Kamal Singh khada

1. BACTERIOLOGICAL ANALYSIS
OF DRINKING WATER BY
MOST PROBABLE NUMBER
AND MEMBRANE-FILTRATION
METHOD
Kamal Education Land
Kamal Singh Khadka
Anji Sherpa

2.  Indicator organisms:
• Indicators are easily detectable organisms whose presence
correlates directly to one or more pathogens contaminating an
environment
• Most indicators are enteric organisms or viruses, which are
commonly found in warm blooded mammalian and avian
gastrointestinal systems, giving a direct connection to fecal
contamination.
• However, many indicators can lack effectiveness due to a poor
correlation with certain pathogens.
• Two of the most widely accepted bacterial indicator organisms
are Escherichia coli and coliforms due to their fecal linkages,
and ease in laboratory analysis.

3. Characteristics of indicator organisms
 Total coliforms
o The term “total coliforms” refers to a large group of
Gram-negative, rod-shaped bacteria that share
several characteristics.
o The group includes thermo tolerant coliforms and
bacteria of fecal origin, as well as some bacteria that
may be isolated from environmental sources.
o In the laboratory total coliforms are grown in or on a
medium containing lactose, at a temperature of 35
or 37 °C. They are provisionally identified by the
production of acid and gas from the fermentation of
lactose.

4.  Thermo tolerant (fecal) coliforms
o The term “fecal coliform” has been used in water microbiology
to denote coliform organisms which grow at 44 or 44.5oc and
ferment lactose to produce acid and gas.
o The presence of thermo tolerant coliforms nearly always
indicates fecal contamination.
o Usually, more than 95 per cent of thermo tolerant coliforms
isolated from water are the gut organism Escherichia coli, the
presence of which is definitive proof of fecal contamination.

5.  Fecal streptococci
o The presence of fecal streptococci is evidence of fecal
contamination.
o Fecal streptococci tend to persist longer in the environment
than thermo tolerant or total coliforms and are highly resistant
to drying.
o It is, therefore, possible to isolate fecal streptococci from water
that contains few or no thermo tolerant coliforms.
o Fecal streptococci grow in or on a medium containing sodium
azide, at a temperature of 37-44 °C.
o They are usually detected by the reduction of a dye (generally
a tetrazolium-containing compound) or
the hydrolysis of aesculin. Routine methods may give
“false positives” and additional confirmatory tests may
be required.

6. MPN test and MF test:
• The principal methods used in the isolation of indicator
organisms from water are the membrane-filtration (MF)
method, the multiple-tube (MT) or most probable number
(MPN) method and presence-absence tests.
[A] Membrane-Filtration Method (MF)
[B] Most Probable Number Method (MPN)

7.  Comparison of methods for analysis of coliform
bacteria
Most probable number technique(MPN) Membrane filter technique
Slower: requires 48 hours for a positive More rapid: quantitative results in or
presumptive positive about 18 hours
More labor-intensive Less labor-intensive
Requires more culture medium Requires less culture medium
Requires more glassware Requires less glassware
More sensitive Less sensitive
Result obtained indirectly by statistical
approximation
(low precision)
Results obtained directly by colony count
(high
precision)
Not readily adaptable for use in the field Readily adapted for use in the field
Applicable to all types of water Not applicable to turbid waters
Consumables readily available in most Cost of consumables is high in many

8. [A] Membrane-Filtration Method
Objectives:
 To determine the microbial quality of
water.
 To enumerate and identify the bacteria
present in
water sample.

9. Introduction
o In the membrane-filtration , a minimum volume of 10ml of the sample
is introduced aseptically into a sterile or properly disinfected filtration
assembly containing a sterile membrane filter.
o A vacuum is applied and the sample is drawn through the membrane
filter.
o All indicator organisms are retained on or within the filter , which is
then transferred to a suitable selective culture medium in a petri dish.
o Petri dish is transferred to an incubator at the appropriate selective
temperature for a suitable time to allow the replication of the indicator
organism.
o Visually identifiable colonies are formed and counted, and the results
are expressed in numbers of “colony forming units” (CFU) per 100ml
of original sample.

11. Advantages
o Permits testing of large sample volumes.
o The membrane can be transferred from one medium to
another for purposes of selection or differentiation of
organisms thus allowing isolation and enumeration of
discrete colonies of bacteria.
o Results can be obtained more rapidly than by the
conventional MPN standard methods.

12. Disadvantages:
o This technique is inappropriate for waters with a
level of turbidity that would cause the filter to
become blocked before an adequate volume of
water had passed through.
o Membrane filters may be expensive in some
countries.

