Introduction

There are several aims or objectives for this experiment. First, to understand the
principle of MPN and apply it into practical. Second, to determine the amount of estimated
number of bacteria in liquid food sample. Third, to learn and get familiar with different media
and test.
Most Probable Number (MPN) method is a technique that is used to estimate number
of microorganisms that present like spread plate or pour plate does. Specific speaking, it is
an estimation of probable population in a liquid using statistics by diluting and determining
final points for microbial growth (Prescott, Harley & Klein, 2002). It is useful in determining
low concentrations of organisms, which is <100/g or ml (McLandsborough, 2005). In other
words, MPN is used in samples which contain too few organisms or when the
organisms do not grow on agar (Black, 2004). In the experiment, the MPN of E.
coli can be estimated by obtaining the number of positive results from Eosin
Methylene Blue (EMB) agar which is then referred to the MPN table. In food area, it is
used especially to estimate microorganisms contained in liquid food products or food
homogenates.
MPN method is usually used to determine the presence of coliform in food in either
liquid or solid form. Coliform is a term used to describe organism that are aerobic,
facultatively anaerobic, non-spore forming, gram-negative, ferment lactose which forms acid
and gas within 48 hours at 35°C (Downes & Ito, 2001). It includes species from genera
Escherichia like Escherichia coli (E. coli), genera Enterobacter, Klebsiella and Citrobacter.
These coliforms are used as an indicator microorganism to measure the faecal
contamination and presence of enteric pathogens like Salmonella. Coliforms are usually
found in intestine of humans and animals. This is supported by Prescott et al. (2002) as
these bacteria make up about 10 percents of the intestinal microorganisms in humans and
animals.
This method is done in series of steps which require several media. First MacConkey
broth is used, it is a purple liquid medium which is normally used to isolate coliforms and
enteric pathogens (Atlas, 2006). Durham tube within the broth is to capture the CO2 as gas
formation has resulted. MacConkey broth is used for its ability to change colour from purple
to yellow when coliforms are present. Only when there is a change of colour, the following
steps will be continued. If not, more time will be needed for the reaction to produce positive
result. After first step, Brilliant Green Lactose Bile (BGLB) broth is used, it is a green liquid
medium which is used to detect coliform microorganisms in foods or other sources. If it is
found turbid and gas has formed, there is positive result of E. coli. Then, Eosin Methylene
Blue (EMB) agar is used, it is in dark red colour which is used to confirm the presence of E.
coli by determining its ability to ferment lactose. If it can ferment lactose, dark colony with
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green metallic sheen will appear as a positive result (Prescott et al., 2002). Then,
biochemical test which is IMVic test can be used on further study positive result on the E.
coli bacteria. IMVic stands for Indole, Methyl red, Voges-Proskauer, Citrate which plays
different roles in detection respectively. Prescott (2002) states that E. coli is distinguished
from other genus when it shows positive result in Methyl Red and indole production while
negative result in Voges-Proskauer. When this reagent is added into peptone water
containing E. coli, a red ring will appear on top of the solution. This shows that the presence
of E. coli in the sample.

Potential Applications of Current Study

The current study of most probable number technique can be applied in many areas
of food sectors that involve liquid food products or food homogenates (Roberts, D. &
Greenwood, M., 2003). The potential usefulness of this technique is that bacteria yet to be
found can be discovered. As this method is useful in determining small number of bacteria,
the dangerous bacteria can be avoided by obtaining the probable number of bacteria.

Materials
a) Equipments
Laminar flow cabinet, Bunsen burner, micropipette, incubator, inoculating loop, universal
bottles, Durham tube, Petri dish
b) Sample
Liquid sample
c) Materials
Saline water, MacConkey broth, Eosin Methylene Blue (EMB) agar, Brilliant Green
Lactose Bile (BGLB) broth, Kovac’s indole reagent, Peptone water, micropipette’s tips,
disposable container, rubber bands

