1

BACTERIOLOGICAL ANALYSIS OF
WATE
 Objective
2

 After completion of this chapter, the student should be able to:

◼ Explain types of water safety and quality
◼ Identify the sources of contamination of water
◼ List the type of organisms which are found in water
◼ Describe different sampling method for water analysis
◼ Explain types of water pollution
◼ Recognize water analysis methods
◼ Perform microbiological analysis of milk and water
◼ Conform with the standard operational procedure of
microbiological analysis of milk and water
 Introduction
3

Microbiology of Water
❑ Water could be classified in to two based on sources.
 Ground water
◼ This kind of water found in deep wells and in
subterranean springs. It is free from microbe due to
filtration action of soils.
 Surface Water
◼ Thisform of water found in lakes, Streams, and Shallow
wells. It has certain number of microbes.
 Introduction………
4

Contaminated water
 Water that is contaminated with chemicals and
microbes.
Polluted water
 Contaminated water and the condition is known exactly
 It
is characterized by unpleasant test, smell and
appearance.
 Potability of water
5

 The term that refers to the drinkability of water (Potable or

unpotable/ non-potable).

 In unpolluted water (Mountains, lakes or streams) the number

and types of bacteria is low and mainly Actinomyces, Bacillus,
Sterptomyces, Clostridium spp are commonly found.

 In polluted water, on the other hand you may have E. coli

forms, Enterobacter, Sterptococcus, Proteus and Pseudomonas
spp may found in high number.
 Types of Water Pollution
6

A. Physical Pollutions:
◼ When a particulate matters, like sand or soil make
water as cloudy.
◼ Some living materials also present.

B. Chemical Pollution:
◼ Thisis due to introduction of organic and inorganic
wastes.

C. Biological Pollution:
◼ When microorganisms contaminate water bodies.
 The Biochemical Oxygen Demand
7

 It is a critical measurement in polluted water .

 It refers to the amount of oxygen that microbes require

to decompose water organic matter.

 The dissolved oxygen is measured at different intervals

usually immediately after collection and after five days
intervals.

 The BOD is reported in PPM.

 Introduction………
8

Sources of contamination of water

 Water receives its bacterial from
 Air − Soil
 Sewage − Organic wastes
 Dead plants & animals

 Streams, rivers, wells and ground water can be contaminated

with feces of humans and animals

 Contamination of water polluted with animal or human feces is

an important source bacterial disease.
 Types of microorganisms found in water
9

❑ Most important pathogens that man acquires through water -

 Bacteria :
◼ V.cholera − Salmonella spp
◼ Shigella , − E.coli
◼ Campylobacter − Leptospira
 Parasites :
 Entameoba spp − Cryptosporidium
 Giardia − Balantidium
 Virus :
 Rota virus − Polio virus
 Hepatitis A
 Sampling methods of water
10

 Samples of water for bacteriological testing must be collected

in sterile bottles and care must be taken to prevent accidental
contamination of the water during its collection( aseptic
technique).
Sampling bottles
 Glass bottles used for water sampling should have a capacity
of at least 200 ml.
 They should be fitted with ground glass stoppers or screw caps.
 Sampling bottles cont’d…
11

 The stopper or cap and neck of the bottle should be protected

from contamination by a suitable cover either of paper or thin
aluminum foil.

 Silicon rubber liners, that will withstand repeated sterilization

at 160 ºC, should be used inside screw caps.

 After being sterilized the bottle should not be opened before

the sample is collected.
 Neutralizing chlorine in water samples
12

 When the water to be examined is likely to contain, sufficient sodium

thiosulphate (Na2S2O3.5H2O) should be added to neutralize
chlorine or chloramine to each bottle as follows:
 Add 100–200 μl (0.1–0.2 ml) sodium thiosulphate 30 g/l (3% w/v)
to each bottle of 200 ml capacity before it is sterilized.

 This will give a concentration of approximately 18 mg/liter of

water.
 Note: Sodium thiosulphate at a concentration of approx. 18 mg/
litter has no significant effect on the coliform or E. coli.
 Information to be supplied with water samples
13

 Each water sample should be given a code number and the

following information should accompany the sample
(preferably using a standardized form):

◼ Reasons for examination, e.g. whether a routine sample or

otherwise.

