Bacteriological Test

It is a method of analysing water to estimate the numbers of bacteria present and, if

needed, to find out what sort of bacteria they are. It represents one aspect of water
quality. It is a microbiological analytical procedure which uses samples of water and from
these samples determines the concentration of bacteria. It is then possible to draw
inferences about the suitability of the water for use from these concentrations. This
process is used, for example, to routinely confirm that water is safe for human
consumption or that bathing and recreational waters are safe to use.

E. coli culture on a Petri dish

The interpretation and the action trigger levels for different waters vary depending on
the use made of the water. Whilst very stringent levels apply to drinking water, more
relaxed levels apply to marine bathing waters, where much lower volumes of water are
expected to be ingested by users.

The examination of water for the presence of bacteria is very important. The bacteria are very
small organisms and it is not possible to detect them by microscopes. Hence they are detected
by circumstantial evidences or chemical reactions.

The growth of bacteria takes place by cell division and there are various classifications of
bacteria depending upon their shapes, oxygen requirements and effects on mankind. The last
classification is important for the water supply engineer from the view point of public health.
The bacteria may be harmless to mankind or harmful to mankind. The former category is
known as the non-pathogenic bacteria and the latter category is known as the pathogenic
bacteria. It is not possible to isolate pathogenic bacteria with the help of laboratory
instruments. Their chances of presence in a sample of water are increased in relation) to the
amount of non-pathogenic bacteria present in the sample of water.
The combined group of pathogenic and non-pathogenic bacteria is designated by bacillus coil
(bacillus – bacterium and coil = intestine) or B-coli group. This group of bacteria is present in
intestines of all living warm-blooded animals.

It should be noted that practically all natural waters contain some variety of bacteria and only
in rare cases, such as the supply from deep boreholes, the water is found without the presence
of any type of bacteria. Even rain before it touches the surface of earth collects dust and
bacteria according to the nearness of dense populations, presence of industries, climatic
conditions, etc. Subsequently numerous bacteria are added to the water by contact with the
soil, vegetation, life and matter of the earth's surface.

The number and species of these bacteria differ to a great extent. Thus many of the bacteria
found in water are derived from the air. soil and vegetation. Some of these are able to multiply
and continue their existence while the remaining die out in due course of time.

Following facts in connection with bacteria in water are to be noted:

(1) A certain number of bacteria cannot be cultivated under laboratory conditions while it is
possible to cultivate some varieties of bacteria under special conditions in laboratory. (2) For
surface waters, the seasonal changes have a marked and complex influence on the bacteria.

(3) Many varieties of bacteria produce colonies of various colours such as yellow-brown, red,
green and violet.
(4) Only few varieties of bacteria can tolerate any degree of acidity or caustic alkalinity.

(5) Some bacteria continue to live and multiply, others find conditions unsuitable and succumb
and there may be many new additions.

(6) The temperature of water has some bearing on bacterial multiplication. Each variety of
bacteria has its own optimum temperature for multiplication.
(7) The amount and remoteness of sewage and manurial pollution are the most important
sanitary considerations with regard to the bacterial contact of water.

(8) The bacterial purity of water varies according to the source of water i.e. whether from river,
stream, lake, shallow well or deep well and these differ greatly among themselves.
(9) The density of a bacterial population in water depends largely on quantity and quality of
food available. It may however be noted that the nutritional requirements of bacteria are very
simple and as a matter of fact, the organically pure deep well waters can support comparatively
large numbers of bacteria period even permit rapid multiplication.
(10) The gaseous constituents of water also affect bacterial activity in water to a certain extent.
For instance, the oxygen is favourable to some bacteria, but not to others
(11)The inorganic constituents of the water can also create favourable or unfavourable
conditions for the growth of bacteria.
(12) The nature of the organic matter as well as its quantity present in water will determine the
degree of bacterial multiplication and also the types or varieties of bacteria that will develop.
(13) The presence of metals in water, especially of copper, silver and aluminium, has usually
detrimental effect on bacteria
(14) The process of sedimentation which takes place during the storage of water is considered
to be a potent factor in the bacterial purification of water.
Apart from the main aim of finding out the sewage or manurial pollution, the bacteriology is
also of great value for the following purposes:
(1)To ascertain the effect of rainfall or drought on sources of water supply.
(2)To ascertain the efficiency of chlorination of sewage works effluent.
(3) to find out the change in quality of water either at source or after passing through the
distribution system of mains and reservoirs.
(4) to study the degree of pollution of river waters.
(5) to study the pollution of sea water near shores by tidal outfalls from sewage works:
(6) to work out the efficiency of purification system of water such as filtration, chlorination, etc.
(7) to work out the pollution by bathers of swimming pool water and to ascertain the efficiency
of purification process, etc.

