DEPARTMENT OF NUTRITION

FACULTY OF MEDICINE AND HEALTH SCIENCES

UNIVERSITI PUTRA MALAYSIA

PKK 3107

FOOD SAFETY FOR CONSUMER HEALTH

TOPIC: Standard qualitative analysis of water

OBJECTIVES:
1) To determine the presence of coliform bacteria in a water sample.
2) To obtain an index indicating the possible number of organism present in the sample
under analysis
3) To confirm the presence of coliform bacteria in a water sample for which the presumptive
test was positive

INTRODUCTION:

The standard analysis of water for the presence of coliform bacteria consist of three parts: the
presumptive test, the confirmed test and the completed test. In the presumptive test, microorganisms
that are capable to ferment lactose will produce gas. This observation is used for the analysis.
Besides, the presumptive test can be used to obtain some ideas of the number of coliform organism
present by means of the most probable number (MPN). On the other hand, the confirmed test
requires that selective and differential media such as eosin-methylene-blue (EMB) be streaked from a
positive lactose broth tube obtained from the presumptive test. Later, the completed test is conducted
to examine the coliform colonies that appeared on the EMB agar plate used on the confirmed test. An
isolated colony is picked and inoculated into a tube of lactose broth and streaked on a nutrient
agar to perform a Gram stain. In summary, these analyses detect the presence of coliform bacteria,
the gram-negative, nonspore-forming bacilli that ferment lactose with the production of acid and gas
that is detectable following a 24-hr incubation period at 37 ºC.

PROCEDURES (Appendix 1):

Presumptive test

1. For each water sample, collect 3 double-strength lactose fermentation broths (Label 10 mL)
and 6 single-strength lactose fermentation broths (Label 1 mL and 0.1 mL). Hence, for one
water sample, you will have 9 lactose fermentation broths.
2. In the double-strength lactose fermentation broths, 10 mL aliquots of water sample is
transferred using a sterile pipette.
3. In the single-strength lactose fermentation broths labelled with “1 mL”, 1 mL aliquots of
water sample is transferred using a sterile pipette.
4. In the single-strength lactose fermentation broths labelled with “0.1 mL”, 0.1 mL aliquots
of water sample is transferred using a sterile pipette.
5. Repeat steps 1 through 4 for other water samples.
6. Incubate all tubes for 48 hours at 37 °C.
7. Following 24 hours of incubation, the production of gas in any tube is examined. The
presence of gas in any tube within 24 hours of incubation period is a positive presumptive
test. In addition, the formation of gas for the 2nd 24 hours incubation period (48 hours of
incubation) is a doubtful test. The absence of gas formation after 48 hours of incubation
indicates a negative test, showing that the water sample is not contaminated.
8. Following 48 hours of incubation, determine the MPN by referring to Appendix 2.

Confirmed Test

1. Using a positive 24-hour lactose broth culture of your water sample from the presumptive test,
streak the surface of EMB agar plate as shown in Appendix 1 to obtain discrete colonies.
2. Repeat Step 1 using the positive lactose broth cultures from other water samples.
3. Incubate all plate cultures in an inverted position for 24 hours at 37 ºC.
4. Examine all the plates for the presence of or absence of coliform colonies. Coliform
organisms on EMB agar plate will form darkish colonies that often, but not always exhibit a
greenish metallic sheen.
5. Determine whether each of water samples is potable (absence of coliform colonies) or non-
potable (presence of coliform colonies).

Completed Test
1. Inoculate one lactose broth and one nutrient agar slant/plate from the same isolated
coliform colony obtained from an EMB plate. [Pick two colonies that you consider most
likely to be organism of the coliform group].
2. Incubate 24 hours at 37 °C.
3. From the agar slant/plate, perform a gram stain. Draw and describe your observation.

The formation of gas in the lactose broth and the demonstration of gram negative, nonspore-
forming bacilli in the agar culture constitute a satisfactory completed test, revealing the presence
of members of the coliform group and thus indicating that the water sample was polluted.

REFERENCES

1. Johnson, T.R. & Case, C.L. (2010). Laboratory experiments in microbiology 9th Edition.
Pearson Education, Inc., San Francisco, CA.
2. Cappucino, J. G. & Sherman, N. (2010). Microbiology: A laboratory manual 7 th Edition.
Pearson Education, Inc., San Francisco, CA.
RESULTS
Preliminary water sample analysis
Water sample Color Smell Clarity

Presumptive test
24 hours of incubation
Presence of Gas
Water sample Tube Labelled Tube Labelled Tube Labelled
“10 mL” “1 mL” “0.1 mL”
1 2 3 1 2 3 1 2 3

48 hours of incubation
Presence of Gas
Water sample Tube Labelled Tube Labelled Tube Labelled
“10 mL” “1 mL” “0.1 mL”
1 2 3 1 2 3 1 2 3

Most Probable Number (MPN) reading

Following 48 hours of incubation, if gas appeared in all 3 tubes labelled “10 mL”, in two of the
tubes labelled “1 mL” and in one labelled as “0.1 mL”, the series would be read as 3-2-1. From
the MPN table, such reading would indicate that there would be approximately 150
microorganisms per 100 mL of water.

Presence of Gas
Water sample Tube Labelled Tube Labelled Tube Labelled Reading MPN
“10 mL” “1 mL” “0.1 mL”
1 2 3 1 2 3 1 2 3
Confirmed Test
Observation of Colonies
Water sample on EMB Plate Potable Non-potable

Completed Test

Water sample Presence of gas in Lactose Gram-stain

Broth (Morphology)
 Appendix 1 – Water analysis

Adapted from: Cappucino, J. G. & Sherman, N. (2010). Microbiology: A laboratory manual 7th
Edition. Pearson Education, Inc., San Francisco, CA
 Appendix 2 – Determination of MPN

No. of Tubes Giving Positive No. of Tubes Giving Positive

Reaction Out of MPN Reaction Out of MPN
3 of 3 of 3 of Index per 3 of 3 of 3 of Index per
10 ml 1 ml 0.1 ml 100 ml 10 ml 1 ml 0.1 ml 100 ml
each each each each each each

0 0 0 <3 3 0 0 23
0 0 1 3 3 0 1 39
0 1 0 3 3 0 2 64
1 2 0 4 3 1 0 43
3 1 1 75
1 0 1 7 3 1 2 120
1 1 0 7 3 2 0 93
1 1 1 11 3 2 1 150
1 2 0 11 3 2 2 210
3 3 0 240
2 0 0 9 3 3 1 460
2 0 1 14 3 3 2 1100
2 1 0 15 3 3 3 >2400
2 1 1 20
2 2 0 21
2 2 1 28

Sources: Standard Methods for the Examination of Water and Wastewaster. 13th ed New York:
American Public Health Association, 1971.