◼ Source and the exact place from where the water has
been collected

◼ State whether the water has been filtered, chlorinated, or

treated in some other way.
 Information to be supplied……
14

❑ Sample from a house tap water:

❖ Mention Whether the water was drawn from
a cistern (tank) or direct from the main.
❑ Sample from a well, Give details of:
◼ The well’s depth
◼ Covered or uncovered
◼ Recently constructed or altered
 Information to be supplied……
15

❑ Sample from a spring:

 Describe the stratum from which it issued and whether
the sample was taken directly from spring or from a
collecting chamber.
❑ Sample from a river or stream
 Mention the depth at which the sample was collected
 Whether from the side or the middle of the stream
 Whether the water level was above or below average
 Whether there had been heavy rainfall or flooding.
 Sample from a lake or reservoir:
16

Give the detail of :

 Exact position and the depth
 Temperature of the source
 Possible sources of pollution in the area
 Their approximate distance from the sampling
point.
 Date and time when the sample was taken (and
dispatched if sent to a testing laboratory).
 How to Sampling?
17

 Hold the sterile bottle by the base in one hand. Use the other
hand to remove the stopper and cover together.

 The stopper and cover should be retained in the hand while

the bottle is filled and then they should be replaced together.

 To prevent contamination, the person collecting the water must

not touch, or allow any surface to touch, the screw thread of
the bottle neck or the inside of the cap.

 If the bottle becomes contaminated, it must not be used.

 Collecting a sample from a tap
18

 Remove any external fittings from the tap, such as an

antisplash nozzle or rubber tube.
 Clean carefully the outside nozzle of the tap, especially any
grease which has collected.
 Turn the tap on full, and allow the water to run to waste for 1
minute.
 This allows time for the nozzle of the tap to be flushed and
any stagnant water in the service pipe to be discharged.
 Sterilize the tap using the flame of a blowlamp or gas torch,
or by igniting a piece of cotton-wool soaked in methylated
spirit and holding it with a pair of tongs close to the nozzle
until the whole tap is unbearably hot to the touch.
19
Sample from the tap cont’d…
 Allow the tap to cool by running the water to waste for a few
seconds.

 Fill the sample bottle from a gentle flow of water, and replace the
cap of the bottle.

 Using a water-proof marker or grease pencil, number the bottle with

the sample code number.

Note:

 Leaking taps may cause contamination of the sample from sources

outside the water pipe and therefore leaks should be reported when
sampling.

 A bacteriological sample should not be taken until the leak is

repaired.
 Collecting a sample from a river, stream,
or other surface water
20

1. Aseptically remove the cap and cover of the sterile bottle, and face
the mouth of the bottle upstream (i.e. towards the flow of water).

Note: To avoid entering the water, the bottle should be clamped to the
end of a stick. One way of doing this is to fix the bottle neck in a
retort stand clamp and mount this on a stick.

2. Plunge the neck downwards about 30 cm below the water surface,

and then tilt the neck slightly upwards to let it fill completely before
carefully replacing the cap and cover.

 Where there is no current, push the bottle forward horizontally until it

is filled.

3. Label the bottle with the sample code number.

 Collecting a sample from a tube well
21

1. Continuously operates the hand-pump for 5 minutes.

2. Heat the mouth of the pump, preferably by means of a blow

lamp or gas torch, and pump several gallons of water to
waste.

3 Aseptically collect a sample of water by allowing the water

from the pump to flow directly into the sterile bottle.
 Collecting a sample from an open well
22

 If the well is one from which water can be raised only by means of a
bucket and rope, use a weighted bottle to collect the sample as
follows:

1. Tie a sterile sample bottle on to a weighted length of rope or strong

string. Use a stone or heavy piece of metal as a weight, and attach the bottle
just above the weight.

2. Aseptically remove the cap from the bottle, and lower the bottle into
the well to a depth of about 1 meter.

3. When no more air bubbles rise to the surface, raise the bottle out of
the well and carefully replace the cap.

4. Label the bottle with the sample code number.

 Transporting water samples to a water
testing laboratory
23

 Immediately after collection, samples should be placed in an

insulated cold box for transport to a water testing laboratory.

 Water samples should be examined as soon as possible on

arrival and always within 6 hours of collection.

 Whenever possible, process water samples in the field.

 Frequency of sampling
24

 In large treatment plants, it should be a routine to sample

water daily at each stage of treatment.

 In many tropical rural areas, however, untreated sources of

water are used.