It may be mentioned that the chemical analysis of water affords valuable information of past or
remote pollution and as such, it cannot be relied upon to reveal all forms of recent pollution.
On the other hand, the bacteriological analysis of water gives less information of the remate
history of the water, but it discloses with ease and with great reliability, the immediate or
recent causes of water pollution.

It is for this reason that the results of the chemical and bacteriological examinations should
always be suitably correlated and an opinion should be formed or expressed regarding the
purity of water on the strength of the results of its chemical analysis only is quite likely that
chemically pure water may reveal the presence of pathogenic bacteria during its bacteriological
analysis and it is for this reason that the bacteriological examination of water has been given
considerable significance and importance.

Following are the two standard bacteriological tests for the bacteriological examination of
water:

(i) Total count or Agar plate count test

(ii) B-coli test

(1)Total count or Agar plate count test: In this test, the bacteria are cultivated on specially
prepared medium of agar for different dilutions of sample of water with sterilised water. The
diluted sample is placed in an incubator for 24 hours at 37°C (ie blood heat) or for 48 hours at
20°C. These represent the so-called hat counts and cold counts respectively. The bacterial
colonies which are formed, are then counted and the results are computed for 1 cc For potable
water, the total count should not exceed 100 per c.c. (2)
B-coli test: This test is divided into the following three parts

(a) Presumptive test

(b) Confirmed test
(c) Completed test
(a) Presumptive Test
The presumptive test is based on the ability of coliform group to ferment the lactose broth and
producing gas. The confirmed test consists of growing cultures of coliform bacteria on media
which suppress the growth of other organisms. The completed test is based on the ability of the
culture grown in the confirmed test to again ferment the lactose broth

(al Presumptive test: Following procedure is adopted in this test:

(1) The definite amounts of diluted samples of water are taken in multiples of ten, such as 0.1
cc. 1.0 cc, 10 c.c. etc.
(2) The water is placed in standard fermentation tubes containing lactose broth.
(3) The tube is maintained at a temperature of 37°C for a period of 48 hours.
(4) If gas is seen in the tube after this period is over, it indicates presence of B-coli group and
the result of test is treated as positive. If reverse is the case, it indicates absence of B-coli group
and the result of test is treated as negative.
(5) A negative result of presumptive test indicates that water is fit for drinking.

(b) Confirmed test:

This test is carried out in one of the following ways:

(1) A small portion of lactose broth showing positive presumptive test is carefully transferred to
another fermentation tube containing brilliant green lactose bile. If gas is seen in the tube after
48 hours, the result is considered positive and the completed test becomes essential.

(2) A small portion of material showing positive presumptive test is marked on the plates
containing Endo or eosin-methylene-blue agar. The plates are kept at 37°C for 24 hours. If
colonies of bacteria are seen after this period, it indicates positive result and the completed test
becomes essential. The colonies are prominent by metallic brightness and dark spots.
(c) Completed test :
This test is made by introducing or inoculating bacterial colonies into lactose broth
fermentation tubes and agar tubes. The incubation is carried out at 37°C for 24 to 48 hours. If
gas is seen after this period, it indicates positive result and further detailed tests are carried out
to detect the particular type of bacteria present in water. The absence of gas indicates negative
result and the water is considered safe for drinking.
The recent development of field measurement of bacteria is that of membrane filter technique.
The membrane contains microscopic pores which are capable of retaining bacteria. The water is
filtered through the membrane and it is then incubated for a period of 20 hours along with
nutrients. The colonies of bacteria, if any, can then be counted by means of microscope.