Methods
1. The food sample is diluted with saline water in a series of decimal dilution until 10-3.
2. 1.0ml from dilution 10-3 is taken and transferred to 3 tubes containing 10ml of
MacConkey broth and Durham tube. It is repeated for dilution 10-2 and 10-1.
3. The tubes were incubated at 35°C for 24-48 hours.
4. After incubation, tubes with acid (color changes) and gas (Durham tube floating)
formation are considered as positive result.
5. A loopful of inoculum from every positive MacConkey tube is transferred to Brilliant
Green Lactose Bile (BGLB) broth with Durham tube. BGLB tubes were incubated at
35°C for 24-48 hours. Only tubes with gas formation are considered coliform positive.
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 True coliform MPN number is calculated by recording the number of positive and
negative BGLB tubes from every set of dilution and based on standard MPN table.
6. One loopful of inoculum from every positive BGLB broth is taken and is streak on
Eosin Methylene Blue (EMB) agar. E. coli gives black color with green metallic sheen
colonies. The number of E. coli present is determined by counting the number of
BGLB tubes which give positive results on EMB and the most probable number of E.
coli is checked from MPN table.
7. For each positive tube, a single colony is inoculated into a universal bottle that
contains peptone water. The sample is then left to be incubated for 24 hours.
Biochemical tests (IMVic) is carried out for confirmation of typical E. coli colonies.

Results
MPN for Different Media at Three Consecutive Dilutions
Media Number of positive tubes MPN

10-1 10-2 10-3

(MPN/ml)

MacConkey 3 3 3 >1100

Brilliant Green 3 3 3 >1100

Lactose Bile (BGLB)

Eosin Methylene Blue 2 1 1 20+

(EMB)

Biochemical test 0 0 1 -
(IMVic)

Table 6.2: Number of positive tubes at different dilutions with its MPN at different media
respectively.

Observations of media before and after incubation

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 Media Before Incubation After Incubation

MacConkey
broth
on first and
second day

• Appear in clear purple • Change to turbid yellow

colour which the Durham colour and gas formation as
tubes are visible a positive result

Brilliant
Green
Lactose Bile
(BGLB) broth
on second
and third day
• Appear in clear green • Change to turbid green
colour which the Durham colour and gas formation as
tubes are visible a positive result

Eosin
Methylene
Blue (EMB)
agar on third
and fourth
• Appear in dark red • Positive result as
day
colour without any colonies appearance of black colour
with green metallic sheen
colonies without medium
changing colour.

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 Biochemical
test (IMVic)
on fifth day

• Red ring in black circle appears on top of peptone water showing

positive result while the rest do not have.
Table 6.1 shows the different media undergone changes after incubation.

