CFB 20303LMPRACTICAL

1.

MALAYSIAN INSTITUTE OF CHEMICAL AND BIOENGINEERING

TECHNOLOGY.

FOOD MICROBIOLOGY (CFB 20303)

UNIKL MICET
JAN
DOC. NO. :
FDTLM2033 LAB MANUAL 07
(REV. NO. 1)

PRACTICAL 5A: ENUMERATION OF COLIFORM AND

ESCHERICHIA COLI

OBJECTIVE:

 To familiarize with the enumeration techniques employed for coliform and E. coli.
 To determine coliform bacteria and E. coli count by using Most Probable
Number (MPN) method.

KEYWORDS

Enumeration, Coliform Bacteria, Escherichia coli, MPN method

1. INTRODUCTION

Coliform bacteria are defined as rod-shaped Gram-negative organisms which

ferment lactose with the production of acid and gas when incubated at 35 °C.
Coliforms are abundant in the feces of warm-blooded animals, but can also be found
in the aquatic environment, in soil and on vegetation. In most instances, coliforms
themselves are not the cause of sickness, but they are easy to culture and their
presence is used to indicate that other pathogenic organisms of fecal origin may be
present. Fecal pathogens include bacteria, viruses, protozoa or parasites.

Escherichia coli (E. coli), a member of the coliform group, can be distinguished
from most other coliforms by its ability to ferment lactose at 44°C, and by its
growth and colour reaction on certain types of culture media. Unlike the general
coliform group, E. coli are almost exclusively of fecal origin and their presence
is thus an effective confirmation of fecal contamination. Typically, E. coli are
about 10% of the coliforms in human feces.

Serial dilution tests measure the concentration of a target microbe in a sample

with an estimate called the most probable number (MPN). The MPN is
particularly useful for low concentrations of organisms (<100/g), especially in
milk and water, and for those foods whose particulate matter may interfere with
accurate colony counts.

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2. MATERIALS AND INSTRUMENTS

2.1 Lake water/ meat as food sample and cultures of

appropriate microorganisms supplied or as advised
2.2 Violet red bile agar (VRBA)
2.3 Mc Conkey broth, 2%
2.4 Peptone water as diluent for food preparation in analysis (Sterilised)
2.5 Numerous Sterilised MacCartney bottles filled up with 9 ml
of Peptone water for serial dilution.
2.6 Stomacher, blender or equivalent
2.7 Compound microscope, slides and coverslips
2.8 Colony counting device (optional)
2.9 Incubator capable of maintaining 35° C
2.10 Autoclave for sterilization
2.11 Sterile utensils
2.12 Durham tubes
2.13 Screw cap test tubes
2.14 Kovac reagent

3. PROCEDURE

3.1 Sample Handling

All sample units are refrigerated (0-5° C) until needed, with exception of shelf-
stable products. The sample units of frozen products shall be kept frozen.
Thawing of frozen samples must be cautiously undertaken under time and
conditions which prevent microbial growth or death. Analyze the sample units as
soon as possible after receipt at the laboratory.

3.2 Preparation for Analysis

Preparation of the violet red bile agar (VRBA) for analysis must be carried out
prior to the analysis according to the manufacturer's instructions. Since the pour
plate technique will be used, molten VRBA tempered to 45 - 48° C will be
required. Clean surface of working station with suitable disinfectant. Mark
clearly the duplicate/triplicate Petri dishes identifying the sample, sample unit,
dilution or date of inoculation as required for identification where appropriate.

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3.3 Preparation of Sample

Prepare Peptone water as diluent (It may be provided.) Conduct serial dilution up
to 10-6 dilution factor for each sample.

3.4 Plating

The pour plate technique will be used.

Molten VRBA tempered to 45 - 48° C is prepared.

Cool it to recommended temperature before pouring it into the plates.

Transfer 1 ml of each dilution into Petri dishes aseptically.

Pour 10 ml VRBA into the plates and swirl plates to mix.

In order to prevent surface growth and spreading of the colonies, overlay

with 5 ml VRBA and allow solidifying.

3.5 Incubation

Invert solidified plates and incubates for 18 - 24 hour at 35° C. If dairy

products being analyzed, incubate at 32° C.

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3.6 Counting of Colonies and Growth Examination

Examine plates under illumination and use of magnification glass may be

required in the process. Count the colonies that have purple-red coloration with
diameter of 0.5 mm or larger, and are surrounded by zone of bile acids
deposition or precipitation. Plates should score around 25 - 250 colonies.

Plate Count

1. Choose a plate that appears to have between 25 and 250 colonies.

For colonies more than 250, report as TNTC (too numerous too count)
For colonies less than 25, report as TSTC (too small to count) or <30
For plate which appears no growth, express as NC (no colony)
If half of the plate surface is covered with spreader , report as
SPR (Spreader)
If the higher dilution factor gives the higher number of colonies than
the lower dilution factor, report as LA (laboratory accident)
If ALL of the plates have more than 300 colonies, choose a plate that
near to the 300 colonies or from the highest dilution factor. Report as
Estimated cell number.
If no colonies appear in ALL plates, even though no antimicrobial agent
has been used, report the cell number as <1 multiply with the lowest
dilution factor.