 In these situations, periodic sanitary surveys of the raw water

should be carried out to establish the level of risk of epidemic
waterborne disease to which the population is exposed.
 Frequency cont’d …
25

 The survey should include an on-site inspection and evaluation

of the water supply system, and a bacteriological analysis of
the water.
 From such a survey, sources of pollution can often be identified
and measures taken to prevent future contamination.
 The frequency of sampling water in distribution pipes,
Unchlorinated water supplies before distribution, and
chlorinated water before distribution is as follows:
 Water in distribution pipes
26

 Water quality deteriorates as a result of corrosion in pipes

allowing leaks and infiltration.

 The larger the population served, the greater the risk of

contamination.

 At least one sample /5000 population / month should be

examined

 Random routine sampling procedure should be established .

 Unchlorinated water supplies before
distribution
27

 According to WHO - maximum interval between successive

samples for bacteriological analysis :
◼ Population served Maximum interval
◼ Less than 20,000 . . . . . . . . . . . 1 month

◼ 20,000–50,000 . . . . . . . . . . . . . 2 weeks

◼ 50,000–100,000 . . . . . . . . . . . . 4 days
◼ >100,000 ……………………… 1day with
continuous chlorine residual recording
 Bacteriological Analysis of Water
28

 Water samples could be contaminated with different bacteria.

 All bacteria may not be detected for quality measure. Hence

indicator organism like Coliforms could be detected in routine
public health laboratory.

Water Sample Collection

 Raw or tap water could be taken us a sample under standard

conditions with appropriate containers.
 Bacteriological testing of water
29

❑ As previously explained, the E. coli count is the most

useful test for detecting faecal contamination of
water supplies in water quality analysis.
❑ Two principal techniques are available for counting
faecal coliforms:
❖ Membrane filter techniques
❖ Multiple tube/Most probable number (MPN) techniques
 I. Membrane filter techniques
30

❑ This method is a popular method, where a measured volume

of water sample (100 ml) is passed in a fine filter that
retains bacteria and the filter with the coliform organisms on
it is plated on different medium and incubated .
❑ The bacterial colony is counted and reported per 100 ml of
water sample.
❑ This gives the presumptive number of E. coli in the 100 ml
water sample.
 Culture media and buffered dilution water
31

 Each part of the test requires a different type of medium. For

example, when enumerating coliforms, lauryl sulphate broth
(lactose sodium lauryl sulphate broth) is used in the first
(isolation or presumptive) part of the test.
 For use, place a sterile cellulose pad in a sterile petri dish and add 2.5 ml of the sterile
broth.
 E. coli colonies appear yellow (lactose fermenting colonies) on both types of broth.

 In the second (confirmation) part, brilliant green lactose bile (BGLB) broth is
used to confirm total coliforms and E. coli medium to confirm faecal
coliforms.
32

 Calculate the presumptive E. coli count/100 ml as

follows:
 100 ml water sample, multiply number of colonies by 1.

 50 ml water sample, multiply number of colonies by 2.

 10 ml water sample, multiply number of colonies

by 10.
 Membrane filter technique procedure
33

1 Adding sterile broth to the cellulose pad in a petri

dish.
2 Aseptically removing the sterile membrane.
3 Placing the membrane on the filter base.
4 Pouring the water sample in the filter unit.
5 Drawing the water through the membrane by suction.
6 Removing the membrane.
7 Placing the membrane on the broth impregnated pad.
8 Labeling the Petri dish before incubation.
 Membrane filter technique procedure….
34

1. Adding sterile broth in 2. Aseptically removing the

cellulose pad in a petridish sterile membrane

3. Placing the membrane on

4. Pouring the water
the filter base
sample In the filter unit
 Procedure cont’d…
35

5. Drawing the water through

the Membrane by suction 6. Removing the membrane

8. Labeling the Petridish

7. Placing the membrane on
before incubation
broth impregnated pad
 II. Multiple tube/Most probable number (MPN)
36

 Water sample of 0.1 ml, 1ml, and 10 ml will be inoculated in

a medium (lactose broth tube) and coliforms will be detected.
 In this technique a 100 ml water sample is distributed (five 10
ml amounts and one 50 ml amount) in bottles of sterile
selective culture broth containing lactose and an indicator.
 After incubation, the number of bottles in which lactose fermentation with
acid and gas production has occurred are counted.
 By comparing the MPN table with the dilution on different
tubes an estimate will be done.
 By reference to probability tables, the most probable number
of coliforms in the 100 ml water sample can be estimated.
 Multiple tube/most probable number (MPN)….
37