Discussion
From the experiment, MPN method is run in a series of steps from dilution of liquid
sample to addition of Kovac’s indole reagent in order to determine the presence of E.coli and
its Most Probable Number (MPN). During the experiment, serial dilution are made for three
consecutive dilutions, they are 10-1, 10-2 and 10-3. Dilutions are done in order to get
acceptable results from MPN test (Bell, Neaves and William, 2005). This is because MPN
method requires low number of microorganisms to be tested on. It is done by diluting the
sample until highest dilution which at some point will contain single or no microorganism
(Black, 2004). For each dilution, triplicates of 3 or 5 universal bottles are prepared which
number of positive and negative results will be referred to MPN table. More universal bottles
like triplicates of 10 can be used to increase the precision at the same can improve the
detection limit of the method (Yousef & Carlstrom, 2003). For this experiment, triplicates of 3
universal bottles which in total are 9 bottles are applied.
There are several media used for MPN test in the four days of experiment. A few
stages of test are done, first presumptive test by MacConkey broth, second confirmatory test
by Brilliant Green Lactose Bile (BGLB) broth and last completed test by Eosin Methylene
Blue (EMB) agar (Yousef & Carlstrom, 2003). During the first day, presumptive test is done.
As other microorganisms can produce similar result under the same conditions with
MacConkey, this is why the test is considered as presumptive (Kleyn & Bicknell, 2003). The
MacConkey broths are a type of differential medium. They are used to detect coliforms and
enteric pathogens based on their ability to ferment lactose (Prescott & Harley, 2001). From
the result, all the MacConkey broths have changed colour from purple to yellow. This
change of colour is due to the production of acid that causes the pH-indicating dye to turn
into yellow (Downes & Ito, 2001). The yellow colour is not bright enough which may caused
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by insufficient time of incubation, which is less than 24 hours where it normally will take 24-
48 hours to be incubated (laboratory manual). Besides that, gas formation is realized from
the opening of universal bottles as not much gas has formed. The gas is not found trapped
in the Durham tube because of inadequate time of incubation. However, the presence of
yellow colour and gas formation is considered as positive result and proceed with BGLB
broth as confirmatory test.
After that, all the positive results of MacConkey broth are transferred to BGLB broth
which are selective selective and differential for coliforms. The purpose of transferring
inoculums into BGLB broth is to confirm the presence of coliforms (Yousef & Carlstrom,
2003). All the 9 broths are seen to be turbid and gas formation in the Durham tube. The
positive result has proved the coliforms are present by becoming turbid and forming gas
after incubation. The turbidity of the broth is caused by the growth of microorganism which is
E.coli. The gas formation is observed that indicating the respiration of the bacteria. These
events that occur in BGLB have provided positive indications of E.coli (Atlas, 2006).
Then, the inoculums in BGLB broth are transferred to Eosin Methylene Blue (EMB)
agar which is selective and differential as well by streaking. The purpose of this step is to
further confirm the positive result from BGLB broth. After streaking and incubation, only
those Gram-negative bacteria that ferment lactose will react on the agar. This is shown by
the bacteria, E.coli, they will appear as dark colour with green metallic sheen. In contrast,
the bacteria which do not ferment lactose will appear as colourless or purple colonies (Atlas,
2006).
As the EMB agar has confirmed the presence of E.coli, confirmation is further done
again with a biochemical test which is IMViC test. As IMViC test is a combination of a few
tests which are I: indole production, M: methyl red test, V: Voges-Proskauer test and C:
citrate test (Prescott & Harley, 2001). If there is reaction to either test of these, it will indicate
certain meanings from the sample. E. coli is a facultatively anaerobic gram-negative rod that
ferments lactose. Among the tests, indole production is used to indicate and confirm the
presence of E. coli. Indole production is done by adding Kovac’s indole reagent into peptone
broth that contains E. coli. When red ring appear on the broth, it fully confirm the presence of
E. coli. Other tests are not observed as no reagents are provided.
The advantage of MPN compared to other techniques like spread plate and pour
plate is it is more sensitive than humans when the amount of microorganisms are low (Bell,
Neaves and William, 2005). For the determination of MPN number, the MPN table especially
for 3 tubes are referred. The number of positive tubes are identified and referred on 3
dilutions. The MPN number is 20+ which is estimated about 20 bacteria per g or ml. The
95% confidence level provides the range of E.coli present which is in between 7 and 60.
There are a few precaution steps to be taken. First, the universal bottles that contain
6
Durham tube should be handled gently so that air will not trap inside the tube. The tubes
should be filled with gas from bacteria. Second, the incubation time should be within the
required time as early observation will not be seen as the reaction or fermentation has not
fully done. Third, care should be taken when serial dilution is done in order to prevent more
or less volume is taken from sample to sample. Fourth, all the steps in MPN method should
be done in Laminar air cabinet to avoid contamination.

Conclusion
The principle and application of MPN method is done and understands. The MPN of
E.coli is estimated 20+/g or ml. The different media used in determining E.coli for a series of
stages from presumptive, confirmatory to completed test and finally confirmation from
biochemical test, IMViC test with red ring appearing on top of broth. The results obtained are
almost close to what was expected because the colour resulted from MacConkey broth
observed there are still a little purplish in the yellow solution that is expected to be bright
yellow. The other results from other media are similar to expected one.

References

Atlas, R.M., 2006. Handbook of Microbiological Media: Food Examination. United States of
America: CRC Press.
Bell, C., Neaves, P. and William, A.P. 2005. Food Microbiology & Laboratory Practice.
USA: Blackwell Publishing.
Black, J.G. 2004. Microbiology, Principles and Explorations. 6th edition. London:
John
Wiley & Sons Inc.
Downes, F.P. & Ito, K. 2001. Compendium of Methods for the Microbiological Examination
of Foods. Washington: American Public Health Association.
Kleyn J. & Bicknell, M. 2003. Microbiology Experiments: A Health Science Perspective 4th
Edition. United States of America: McGraw-Hill Higher Education.
McLandsborough, L. A. 2005. Food Microbiology Laboratory. United States of America:
CRC Press.
Prescott, L.M. & Harley, J.P. 2001. Laboratory Exercises in Microbiology 5th Edition.United
States of America: McGraw-Hill Higher Education.
Prescott, L.M., Harley, J.P. & Klein, D.A. 2002. Microbiology 5th Edition. United States of
America: McGraw-Hill Higher Education.
Roberts, D. & Greenwood, M., 2003. Practical Food Microbiology 3rd edition. United
States of America: Blackwell Publishing Ltd.

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Yousef, A. E. and Carlstrom, C. 2003. Food Microbiology: A Laboratory Manual. New
Jersey: John Wiley & Sons, Inc.