2. Count the exact number of colonies on that plate using the colony counter
(as demonstrated by your instructor). Then record your result as in Table 1

Table 1 : total number of colonies

Microorganism:

Dilution No of colonies

factor 10-1 10-2 10-3 10-4 10-5 10-6 10-7

Plate 1

Plate 2

Average

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3. Calculate the number of CFUs per ml of original sample as follows:

The number of CFUs per ml of sample =

The number of colonies (25-250 plate) X

The dilution factor of the plate counted X

1/ amount of sample in the plate

____________ = Number of colonies

____________ = Dilution factor of plate counted

____________ = Number of CFUs per ml

In the case of the example above, 150 x 1,000,000 = 150,000,000 CFUs per ml.

For a more accurate count it is advisable to plate each dilution in duplicate

or triplicate and then find an average count.

4. If there are more than one plate has total colonies in the range of 30 – 300, the
following rules should be followed:

a) If the highest number of cells from the countable plate is two times or less
than two times from the lowest number of cells, report the total average of all
plates as the amount of cells in the sample.

b) If the highest number of cells from the countable plate is more than two
times the lowest number of cells, choose the LOWEST counts. The lowest
count represents the number of bacterial cells in the undiluted original sample.

5. Record your results .

If the test organisms are detected at counts of 100 or higher per gram, report
with one figure before and one figure after the decimal point expressed to the
power of 10 in the form of :

a x 10b cfu/ g or mL

where a is never less than 1.0 or greater than 9.9 and b represents the
appropriate power of ten.

Round counts up if the last figure is 5 or more and down if the last figure is 4 or less

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e.g. 11,300 cfu/g reported as 1.1 X 104 cfu/g

235,000 cfu/g reported as 2.4 X 105 cfu/g

3.7 Most Probable Number (MPN) for Coliform enumeration

The sample should be diluted by serial dilution manner up to 10-6 dilution
factor. Then transfer each to a tube of Mc Conkey broth that comes with
inverted Durham tube and incubate the tubes at 37° C. Examine the tubes at 48
hour for gas production. Label the tubes for positive sign if the broth color
change to yellow with gas production occurs in the tube, while negative sign
should be labeled to the tubes with no gas production as in Table 2. By using 3 –
tubes MPN table, we can determine the estimated coliform number in our
sample. (Please refer to Appendix 1).

3.8 Record of Results and Discussion

You are required to compute the coliform bacteria count and present your results
in colony forming units (cfu) per gram or ml of product. Round off counts to
TWO(2) significant figures, to the nearest whole number.

You should compare the result total plate count of coliform by using two
different kinds of method (pour plate technique and MPN technique). Discuss
the factors that may affect the result.

Table 2 : Observation of gas production in Mc Conkey broth after 48 hours

Microorganism:

Positive/negative (+/-) of gas production

Dilution factor
10-1 10-2 10-3 10-4 10-5

Tube 1

Tube 2

Tube 3

Number of positive tube(s)

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3.9 Confirmation test on Escherichia coli

(a) From 3.7, transfer 1 mL from each gas positive tube of Mc Conkey broth to a
separate replicate tubes of new Mc Conkey broth (3 tubes MPN) that was
previously warmed at 44.5°C.

Eg. If you obtained 3-2-1 positives tubes from 10-1, 10-2 and 10-3 dilutions,
replicates the same positive tubes into the new Mc Conkey broth with the same
label.

(b) Repeat (a) but now replace Mc Conkey broth with sterile peptone water (3 tubes
MPN) that was previously warmed at 44.5°C.

(c) Incubate all tubes for 48 hours at 44.5°

(d) After 48 hours, label the tubes for positive sign if the broth color of Mc Conkey
change to yellow with gas production occurs in the tube, while negative sign
should be labeled to the tubes with no gas production as in Table 2.

(e) As for samples in sterile peptone water, put few drops of Kovac reagent into the
tubes. Label the tubes for positive sign if there is red ring occurred in the tubes.

(f) The final result of E. coli confirmation should only be counted if both test from
Mc Conkey broth and sterile peptone water give positive sign from each dilution
factors.

Eg. If you obtained 3 positive tubes from 10-1 of Mc Conkey broth and 2
positive tubes from 10-1 from sterile peptone water, the final positive tubes of
E.coli from 10-1 are two (2) tubes only.

(g) Calculate the estimated E. coli number in your sample by using 3 – tubes MPN
table.

4. REFERENCE
1. Bell C., Neaves P. and Williams A.P. (2005). Food Microbiology and
Laboratory Practice, Wiley-Blackwell, Oxford.
2. Garg N. and Garg K.L. (2010). Laboratory Manual of Food Microbiology, IK
International Publishing House Pvt. Ltd.