 Specific tests are also available for individual

indicator organisms.
 MPN analysis is based on certain assumption

and formula
38
 Multiple tube/most probable number (MPN)….
39

 Bottles of sterile MacConkey broth (purple). Depending on the

type of water sample (treated or untreated), the following are
required:
Numbers of Ml of broth Strength of
bottle broth
Treated Water 1 50 Double
5 10 Double
Untreated water 1 50 Double

5 10 Double
5 5 Single

 *Double strength broth refers to broth made up using twice

40

 Table 10.3 Typical sample volumes and number of tubes for multiple
fermentation tube analysis

 Volumes of 0.1 and 0.01 ml of sample are obtained by addition of

1 ml of a 1/10 and 1/100 dilution, respectively, of the sample to
10 ml of single-strength culture medium.
41
 Procedures of MPN technique
42

1. Add 50 ml of water to the bottle containing 50 ml of broth.

This is most easily done by pouring direct into the bottle of
broth up to a 100 ml graduation mark scratched previously on
the bottle.
2. Using a sterile pipette, add 10 ml of water to each of the five
bottles containing 10 ml of broth.
Caution: Do not mouth pipette the water sample. Use a pipette filler.
3. If required (for untreated samples), pipette 1ml of water into
each of five bottles containing 5 ml of broth.
Note: the total volume of water inoculated is 100 ml for treated samples, and
105 ml for untreated poor quality samples.
43

4. Incubate the inoculated broths in a water bath or incubator at

44 ºC for 24 hours with the bottles loosely capped.
5. After incubation, examine and count each bottle which has
produced both acid and gas.
 Note: Acid production will be shown by a change in colour of
the MacConkey broth from purple to yellow, and gas
production by the collection of a bubble in the Durham tube.
 Refer to the probability tables shown below to
determine the most probable number (MPN) of faecal
coliform bacteria in the 100 ml or 105 ml water sample
 III. STANDARD PLATE COUNT METHOD
44

A sample of water is diluted in a sterile buffer

solution and specified amount will be inoculated in
agar medium and incubated.

 The total colony will be enumerated and multiplied

by the dilution factor for final report.
 Analysis of milk
45

❑ Milk is extremely nutritious food.

❑ It is an aqueous solution of proteins, fats, and carbohydrates.
❑ The pH of fresh milk is about 7.0 and this pH is an excellent
medium for growth of microorganism.
❑ More than 87% of milk is water and 2.5 % is Caseins.
❑ In addition Lacto albumin is present.
❑ Lactose is the commonest carbohydrates in milk and called Milk
sugar.
 Spoilage of Milk
46

 Lactobacillus and Streptococcus can ferment lactose in milk that

will result in sour curd.

 Sweat Curdling: This happens when Bacillus, Proteus,

Micrococcus attacks caseins enzymatically.

 It can be also contaminated by gram negative rods: E.coli,

E.aerogens

 Ropiness: Formed from Enterobacter, Klebsiella and Alcalegens.

 Milk Born infection
47

 Theses are mainly caused by M. bovis, Brucella spp, Coxiella

burnetti (Q-Fever), Campylobacter, Salmonella, Listeria spp.

Pasteurization

 It is the process of eliminating the least microorganism to make

sterile milk ( Relative sterility or commercial sterility).

Methods

 LTLT (Low temperature, long Time) pasteurization : This is

pasteurization of milk is at 62.80C for 30 minutes.
 Methods of pasteurization cont’d…
48

 HTST (High temperature Short Time) pasteurization: This

is a method of pasteurization at 71.7 0C for 15 s.
 Ultra-pasteurization: Pasteurization at 82 0C for 3 minutes.
The shelf life of this type of milk is long.
 Ultra high temperature (UHT) may be used for commercial
sterility method or for sterile foods preparation necessary for
immunosuppressant individuals in order to avoid any
complications from the Mos that are normally present in
heated but non-sterile foods.
 Milk heated to about 150oC for 2 - 3 sec can be stored at
room temperature and the products generally have a 3
months shelf life.
 Milk Standards
49

 The quality of milk may vary from place to place

(state to state) in terms of temperature, bacterial
load and other chemical tests.

Grade A milk: Pasteurized grade A milk

 This type of milk is pasteurized and the total

bacterial limit is 20,000/ml.

