DETECTION AND ENUMERATION OF THE MOST PROBABLE NUMBER (MPN) OF COLIFORM IN WATER

T.L.V. PEIRIS

GS/MSC/FOOD/3630/08

2008/2010

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4.0 Introduction

The MPN procedure involves a multiple tube fermentation technique where three or more decimaldilutions of the sample are inoculated into tubes of broth medium and incubated at a specifictemperature and for a specific time. The method is progressive; i.e., first determining the presenceof coliforms in the tubes, then determining if these tubes also contain faecal coliforms, and thenconfirming whether E. coli is present. Based on the number of tubes indicating the presence /absence of the three groups of organisms, the most probable number present can be estimatedfrom a standard statistical MPN table.This method has been shown to produce satisfactory results with naturally-contaminated andartifically-contaminated water in sealed containers (including mineral and spring water) andprepackaged ice.The presence of coliforms, faecal coliforms and aerogenic E. coli in water may be determined bymeans of the MPN procedure. Briefly, this method involves serially diluting out the targetorganisms in the sample, in 5-replicate aliquots, to extinction .The probable level of the target organisms is then statistically estimated from an MPN table.Gas production is used as an indication of ability to ferment lactose from LST broth (presumptivecoliform test); gas production from BGLB broth is considered confirmation of coliform presence;gas production at 45o C from EC broth is used as confirmation of faecal coliform presence; andappearance of typical nucleated, dark-centred colonies with or without metallic sheen whenpositive EC broths are streaked onto L-EMB agar are indicative of E. coli. The typical colonies onL-EMB agar must be confirmed by further biochemical tests to prove the presence of E. coli.

Most probable number (MPN) test

Most probable number (MPN) test tubes of lactose containing Macconkey broth are inoculated

with the samples of interest (usually water) measuring 10 ml, 1 ml, and 0.1 ml. During incubation,

coliform organisms produce gas. Depending upon which tubes from which water samples display

gas, an MPN table is consulted and a statistical range of the number of coliform bacteria is

determined. The MPN test is very easy to perform and interpret, but it does not determine the

exact number of bacteria as the standard plate count does.

4.1.1 Materials :

Beaker (1L), Pipettes (1ml), Pipettes (10 ml), Tubes containing Macconkey Broth (sterilized)

4.1.2 Procedure :

100 ml of tap water and pond water was measured. Two set of series consisting of 5 tubes filled

with 10.0 ml of double strength Macconkey broth and another ten tubes (two sets) filled with 10.0

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ml of single strength Macconkey broth, with inverted Durham tubes was prepared. (it was

ensured-that Durham tubes do not contain air bubbles.)

5 tubes with double strength Macconkey broth was inoculated with 10.0 ml aliquots of the

sample to be tested and another 5 tubes with single strength medium with 1.0 ml aliquots of the

sample and the other 5 tubes with 0.1 ml of the sample using sterile pipettes. They were then

mixed well and kept at 370C for 24 hours and observed the tubes at the end of 24 hours for acid

and gas production. Negative tubes were re incubated for additional 24 hours. Positive tubes were

recorded. These are considered as positive for presumptive coliforms and estimate the microbial

content using the MPN table.

"Note: For the first five tubes (tube with double strength Macconkey broth), inoculum can be

prepared through a serial dilution as direct inoculums may contain immeasurable load of

microorganisms. This is applicable if the sample is contaminated water but not samples like milk.

4.1.3 Results

Type of Sample Tap water Pond water

Quantity of water put up in each tube 10 ml 1 ml 0.1 ml 10 ml 1 ml 0.1 ml

Number of tube used 5 5 5 5 5 5

Number of tubes giving positive

reaction

0 0 0 5 2 2

4.1.4 Conclusion

According to the MPN Table tap water has not been contaminated by coliforms and pond water

has been contaminated by micro organism. MPN of coliforms 100 ml of the pond water 95.

MPN Table - for Coliforms / E.coli /100 ml water.

Quantity of water put up in each tube

10 ml 1 ml 0.1 ml Most probable number of coliforms organisms in 100 ml of the original water Number of tubes used 5 5 5

000

000

012

0 *24

3

Number of tubes giving positive

reaction

555555

55555

55555

55555

555

222222

33333

34444

44555

555

012345

01234

50123

45012

345

5070

95 *120150175

80110150175200

250130170225275

350425250350550

9001 6001 800

4.2 Conformation of coliform

Conformation of the results is necessary since positive presumptive tests may be the result of

organisms of non coliform origin that are not recognized as indicators of faecal pollution. The

confirmed test requires that selective and differential media such as eosin methylene blue (EMB)

streaked from a positive lactose broth tube obtained from the presumptive media. Eosin

methylene blue contains the dye methylene blue, which inhibits the growth of gram - positive

organisms. In the presence of an acid environment EMB forms a complex that precipitates out on

the coliform colonies producing dark centers and a green metallic sheen. This reaction is

characteristic for Eschirichia coli, the major indicator of faecal pollution.

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4.2.1 Materials:

EMB agar plates, Inoculating loops, lamps

4.2.2 Procedure :

Labeled EMB agar plates was inoculated by streaking Using lactose broth culture which gave

positive results in 24 hours and Incubated the plates in an inverted position for 24 hours at 37 0C

Examined the colony characters.

4.2.3 Results & Conclusion

Type of Sample Results Conclusion

Tape water no dark cerves and agreen metallic

sheen

no faecal pollution in tape

water

Pond Preducing dark centres and a green

metallic sheen

There is a facal pollution in

pond water

4.3 Completed test :

The completed test is the final analysis of the sample. It is used to examine the coliform colonies

that papered on the EMB plates used in the confirmatory test. An isolated colony is picked from

the confirmatory test plate and inoculated into a tube of lactose broth and streaked on a nutrient

agar slant to perform Gram stain. The test tubes that shows acid and gas in the lactose broth and

the presence of gram negative bacilli on microscopic examination after inoculation and incubation

are further confirmation of the presence of E.coli and they are indicative of a positive completed

test.

4.3.1 Result

When examined, slide which prepared from the EMB Culture plate (belongs to sample of pond

water) by a microscope it contained pale to dark red coloured rod shaped bacteria, when spore

stain was done it got clared that these are non sporing rods.

Therefore this sample is bacteriologically unsatisfactory andit is contaminated by faecal bacteria (E-Coli)

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4.4 Discussion

The test methods described in this Practical provide guidelines for detection and enumeration of

bacteria of food and water origin. Then we can prevent food borne infections and diseases

occurring more commonly in Sri Lanka.

Disadvantages of MPN Techniques 1. MPN procedure takes very long time for the confirmed test result.2. In MPN the results are probability calculations and cannot be accurate.3. MPN requires more glass wares and media.4. False positive results are of common occurrence.

Advantages of MPN Techniques

1. Interpretation of the results requires minimal experience and training as results can be got by simply observing for the presence of gas or no gas.2. Water samples with high turbidity can be analyzed, since there is no apparent deleterious effect.3.Because of the dilutions used in the range of 1:0 or 1:100, toxic substances present in the sample can be diluted out.4. MPN technique is the effective method for analyzing samples such as muds, sludges, sediments etc.

References :

01. Food and Agriculture organization of the united nations 1992 Mannal of food quality

control 14/4 microbiological analysis FAO, Rome I taly.

02. Sri Lanka standard 614 Part 2 (1983) specification for potable water (Part 2)

Bacteriological Requirements.

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