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2.
SEM Plate of a Coliform bacteria

Durham tube

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APPENDIX 1- MOST PROBABLE NUMBER (MPN) METHOD

FOR COLIFORMS ENUMERATION

To envision the theoretical basis for the Most Probable Number (MPN) Method, think
of a ten-fold dilution series being made of a water sample with one ml of each dilution
being inoculated into a separate tube of an all-purpose broth medium.

After incubation, the broth tubes are observed for the presence or absence of growth.
Theoretically, if at least one organism had been present in any of the inocula, visible
growth should be seen for that particular tube. If the broth inoculated from the 10 –3 dilution
shows growth, but the broth from the 10 –4 does not, it is then possible to say that there were
greater than 1X103 organisms per ml of the original sample but less than 1X10 4 per ml.

Bacteria are rarely, if ever, distributed evenly in a sample. For example, if a 10 ml

sample contains a total of 300 organisms, not every one ml aliquot will contain 30
organisms; some will contain more or fewer, but the average of all ten aliquots in the
entire 10 ml sample will be 30. This also applies to any of the dilution tubes from which
inocula are taken.

To increase the statistical accuracy of this type of test, more than one broth tube can be
inoculated from each dilution. Standard MPN procedures use a minimum of 3 dilutions
and 3, 5 or 10 tubes per dilution. The statistical variability of bacterial distribution is
better estimated by using as many tubes as possible or practical. After incubation, the
pattern of positive and negative tubes is noted, and a standardized MPN Table is
consulted to determine the most probable number of organisms (causing the positive
results) per unit volume of the original sample.

In the following example, a set of 3 tubes of an all purpose broth medium is inoculated
from each of the ten-fold dilutions, with each tube being inoculated with one ml.

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3-tube MPN Table as below will be used to determine the coliform numbers in the
sample. The heading of the last column tells us that this combination of results (in order:
3-2-0) suggests an average of 0.93 organisms being inoculated into each of the tubes of
the middle set (of the three sets of tubes chosen) – i.e., those inoculated with one ml of a
10–3 dilution. Therefore, the most-probable number of organisms per one ml of the
original, undiluted sample would be 0.93 X 103 or 9.3 X 102.

This method, with the associated table, only works if there is a succession of 1/10
dilutions being tested (in an appropriate medium) for growth, and the tubes chosen to
compare with the table are in consecutive order of increasing dilution.

Some things to keep in mind:

 Other amounts than one ml can be inoculated into the broth tubes. For example,
inoculating 0.1 ml of a 10–3 dilution is equivalent to inoculating 1 ml of a 10–4
dilution; the "plated dilution" (10–4) is the same in each case.
 It doesn't matter what amount of medium there is in the tubes being inoculated.
For example, the same growth response should be evident if we doubled the
amount of broth in each tube.
 Also, we don't have to inoculate our tubes from dilutions. For example, one can
set up a series of tubes where 10 grams are inoculated into each tube in the first
set, 1 gram is inoculated into each tube in the second set, and 0.1 gram is
inoculated into each tube in the third set. The sequence of decimally-decreasing
amounts is maintained. (Recall how the so-called "plated dilution" represents the
actual amount of sample being inoculated.)

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Appendix 1: 3-tubes MPN Table

No. of Tubes Positive in MPN in the

inoculum of the 2 0 0 0.091
first middle last middle set of 2 0 1 0.14
set set set tubes 2 0 2 0.20
0 0 0 ‹0.03 2 0 3 0.26
0 0 1 0.03 2 1 0 0.15
0 0 2 0.06 2 1 1 0.20
0 0 3 0.09 2 1 2 0.27
0 1 0 0.03 2 1 3 0.34
0 1 1 0.061 2 2 0 0.21
0 1 2 0.092 2 2 1 0.28
0 1 3 0.12 2 2 2 0.35
0 2 0 0.062 2 2 3 0.42
0 2 1 0.093 2 3 0 0.29
0 2 2 0.12 2 3 1 0.36
0 2 3 0.16 2 3 2 0.44
0 3 0 0.094 2 3 3 0.53
0 3 1 0.13 3 0 0 0.23
0 3 2 0.16 3 0 1 0.39
0 3 3 0.19 3 0 2 0.64
1 0 0 0.036 3 0 3 0.95
1 0 1 0.072 3 1 0 0.43
1 0 2 0.11 3 1 1 0.75
1 0 3 0.15 3 1 2 1.2
1 1 0 0.073 3 1 3 1.6
1 1 1 0.11 3 2 0 0.93
1 1 2 0.15 3 2 1 1.5
1 1 3 0.19 3 2 2 2.1
1 2 0 0.11 3 2 3 2.9
1 2 1 0.15 3 3 0 2.4
1 2 2 0.20 3 3 1 4.6
1 2 3 0.24 3 3 2 11
1 3 0 0.16 3 3 3 ›24
1 3 1 0.20
1 3 2 0.24
1 3 3 0.29