Ex.

20 Testing for Coliforms using MPN

Introduction
In the final coliforms exercise, you will combine techniques of qualitative and
quantitative data collection to obtain a definitive number of colony forming units (CFU) within a
contaminated sample.
In many testing laboratories, coliforms are grown in selective liquid media using dilution
series and replicates. The liquid medium used for this method is Lauryl Tryptose Broth (LTB), and
microbes are incubated in test tubes with a Durham tube inside. The lauryl sulfate present in
the medium inhibits the growth of Gram-positive microbes.
After incubation, a positive (coliform present) is noted when tubes show signs of
turbidity and gas production, a qualitative positive result. This positive result is then made
quantitative by counting combinations of positives and comparing them to a reference
statistical index to then calculate the final MPN/mL. The MPN/mL of a sample is calculated
based on the presence or absence of growth across replicates, the dilution factor, and the
volume inoculated.
Standard Most Probable Number (MPN) procedures require a minimum of 3 dilutions of
a sample, with 3, 5, or 10 replicates per dilution for incubation. In this exercise, 4 dilutions will
be used, with 5 replicates per dilution.

Learning Objectives
- Identify the correct combination of positives by reading LTB tube results
- Interpret growth results correctly using the MPN Index chart
- Calculate MPN/100mL using the provided equation (with units)
- Explain whether MPN provides qualitative or quantitative data, or both, and explain why

Materials and Supplies

– Contaminated water sample
– Four 9 mL dilution test tubes
– 20 tubes of Lauryl Tryptose Broth (LTB), each containing one Durham tube
– Micropipettes, pipette tips, and disposal containers for the tips

Procedure

Serial dilutions
1) Label four dilution tubes: 10-1, 10-2, 10-3, and 10-4
2) Mix the contaminated sample using the vortexer, and then use a P-1000 micropipette to
transfer 1 mL of the sample to the 10-1 dilution tube, creating a 1/10 dilution.
3) Mix the 10-1 dilution by vortexing. Using a fresh, sterile pipette tip, transfer 1 mL of the
10-1 dilution to the 10-2 dilution tube, and mix well.
4) Using a new sterile pipette tip each time, and vortexing each time, repeat the
procedure to create the full dilution series (10-1 to 10-4).
LTB tube inoculations
1) Obtain 20 LTB tubes, and separate them into four groups of five. Each dilution made
made above will be used to inoculate five replicates. Label each LTB tube individually so
that you will be able to pick them up and examine them after incubation and still know
the dilution they contain.
2) Aseptically transfer 0.5 mL (500μL) from the 10-1 dilution tube into five LTB replicate
tubes labeled 10-1.
3) Repeat with the 10-2, 10-3, and 10-4 dilutions, so that all 20 LTB tubes are inoculated (five
per dilution).
4) Incubate until next class.

Lab Period 2 (confirming growth and calculating MPN)

Observations
Growth of lactose fermenters will lead to turbid cultures, along with formation of a gas
bubble in the Durham tube. Both results together give positive evidence of coliforms. Gas
formation can be difficult to see, since the Durham tube inside the test tube is small. It can be
helpful to hold the tube pointed away from you at an angle – if the culture has produced gas,
the Durham tube will float toward you. You can gently shake the tube to encourage any of the
gas stuck at the bottom of the tube to rise to the top, but do not invert or shake the tubes hard
enough to spill the inoculated media out.
Record your observations in Table 20.3. Compare your combination of positives to the
MPN table (Table 20.2).

Calculation of MPN
To calculate the MPN index number, first study the example of Table 20.1, which gives
the results from three different hypothetical experiments (water sources A, B, and C). For each
sample, (A, B, or C) the table gives the number of tubes showing positive results for each
dilution (in the numerator), and the total number of tubes per each dilution (equals five in the
denominator).
Only three dilutions are used to determine the number applied to the MPN index table.
These three dilutions are taken in consecutive order of increasing dilution factor. For this
exercises, you will choose positives from 10-1, 10-2, and 10-3, or 10-2, 10-3, and 10-4. To select the
correct sequence of dilutions you should avoid using repeated or redundant data as much as
possible. The series of three that you choose should best illustrate when the number of
positives change from one dilution to the next.
For example, if the results from sample A in Table 20.1 are:
5/5 positives for 10-1 dilution
 5/5 for 10-2 dilution
2/5 for 10-3 dilution
0/5 for 10-4 dilution
Then, the three values selected are 5/5, 2/5 and 0/5 (5-2-0 for the index), representing
the dilutions 10-2, 10-3 and 10-4. The data showing 5/5 positives from both the 10-1 and 10-2
dilutions are redundant, and thus this dilution step is not informative.
See the examples provided in Table 20.1 as well as the math required for calculating the
final MPN/mL. Your own experiment may vary in its combination of positives, which reflects the
fact that real world samples often give unusual results.
Keep in mind that you will need to know how to do these calculations for quizzes and
the practical.

Table 20.1 Example of different LTB results.

Dilutions 10-1 10-2 10-3 10-4 MPN Index

Sample A 5/5 5/5 2/5 0/5 5-2-0

Sample B 5/5 4/5 2/5 0/5 5-4-2

Sample C 0/5 1/5 0/5 0/5 0-1-0

Use the results from Table 20.1 (the “Combination of positives”) and the conversion from Table
20.2 to determine the MPN Index for the hypothetical water samples A, B, and C using Eq. 1:

MPN/100mL = (MPN Index * 10) / [ (Most concentrated Dilution factor considered) *

( Inoculated volume) ]

1) Example for calculating MPN in sample A:

A 5-2-0 ‘Combination of positives’ in Table 20.1 corresponds with MPN Index = 50
in Table 20.2.
MPN/100 mL = (50*10) / (0.1*10-2) = 5 • 104 MPN/100 mL

2) Example for calculating MPN in sample B:

A 5-4-2 ‘Combination of positives’ in Table 20.1 corresponds with MPN Index =
220 in Table 20.2.
MPN/100 mL = (220*10) / (0.1*10-1) = 2.2 • 104 MPN/100 mL

3) Example for calculating MPN in sample C:

A 0-1-0 ‘Combination of positives’ in Table 20.1 corresponds with MPN Index = 2
in Table 20.2.
MPN/100 mL = (2*10) / (0.1*10-1) = 2 • 102 MPN/100 mL
Table 20.3 Results and combination of positives from LTB

Dilutions Combination of MPN Index

Positives Chosen

10-1 10-2 10-3 10-4

Water
Sample