spores are
destroyed.
4.Sterilized milk- the milk is heated at 100˚ c for long period .
33. Bacteriological examination of milk
• Chemical tests-
Methylene blue reduction test
Phosphate test-
it is based on the principle that if pasteurization is effective , it should
inactive the enzyme alkaline phosphate which is normally present.
Turbidity test
34. Methylene blue test
• Rapid ,inexpensive way of indicating poor quality of milk that had
been unrefrigerated .
• The basis of test is that the length of time for bacterial
dehydrogenases to reduce methylene blue dye and decolorize it is an
indicator of the number of viable bacteria.