B-coli Index:
This is an index or number which represents approximately the number of B-coli per cc. of
sample of water under consideration. The presumptive tests are carried out with different
ratios of the sample of water with sterilized water. A number of tests. is carried out for each
proportion and percentage of positive results is recorded. The difference between successive
percentages is worked out and it is multiplied by the reciprocal of quantity of solution. The Sum
of such values indicates B-coli index. For potable water, the B-coll index should be preferably
less than 3 and it should not exceed 10 in any event.
In order to arrange correctly a number of analytical results, the laws of probabilities are applied
and from the available statistics of test data, the most probable number or [Link] is determined
by applying the laws of statics to the results of the test. This number removes the drawback of
B-coli index and it indicates the bacterial density which is most likely to be present in water. It
should however be remembered that the M.P.N. is not the absolute concentration of organisms
that are present, but it only indicates a statistical estimate of that concentration.
For illustration of working out B-coli index, typical test recorded in table 5-4 and B-coli index of
the case comes to 5.68.

TABLE 5-4 B-COLI INDEX CALCULATION

No Proportion No. of Positive Difference Reciprocal Multiplication

tests result in of column of columns 5
percent 2 and 6
1 2 3 4 5 6 7
1 10.c.c 100 1 0.30 0.10 0.03
 2 1 c.c 100 0.70 0.55 1 0.55
3 0.1 c.c 100 0.15 0.11 10 1.10
4 0.01 c.c 100 0.04 0.04 100 4.00
5 0.001 c.c 100 Nil 1000 —
Total 5.68

Maintenance of purity of waters:

Some of the measures which can be taken to maintain the purity of waters are as follows:
The content of dissolved oxygen present in water should be high.
The entry of undesirable elements such as acidic and alkaline discharges, hot discharges from
thermal power stations, etc. into source of water supply should be prevented by suitable
means.
The presence of algae supplying oxygen to the water is desirable. (4) The quantity of total
dissolved solids, especially the chloride contents, should be very low.
There should be absence of organic wastes exerting oxygen demand on water. (6) There should
be enough reaeration for the purpose of reoxygenation of water.
The temperature of water should be maintained low.
The turbulence of water should be encouraged to make available more oxygen from air.

Water-borne diseases:

The water is considered to be an important agent for carrying and spreading some of the
dangerous diseases. The usual diseases carried by water are those of intestinal disorders such
as cholera, dysentery, typhoid, etc.

The patients suffering from such diseases give out the organisms responsible for such diseases
from their intestinal discharges. Such organisms then somehow obtain entrance to the sources
of drinking water and the disease is thus further carried forward.
The water is also considered partly responsible for other diseases such as hepatitis,
tuberculosis, gastro-enteritis, polimyelitis, etc.
The water-borne diseases are caused by pathogenic or harmful bacteria. The bacteria are found
everywhere on surface of earth and they are in great excess even in natural unpolluted water
bodies. However the water contaminated with sewage provides an excellent medium for the
growth of bacteria.

Out of all bacteria, the pathogenic bacteria form only a very small quantity. Further, the
pathogenic bacteria have only a short life in water and they offer less resistance to adverse
influences. If the source of pathogenic bacteria is known and if water treatment plant is
properly operated, it is possible to detect and to destroy the pathogenic bacteria and thus the
danger of water-borne diseases may be avoided.
The most important water-borne bacteria responsible for water-borne diseases are Salmonella
typhi, Salmonella paratyphi, Shigella dysenterial and Vibrio cholerae. The respective diseases
spread by them are typhoid, paratyphoid, dysentery and cholera.

It is significant to note that the water-borne diseases are predominant even in developing
countries and it is estimated that about five million babies die annually from water-borne
diseases in such countries and that one in six of the world's population suffer every year from
water borne diseases.

The surveys conducted by World Health Organization indicate that about 86% of the rural
populations in developing countries are without reasonable access to safe water. It is therefore
very clear that the battle with water-borne diseases will be finally won only by continued.
attention to the pollution prevention and by maintaining quality of water upto the required
standard. Thus the public health engineer in close co-operation with chemists and biologists will
play a great role in preventing the spread of water-borne diseases

Suitability of water for trade purposes:

The quality of water required for some of the common trades is as follows:
(1) Artificial silk: For the process of preparing artificial silk, the water to be used should be clear,
bright and absolutely free from colour. It should also be soft and should contain only a small
amount. of saline constituents in solution.
(2) Concrete products: The water which is fit for drinking purposes is considered suitable for the
preparation of cement concrete products. It should be clean and free from excess of sulphates
and chlorides which are detrimental to the setting and solidity of the concrete.