 Coliforms should not be more than 10 /ml and

Phosphatase activity must be less than 1 µg/ml.
 Milk grade cont’d…
50

Grade B milk/ Pre pasteurized Milk

 This type of milk may not be pasteurized and

the total plate count should not exceed
1,000,000/ml.

 The coli form count should not exceed 10/ml

and there should be no inhibitory substances.
 Some Useful Laboratory Tests For Milk
Analysis
51

I. Somatic cell count (SCC):

 SCC is a measure of the white blood cell count in milk.

 The SCC in milk of an individual ewe indicates her udder health

status, and

 Bulk tank milk SCC can indicate the general state of udder health in
a cow/ sheep .

 Somatic cells are always present in milk, but the SCC will rise when
an infectious agent enters the udder or when the udder has been
injured.
 SCC cont’d …
52

 A major consequence of rising SCC is a decrease in raw

milk quality, which has implications for milk processing.

 Most studies have found SCC levels for a healthy udder

could range 250,000 to 1,600,000 cells/ml.

 There are negative economic consequences for the

producer if somatic cell counts are elevated in the
animals.
53

❑ A high SCC is a strong signal of an udder infection, referred to

as mastitis.

❑ Common clinical signs of mastitis are a swollen udder half,

abnormal color of milk, or clots present in the milk.

❑ High SCC also has a negative effect on milk processing through

decreased yields and off-flavor development in finished
products.

❑ Milk with a SCC>1,000,000 decreased the cheese yield and

increased the development of rancid flavors in the cheese
 II. The phosphatase test
54

❑ Phophatase normally present in milk.

❑ The enzyme is heat tolerant similar with the phosphatase of

M.bovis and Coxelella burnetii.

❑ Milk sample is incubated with substrate like Sodium diphenyl

phosphate and the mixture is incubated.

❑ The phosphate released by the enzymes result in color change

and detected with chromogen.

❑ In properly pasteurized milk the test is negative and the color

change is absent and vice versa.
55
 III. The Standard Plate Count
56

 It is based on the total bacterial colonies grow on a culture

medium in 1 ml of sample.

 A serial dilution of milk sample is done as 1;10, 1;100,

1;1,000, 1;100,000 and incubated.

 The colony falls between 30-300 is selected and the total

bacterial count is estimated by multiplying the dilution
factor.
57

 Disadvantages

 Do not detect Psychrophilic and Thermophilic bacteria

 Do not detect anaerobic bacteria, viruses and molds

 The coliform bacteria is used as an important indicator

of the sanitary quality of milk that is isolated on a
violet red bile agar.
 IV. The Dye Reduction Test
58

 It tell us the relative amount of bacteria in the sample.

 Test principle:
◼ A sample of milk usually 1 ml is mixed with dyes (methyline blue or Resazurin)
and incubated at 37 0C.

◼ Up on growth of bacteria the blue color is reduced to colorless product in

case of methyline blue or pink to colorless in case of resazurin.

 Poor quality milk may have complete reduction of methyline blue

after 2 hrs.
 While good quality milk has minimal colour changes.
 The test has to be done on lactose fermented organism and most
useful if the CFU is more than 100,000/ml of milk sample.
 V. THE BREED COUNT METHODS
59

 In this test, 0.01 ml of milk sample is taken using a breed

pipettes and spread on a microscopic slide.

 One square c.m. area is taken and after staining with

Newmann-Lampart (Methylne blue) stains, the average
number of bacteria per field is reported.

 One of the disadvantage of breed counting method is it does

not differentiate live and dead cells, debris and the procedure
is tiresome.
 VI. Antibiotic detection test
60

 The test is useful for animals that are treated for M.bovis
Q fever and Brucellosis.

 The milk may have the antibiotic and inhibit a certain

indicator organism (B. subtils) (zone of inhibition).

 Penicillin antibiotic can be detected using Penicilinase

test
 Summary
61

 Milk is contaminated by gram positive rods, E. coli and E.

aerogens

 There are three methods for pasteurization of milk

 Based on coliform count and some chemical activity, milk is

graded as grade A and B.

 water can be polluted/unpolluted based on the number and

type of bacteria present

 There are three types of water pollution.

 Summary cont’d….
62

 Different methods of microbiological analysis of water

 Membrane filter method
 Standard plate count
 Most probable number
 Different laboratory tests for milk analysis
 Somatic cell count
 Phosphatase test
 Standard plate count
 Dye reduction test
 breed count methods
 Antibiotic detection test