MPN Dilution Guidelines

MPN Dilution Guidelines DOC316.53.01231

Non-potable water samples must be diluted so that a MPN test will have three consecutive
dilutions that contain both positive and negative tubes.
If all of the tubes in a MPN test are positive, the sample must be diluted several more times and
the test must be repeated. If all of the tubes in a MPN test are negative, the sample was diluted too
many times. Repeat the test with less serial dilutions.

Sterile buffered dilution water

Use dilution water that is buffered to a neutral pH and sterilized for microbiological testing. Hach
dilution water is recommended for dilution of most non-potable and wastewater samples. This
solution is packaged in 99 mL bottles. Each bottle contains 99 mL of sterile buffered dilution water.
When 11 mL of sample is added to a 99-mL bottle of dilution water, the sample is diluted by a
factor of 10 (10-fold or 10x dilution). Be sure to mix the bottles thoroughly before and after the
sample is added. The dilution factor of an undiluted sample is 1.

Serial sample dilutions

Complete the following procedure to make serial dilutions of the sample. Refer to Serial dilutions
for non-potable water, MPN test for an illustrated dilution procedure.
Procedure

1. Wash hands.

2. Open a bottle of sterile Buffered Dilution Water.

3. Invert the sample container vigorously for 30 seconds, approximately 25 times, using a waist-
to-ear range of motion.

4. Use a sterile pipet to add 11 mL of sample into the dilution water bottle.

5. Put the cap on the dilution water bottle and invert (for 30 seconds) 25 times. This is a 10-fold
or 10x dilution (sample is diluted by a factor of 10).

6. Add 11 mL of the 10x dilution to another dilution bottle and mix well (100x dilution).

7. Add 11 mL of the 100x dilution to a third bottle and mix well (1000x dilution).

8. Continue to make dilutions until there are three bottles that contain the dilutions listed under
Serial dilutions for non-potable water, MPN test.
Note: Shaking the sample too vigorously will injure or stress the organisms.

MPN Dilution Guidelines

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MPN Dilution Guidelines

Serial dilutions for non-potable water, MPN test

Swimming pool water, chlorinated—Lowest dilution factor = 1
A B C
Undiluted Dilution of 10x Dilution of 100x

99 mL 99 mL
Undiluted pipet 11 mL pipet 11 mL
dilution dilution
sample water water

Inoculate 5 tubes Inoculate 5 tubes Inoculate 5 tubes

Bathing beach water; lake water, unpolluted river water—Lowest dilution factor = 10
B C D
Dilution of 10x Dilution of 100x Dilution of 1000x

Prepared 99 mL 99 mL
pipet 11 mL pipet 11 mL
as B dilution dilution
above water water

Inoculate 5 tubes Inoculate 5 tubes Inoculate 5 tubes

Final effluent, chlorinated—Lowest dilution factor = 100

C D E
Dilution of 100x Dilution of 1000x Dilution of 10,000x

Prepared 99 mL 99 mL
pipet 11 mL pipet 11 mL
as C dilution dilution
above water water

Inoculate 5 tubes Inoculate 5 tubes Inoculate 5 tubes

MPN Dilution Guidelines

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 MPN Dilution Guidelines

Serial dilutions for non-potable water, MPN test (continued)

River water, polluted—Lowest dilution factor = 1,000
D E F
Dilution of 1000x Dilution of 10,000x Dilution of 100,000x

Prepared 99 mL 99 mL
pipet 11 mL pipet 11 mL
as D dilution dilution
above water water

Inoculate 5 tubes Inoculate 5 tubes Inoculate 5 tubes

Storm water, unchlorinated final effluent—Lowest dilution factor = 10,000

E F G
Dilution of 10,000x Dilution of 100,000x Dilution of 1,000,000x

Prepared 99 mL 99 mL
pipet 11 mL pipet 11 mL
as E dilution dilution
above water water

Inoculate 5 tubes Inoculate 5 tubes Inoculate 5 tubes

Raw sewage—Lowest dilution factor = 100,000

F G H
Dilution of 100,000x Dilution of 1,000,000x Dilution of 10,000,000x

Prepared 99 mL 99 mL
pipet 11 mL pipet 11 mL
as F dilution dilution
above water water

Inoculate 5 tubes Inoculate 5 tubes Inoculate 5 tubes

MPN Dilution Guidelines

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© Hach Company, 2007. All rights reserved. Printed in the U.S.A. Updated March 2011, Edition 5