13. Uses:
o They are used extensively in the lab to sterilize heat
labile fluid materials.
o Effective and acceptable method to monitor drinking
water .
o Useful for bacterial monitoring in the pharmaceutical,
cosmetics and food beverage industries.
o Allows for removal of bacteriostatic or cidal agents that
would not be removed in pour plate , spread plate or
MPN techniques.

14. [B] Multiple tube method or MPN method
Objectives:
 To identify the bacteria present in
given sample of water.
 To enumerate the number of bacteria present in the
drinking water by MPN method.

15. INTRODUCTION :
o The multiple tube method is also referred to as the most probable
number (MPN) method.
o MPN method is used to estimate the concentration of viable
microorganisms in a sample by means of replicate liquid broth growth
in ten-fold dilutions.
o Water to be tested is diluted serially and inoculated in lactose broth,
coliforms if present in water utilize the lactose present in the medium
to produce acid and gas.
o The media receiving one or more indicator bacteria shows growth
and characteristic color change

16. o The number of total coliform is determined by counting the
numbers of tubes giving positive reaction and comparing the
pattern of positive results with standard statistical tables.
o From the number and distribution of tubes giving positive and
negative reactions, the organism in sample may be estimated
by reference to statistical tables.
o MPN test is completed in three major steps ;
a) Presumptive test
b) Confirmed test
c) Completed test

17.  PRESUMPTIVE TEST
o It is used to estimate and detect coliforms present in
water sample.
o Commonly used medium in MacConkey broth containing
indicator bromocresol purple.
o An inverted Durham’s tube is placed in each tube of the
medium.
o Yellow coloration and collection of gas in Durham’s tube
is assumed as presence of coliforms.
o The number of positive tubes are counted and referred to
the standard chart to find MPN of total coliforms per
100ml water sample.

18.  CONFIRMED TEST :
o Some spore forming bacteria gives false-positive test in
presumptive test.
o Confirmed test is performed to determine whether the
coliforms are of fecal origin or not and they are E. coli or
not.
o To carry this test, the cultures from presumptive test are
inoculated in selective media like Eosin Methylene
Blue(EMB) agar and the subcultures are incubated at
44.5oc and 37oc.
o Presence of typical colonies at 37oc confirms positive
coliform test and those at 44.5oc confirms the presence of
E. coli.

19.  Completed Test:
o The typical colonies in plate cultures are then sub-
cultured in MacConkey broth and incubated at 37
and 44.5.
o The presence of E. coli is confirmed by the
production of gas at 44.5 .

21. Advantages:
o Ease of interpretation, either by observation or
gas emission.
o Sample toxins are diluted.
o Effective method of analyzing highly turbid
samples such as sediments, sludge, mud, etc.
that cannot be analyzed by membrane filtration.

22. Disadvantages:
o It takes a long time to get the results.
o Results are not very accurate.
o Requires more hardware (glassware) and
media.
o Probability of false positives

23. Requirements:
 Petridishes
 Test tubes
 Sampling bottle ( sterile)
 MacConkey or Lactose broth
 EMB agar, Nurtient agar
 Durham’s tube
 Test tube stand
 Water sample

24. Procedures:
For Presumptive test:
1. Prepare MacConkey purple media of single and double strength in test
tubes with Durham’s tube and autoclave it.
2. Take three sets of test tubes containing five tubes in each set; one set with
10 ml of double strength (DS) other two containing 10ml of single strength
(SS).
3. Using sterile pipettes , transfer 10 ml of water to each of DS broth tubes.
Transfer 1ml of water sample to each 5 tubes of one set of SS broth and
transfer 0.1 ml water to five tubes of remaining last sets of SS broth tubes.
4. Incubate the tube at 37oc for 24 hours

25. 5. After incubation, observe the gas production in Durham’s tube and color
change of the media.
6. Record the number of positive results from each set and compare with
standard chart to give presumptive coliform count per 100ml water sample.

26. FOR CONFIRMED TEST:
1. Take the positive tube from the presumptive test and using EMB in
duplicate.
2. Incubate one plate at 37°C for 24 hours and another at 44.5°C for 24 hours.
3. Look for typical colonies in the media ; blue black with green metallic sheen
colonies are of E. coli in EMB agar.

27. For COMPLETED TEST :
1. Inoculate the colony in a tube of Lactose broth with Durham’s tube .
2. Subculture the colony on Nutrient agar plate. This subculture is considered
optional.
3. Incubate the broth cultures at 37°C and 44.5°C and Nutrient agar at 37°C.
4. Examine for acid and gas production in Lactose broth . The nutrient agar is
used for Gram staining and for IMViC test.