MODULE IN

CHEMICAL ENGINEERING

CHE 3131L

Department of Chemical Engineering

SCHOOL OF ENGINEERING AND ARCHITECTURE

 REF SEA-BSCHE-CHE3131L-2022

WATER/WASTEWATER MICROBIOLOGICAL TEST BY MULTIPLE TUBE FERMENTATION

TECHNIQUE (MTFT)

I. OBJECTIVES

To test the presence of microorganisms in water/wastewater by Multiple Tube Fermentation

Technique (MTFT)

II. THEORETICAL BACKGROUND

Water microbiology is concerned with the microorganisms that live in water, or can be
transported from one habitat to another by water. Water can support the growth of many
types of microorganisms. This can be advantageous. For example, the chemical activities of
certain strains of yeasts provide us with beer and bread. As well, the growth of some bacteria
in contaminated water can help digest the poisons from the water.

However, the presence of other disease causing microbes in water is unhealthy and even
life threatening. For example, bacteria that live in the intestinal tracts of humans and other
warm blooded animals, such as Escherichia coli, Salmonella, Shigella, and Vibrio, can
contaminate water if feces enter the water. Contamination of drinking water with a type of
Escherichia coli known as O157:H7 can be fatal. The intestinal tract of warm-blooded animals
also contains viruses that can contaminate water and cause disease.

Bacteria are introduced into waters from many sources naturally or by man and his activities.
Feces from warm-blooded animals, including humans, may, at any time, contain disease-
producing microbes consisting of bacterial pathogens, viruses, or internal parasites. Most
bacteria present in surface waters are not harmful to health, but if pathogenic organisms
are ingested, disease or sickness may occur. Some of these bacteria are the E. coli, total
coliform and fecal coliform.

Escherichia coli (E coli) refers to one of the species of bacteria in the fecal coliform group. It
is found in large numbers in the gastrointestinal tract and feces of humans and warm-
blooded animals. Its presence is considered indicative of fresh fecal contamination, and it is
used as an indicator organism for the presence of less easily detected pathogenic bacteria
(similar to fecal coliform – typically used in assessment of drinking water).

Fecal coliform are bacteria found in the bodily waste of all warm-blooded humans and
animals, most species are not capable of survival outside the body for a long period of time.
Their presence in water indicates contamination by human sewage or animal droppings
(similar to E coli – typically used in assessment of wastewater).

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 Total coliform are a group of bacteria found in soil, on vegetation, and in large numbers in
the intestine of warm-blooded animals, including humans. Water is not natural medium for
coliform organisms and their presence in water is an indication of some type of
contamination. Most coliform bacteria are not disease-causing organisms, but they serve as
an indicator of the sanitary conditions of the water supply.
One of the more important laboratory tests to determine water quality is the bacteriological
test. This test indicates whether or not a given water is bacterially contaminated, and the
extent of such contamination. The test is critically in public supply systems where bacteria
may cause an outbreak of disease, however, in surface waters the test is usually not quite as
critical, though it may be equally as important to the user. In addition to the laboratory test,
other information concerning the probable source and significance of the count must also
be obtained in order for the analysis to be meaningful.

Multiple Tube Fermentation Technique (MTFT)

The multiple-tube procedure is one of the best tested and most authenticated microbial test
procedures and is used as a basis for water quality standards. This procedure involves a series
of preliminary and confirmatory tests in which gas bubbles are formed in small glass vials by
the action of coliform bacteria in a lactose broth medium. A statistical analysis, called the
most-probable-number (MPN), is then made. The MPN is not an actual count of organisms,
but merely on ideas of the number of coliform bacteria which, more probably than any other
number, would give results shown by the laboratory examinations.

III. APPARATUS AND REAGENTS

APPARATUS

Incubator Medicine dropper

Autoclave Bunsen burner
Culture tube with Durham tube Iron ring
Pipet Wire Gauze
Pipetol Wash Bottle
Beaker

REAGENTS

1. Lactose Broth:

For the lactose broth which is a base for the cultivation of salmonella and coliform organisms,
suspend 13.0 grams powder in a 500 mL of distilled water and mix thoroughly. Warm gently
until the solution was completed. Distribute 20 mL each in culture tubes containing inverted
Durham tube and sterilize by autoclaving the solution at 121oC for 15 minutes. Afterwards,
cool the broth quickly.

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 2. Brilliant Green Bile Broth:

Suspend 40.01 grams of the BGBB powder in 1000 mL of distilled water and heat to dissolve
the medium completely. Distribute 20 mL each in culture tubes containing inverted Durham
tube and sterilize by autoclaving the solution at 121oC for 15 minutes. Afterwards, cool the
broth quickly.

3. EC Broth:

Suspend 37.0 grams of the powder in a 1500 mL and heat to dissolve the medium
completely. Distribute 20 mL each in culture tubes containing inverted Durham tube and
sterilize by autoclaving the solution at 121oC for 15 minutes. Afterwards, cool the broth
quickly.

IV. METHODOLOGY

1. Pour 10 mL of the water sample in each of the culture tube containing 20 mL lactose
broth (presumptive broth). 5 culture tubes are used per sample.
2. Shake gently until the sample was uniformly distributed throughout the medium. Make
sure that there is no bubble inside the inverted Durham tube after shaking the solution.
3. Incubate the tubes for 24 hours at 35oC.
4. After 24 hours, examine the tubes for the presence of gas seen in the Durham tube or
effervescence which denote positivity of result. Repeat the incubation for another 24 hours
when the initial outcome was vague.
5. After 48 hours, transfer 2 drops from each presumptive tube into EC medium broth
and BGBB using sterilized pipette.
6. For the confirmation of total coliforms, incubate the BGBB from each of the
presumptive positive tube for 24 hours at 35oC. After the incubation period, check the tubes
for the presence of gas production and/or effervescence indicating positivity of result.
7. To confirm for the presence of thermotolerant coliforms, incubate the EC tubes from
each of the presumptive broth for 24 hours at 44oC and check for the positivity of results
basing from gas production after the incubation period.

Calculation

Compute using Thomas’ simple formula the Most Probable Number (MPN) of coliform
organisms present in the water sample using the MPN index.

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 V. REFERENCES

Standard Methods for the Examination of Water and Wastewater, 20th Edition.
EDWARDS, G.P., A.H. MOLOF & R.W. SCHNEEMAN. 1965. Determination of
orthophosphate in fresh and saline waters. J. Amer. Water Works Assoc. 57:917.
MURPHY, J. & J. RILEY. 1962. A modified single solution method for the determination of
phosphate in natural waters. Anal. Chim. Acta 27:31.
SLETTEN, O. & C.M. BACH. 1961. Modified stannous chloride reagent for
orthophosphate determination. J. Amer. Water Works Assoc. 53: 1031.
STRICKLAND, J.D.H. & T.R. PARSONS. 1965. A Manual of Sea Water Analysis, 2nd ed. Fisheries
Research Board of Canada, Ottawa.

Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any
means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.