(3) Dairies: The bacterial purity of water is essential and the quantity of saline constituents is of
secondary importance. As a matter of fact, the water which may be excessively hard or may
contain an amount of salt perceptible to the taste, can be used, if it is bacteriologically pure.

(4) Dyeing : For producing delicate tints in dyeing, extreme care should be taken to use the
water of desired quality. It should be free from iron, colour and turbidity and should have the
smaller quantity of the salts.
(5) Laundries: The water must be clear, soft and free from iron.

(6) Manufacture of ice: The water should be free from colour and suspended matter and should
not contain metallic impurities such as iron and manganese. It should possess high degree of
organic and bacterial purity as expected for drinking water.

(7) Paper industry. For manufacturing paper of good quality, the water must be free from
colour and sedimentary matter and from every trace of iron and manganese. The saline
constituents should be present in such quantity that no appreciable amount is deposited in the
drying paper. However the water of any quality can be used for the manufacture of coarse
brown paper.

(8) Photographic films: There should be sufficient supply of good water which must be
colourless and absolutely free from suspended matter and metals such as iron, zinc and
manganese.
 Most Probable Number (MPN) Test:

Most Probable Number (MPN) is used to estimate the concentration of microorganisms in a

sample by means of replicating liquid broth growth in ten-fold dilutions. It is commonly used in
estimating microbial populations in soils, waters, and agricultural products. MPN test is
particularly useful with samples that contain particulate material that interferes with plate
count enumeration methods.

MPN is most commonly applied for quality testing of water i.e to ensure whether the water is
safe or not in terms of bacteria present in it. A group of bacteria commonly referred to as fecal
coliforms act as an indicator of fecal contamination of water. The presence of very few fecal
coliform bacteria would indicate that water probably contains no disease-causing organisms,
while the presence of large numbers of fecal coliform bacteria would indicate a very high
probability that the water could contain disease-producing organisms making the water unsafe
for consumption.

Principle

Water to be tested is diluted serially and inoculated in lactose broth, coliforms if present in
water utilizes the lactose present in the medium to produce acid and gas. The presence of acid
is indicated by the color change of the medium and the presence of gas is detected as gas
bubbles collected in the inverted Durham tube present in the medium. The number of total
coliforms is determined by counting the number of tubes giving positive reaction (i.e both color
change and gas production) and comparing the pattern of positive results (the number of tubes
showing growth at each dilution) with standard statistical tables.

MPN test is performed in 3 steps

1. Presumptive test
2. Confirmatory test
3. Completed test

Presumptive test
The presumptive test is a screening test to sample water for the presence of coliform
organisms.

If the presumptive test is negative, no further testing is performed, and the water source is
considered microbiologically safe. If, however, any tube in the series shows acid and gas, the
water is considered unsafe and the confirmed test is performed on the tube displaying a
positive reaction.

The method of the presumptive test varies for treated and untreated water.

Requirements

 Medium: Lactose broth or MacConkey broth or Lauryl tryptose (lactose) broth

 Glassware: Test tubes of various capacities (20ml, 10ml, 5ml), Durham tube
 Others: Sterile pipettes

Preparation of the Medium

 Prepare medium (either MacConkey broth or lactose broth) in single and double
strength concentrations.
 For untreated or polluted water :
o Dispense the double strength medium in 10 tubes (10mL in each tube) and single
strength medium in 5 tubes (10 mL in each tube)and add a Durham tube in an
inverted position.
 For treated water:
o Dispense the double strength medium in 5 tubes (10mL in each tube) and 50 mL
single strength medium in 1 bottle and add a Durham tube in an inverted
position.
 Examine the tubes to make sure that the inner vial is full of liquid with no air bubbles.

Sterilize by autoclaving at 15 lbs pressure (12

 1. Take 5 tubes of double strength and 10 tubes of single strength for each water sample
to be tested.
2. Using a sterile pipette add 10 mL of water to 5 tubes containing 10 mL double strength
medium.
3. Similarly, add 1 mL of water to 5 tubes containing 10 mL single strength medium and 0.1
mL water to the remaining 5 tubes containing  10 mL single strength medium.
4. Incubate all the tubes at 37°C for 24 hrs. If no tubes appear positive re-incubate up to 48
hrs.
5. Compare the number of tubes giving a positive reaction to a standard chart and record
the number of bacteria present in it.
For example, a water sample tested shows a result of 3–2–1 (3 × 10 mL positive, 2 × 1
mL positive, 1 × 0.1 mL positive) gives an MPN value of 17, i.e. the water sample
contains an estimated 17 coliforms per 100 ml

For treated (unpolluted) water

Takeake 1 tube of single strength (50mL) and 5 tubes of double strength (10mL) for each water
sample to be tested.

(2) Using a sterile pipette add 50 mL of water to the tubes containing 50 mL single strength
medium.

1. Similarly, add 10 mL of water to 5 tubes containing 10 ml double strength medium.

2. Incubate the tubes at 37°C for 24 hrs. If no tubes appear positive re-incubate up to 48 hrs.
3. Compare the number of tubes giving a positive reaction to a standard chart and record the
number of bacteria present in it.
For example, a water sample tested shows a result of 1-4 (1 × 50 mL positive, 4 × 10 mL positive)
gives an MPN value of 16, i.e. the water sample contains an estimated 16 coliforms per 100 mL.

Confirmatory Test
Some microorganisms other than coliforms also produce acid and gas from lactose fermentation.
In order to confirm the presence of coliform, a confirmatory test is done.

From each of the fermentation tubes with positive results transfer one loopful of medium to:

1. 3 mL  lactose-broth or brilliant green lactose fermentation tube,

2. to an agar slant and
3. 3 mL tryptone water.
Incubate the inoculated lactose-broth fermentation tubes at 37°C and inspect gas formation after
24 ± 2 hours. If no gas production is seen, further incubate up to a maximum of  48 ±3 hours to
check gas production.

The agar slants should be incubated at 37°C for 24± 2 hours and Gram-stained
preparations made from the slants should be examined microscopically.

The formation of gas in lactose broth and the demonstration of Gram-negative, non-spore-
forming bacilli in the corresponding agar indicates the presence of a member of the coliform
group in the sample examined.

The absence of gas formation in lactose broth or the failure to demonstrate Gram-negative, non-
spore-forming bacilli in the corresponding agar slant constitutes a negative test (absence of
coliforms in the tested sample).

Tryptone Water Test 

1. Incubate the tryptone water at (44.5 ±0.2°C) for 18-24 hours

2. Following incubation, add approximately 0.1mL of Kovacs reagent and mix gently.
3. The presence of indole is indicated by a red color in the Kovacs reagent, forming a film over the
aqueous phase of the medium.
a. Confirmatory tests positive for indole, growth, and gas production show the
presence of thermotolerant [Link].

b.
b. Growth and gas production in the absence of indole confirm thermotolerant
coliforms.

Completed Test.

Since some of the positive results from the confirmatory test may be false, it is desirable to do
completed tests. For this inoculum from each positive tube of the confirmatory test is streaked on
a plate of EMB or Endo agar.

In this process, a loopful of a sample from each positive BGLB tube is streaked onto selective
medium like Eosin Methylene Blue agar or Endo’s medium. One plate each is incubated at
37°C and another at 44.5± 0.2°C for 24 hours.

High temperature incubation (44.5 ±0.2) is for detection of thermotolerant [Link].

Following incubation, all plates are examined for the presence of typical colonies.

 Coliforms produce colonies with a greenish metallic sheen which differentiates it from non-
coliform colonies (show no sheen). The presence of typical colonies on high temperature (44.5
±0.2) indicates the presence of thermotolerant [Link].
 Advantages of MPN

 Ease of interpretation, either by observation or gas emission

 Sample toxins are diluted
 Effective method of analyzing highly turbid samples such as sediments, sludge, mud, etc.
  that cannot be analyzed by membrane filtration.

Disadvantages of MPN

 It takes a long time to get the results

 Results are not very accurate
 Requires more hardware (glassware) and media
 Probability of false positives

IMVIC Tests

Each of the letters in “IMViC” stands for one of these tests. “I” is for indole; “M” is for methyl
red; “V” is for Voges-Proskauer, and “C” is for citrate,  lowercase “i” is added for the ease of
pronunciation. IMViC is an acronym that stands for four different tests.

To obtain the results of these four tests, three test tubes are inoculated: tryptone broth (indole test),
methyl red – Voges Proskauer broth (MR-VP broth), and citrate. IMViC tests are employed in the
identification/differentiation of members of the family Enterobacteriaceae.

General procedure for performing IMViC Tests and their interpretations:

Cultures of any members of Enterobacteriaceae have to grow for 24 to 48 hours at 37°C and
the respective tests can be performed.

Indole Test:

It is performed on sulfide-indole-motility (SIM) medium or in tryptophan broth, or in motility

urease indole (MIU) medium. Result is read after adding Kovac’s reagent.
The positive result is indicated by the red layer at the top of the tube after the addition of
Kovac’s reagent.

1. A negative result is indicated by the lack of color change at the top of the tube after the
addition of Kovac’s reagent.
2. nodule Test Results: Positive-development of Red-ring.

Methyl red test and Voges-Proskauer test both are done in methyl red–Voges-Proskauer
(MR-VP) broth, but the reagents that are added varies according to the tests.

Methyl Red (MR) test

Positive methyl red test is indicated by the development of red color after the addition of methyl red
reagent.

A negative methyl red test is indicated by no color change after the addition of methyl red reagent voges.

1. Negative VP test is indicated by a lack of color change after the addition of Barritt’s A
and Barritt’s B reagents.
2. A positive Voges-Proskauer test is indicated by the development of red-brown color after
the addition of Barritt’s A and Barritt’s B reagents.

Citrate Utilisation Test:

The test is performed on Simmons citrate agar:

1. Negative citrate utilization test is indicated by the lack of growth and color change in the
tube
2.  A positive citrate result is indicated by growth and a blue color change. 

IMViC tests result of some genera of Escherichiaceae

IMViC tests of Escherichia coli

  Indole: Positive
 Methyl-Red: Positive
 Voges-Proskauer test: Negative
 Citrate test: Negative

IMViC Test of Ecoli: ++–

IMViC tests of Klebsiella (formerly Enterobacter) aerogenes.

1. Indole: Negative
2. Methyl-Red: Negative
3. Voges-Proskauer test: Positive
4. Citrate test: Positive

IMViC tests of Proteus vulgaris

1. Indole: Positive
2. Methyl-Red: Positive
3. Voges-Proskauer test: Negative
4. Citrate test: Negative

IMViC tests of Citrobacter freundii

1. Indole: Negative
2. Methyl-Red: Positive
3. Voges-Proskauer test: Negative
4. Citrate test: Positive

Population equivalent
Population equivalent (PE) or unit per capita loading, or equivalent person (EP), is a parameter
for characterizing industrial wastewaters. It essentially compares the polluting potential of an
industry (in terms of biodegradable organic matter) with a population (or certain number of
people), which would produce the same polluting load.[1]: 65  In other words, it is the number
expressing the ratio of the sum of the pollution load produced during 24 hours by industrial
facilities and services to the individual pollution load in household sewage produced by one
person in the same time. This refers to the amount of oxygen-demanding substances in
wastewater which will consume oxygen as it bio-degrades, usually as a result of bacterial
activity.[

Equation and base value

A value frequently used in the international literature for PE, which was based on a German
publication, is 54 gram of BOD per person (or per capita or per inhabitant) per day.[1]: 65 [2] This
has been adopted by many countries for design purposes but other values are also in use. For
example, a commonly used definition used in Europe is: 1 PE equates to 60 gram of BOD per
person per day, and it also equals 200 liters of sewage per [Link] the United States, a figure of
80 grams BOD per day is normally used.

If the base value is taken as 60 grams of BOD per person per day, then the equation to
calculate PE from an industrial wastewater is: