Standard Coliform Most Probable Number (MPN) Test and Presence-Absence Coliform Test

Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



VIII. Environmental and

Food Microbiology



© The McGraw−Hill

Companies, 2002



46. Stand. Coliform Most

Prob. Num. (MPN) Test &

Presence Absence

Coliform Test



Figure 46.1 The Most Probable Number (MPN) Procedure for Water Examination for the Presence of Coliforms by the

Presumptive, Confirmed, and Completed Tests.



Water

sample



Inoculate 15 tubes: 5 with 10 ml of sample, 5 with 1.0 ml of sample, and 5 with 0.1 ml of sample.



Single-strength broth



Presumptive



Double-strength broth



10



10



10



10



10



1.0



1.0



(ml)



1.0



1.0



1.0



0.1



(ml)



0.1



0.1



0.1



(ml)



Lactose or lauryl tryptose broth



Negative presumptive.

The absence of gas in

broth tubes indicates

coliforms are absent.

Incubate an additional

24 hours to be sure.



24 + 2 hours

–

35°C



Confirmed



Negative



After 24 hours of

incubation, the tubes of

lactose broth are examined

for gas production.



Positive



No gas produced.

Negative test.

Coliform group absent.

All positive presumptive

cultures used to inoculate

tubes of brilliant green lactose

bile broth. Incubation for 48 + 3

–

hours at 35°C.



Positive test: gas production —

use positive confirmed

tubes to determine MPN.



Negative



Completed



Positive



Plates of Levine's EMB or LES Endo

agar are streaked from positive

tubes and incubated at 35°C

for 18–24 hours.

Brilliant

green

lactose

bile broth

or lauryl

tryptose

broth



Nutrient

agar slant

Use coliform colonies

to inoculate nutrient

agar slant and a

broth tube.



286



Environmental and Food Microbiology



After 24 hours of incubation make a

Gram-stained slide from the slant.

If the bacteria are gram-negative,

nonsporing rods and produce gas from

lactose, the completed test is positive.



0.1



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



VIII. Environmental and

Food Microbiology



© The McGraw−Hill

Companies, 2002



46. Stand. Coliform Most

Prob. Num. (MPN) Test &

Presence Absence

Coliform Test



46.1



Table

Most Probable Number (MPN) Index for Various Combinations of Positive and Negative Results When

Five 10-ml Portions, Five 1-ml Portions, and Five 0.1-ml Portions Are Used

No. of Tubes Giving Positive Reactions Out of

5 of 10 ml

Each



5 of 1 ml

Each



5 of 0.1 ml

Each



0

0

0

0

1

1

1

1

1

2

2

2

2

2

2

3

3

3

3

3

3

3

4

4

4

4

4

4



0

0

1

2

0

0

1

1

2

0

0

1

1

2

3

0

0

1

1

2

2

3

0

0

1

1

1

2



0

1

0

0

0

1

0

1

0

0

1

0

1

0

0

0

1

0

1

0

1

0

0

1

0

1

2

0



No. of Tubes Giving Positive Reaction Out of

MPN Index per

100 ml

<2

2

2

4

2

4

4

6

6

5

7

7

9

9

12

8

11

11

14

14

17

17

13

17

17

21

26

22



showed growth and gas production into brilliant green

lactose bile broth, which is selective and differential

for coliforms. The tube is incubated for 48 ± 3 hours

at 35°C. Gas formation in the Durham tube is a confirmed test for total coliforms.

In the completed test, a sample from the positive

green lactose bile broth is streaked onto Levine’s

EMB or LES Endo agar and incubated for 18 to 24

hours at 35°C. On EMB agar, coliforms produce small

colonies with dark centers. On LES Endo agar, coliforms produce reddish colonies. Samples are then

inoculated into brilliant green lactose bile broth and

onto a nutrient agar slant. These tubes are incubated

for 24 hours at 35°C. If gas is produced in the lactose

broth (see figure 20.2), and the isolated bacterium is a

gram-negative (based on a Gram stain) nonsporing

rod, the completed test is positive.

An estimate of the number of coliforms (most

probable number) can also be done in the presumptive

test. In this procedure, 15 lactose broth tubes are inoculated with the water sample. Five tubes receive 10 ml

of water, 5 tubes receive 1 ml of water, and 5 tubes receive 0.1 ml of water. A count of the number of tubes

showing gas production is then made, and the figure is



5 of 10 ml

Each



5 of 1 ml

Each



5 of 0.1 ml

Each



MPN Index

per 100 ml



4

4

4

4

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5



2

3

3

4

0

0

0

1

1

1

2

2

2

3

3

3

3

4

4

4

4

4

5

5

5

5

5

5



1

0

1

0

0

1

2

0

1

2

0

1

2

0

1

2

3

0

1

2

3

4

0

1

2

3

4

5



26

27

33

34

23

31

43

33

46

63

49

70

94

79

110

140

180

130

170

220

280

350

240

350

540

920

1,600

≥2,400



compared to a table (table 46.1) developed by the

American Public Health Association. The number is

the most probable number (MPN) of coliforms per 100

ml of the water sample. (It should be noted that the

MPN index usually comes from the presumptive test if

raw sewage is being tested and comes from confirmed

or completed tests for other types of samples.)

More recently, a simple and very sensitive alternative to the classical MPN procedure has been developed: the presence-absence (P-A) coliform test. The

P-A test is a modification of the MPN procedure in

which a large water sample (100 ml) is incubated in a

single culture bottle with triple-strength broth containing lactose, sodium lauryl sulfate, and bromcresol

purple indicator. The P-A test is based on the assumption that no coliforms should be present in 100 ml of

drinking water. Sodium lauryl sulfate inhibits many

bacteria, but not coliforms. A positive test results in

the production of acid from lactose fermentation

(bromcresol purple changes from purple to yellow)

and constitutes a positive presumptive test. As with

the MPN test, it requires confirmation. If there is no

color change, the results are negative for coliforms in

the 100-ml water sample.



Standard Coliform Most Probable Number (MPN) Test and Presence-Absence Coliform Test



287



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



VIII. Environmental and

Food Microbiology



46. Stand. Coliform Most

Prob. Num. (MPN) Test &

Presence Absence

Coliform Test



Procedure for the MPN Test

First Period

Presumptive Test



1. Mix the bottle of water to be tested 25 times.

Inoculate five of the double-strength lactose (or

lauryl tryptose) broth tubes with 10 ml of the

water sample; five single-strength tubes with 1 ml

of the water sample; and five single-strength

tubes with 0.1 ml of the water sample. Carefully

mix the contents of each tube without spilling any

of the broth by rolling the tubes between the

palms of your hands. Using the wax pencil, label

all tubes with your name, date, and the amount of

water added.

2. Incubate the three sets of tubes for 24 to 48 hours

at 35°C.

3. Observe after 24 ±2 and 48 ±3 hours. The

presence of gas in any tube after 24 hours is a

positive presumptive test. The formation of gas

during the next 24 hours is a doubtful test. The

absence of gas after 48 hours is a negative test.

4. Determine the number of coliforms per 100 ml of

water sample (see Principles section and use table

46.1). For example, if gas was present in all five

of the 10-ml tubes, only in one of the 1-ml series,

and none in the 0.1-ml series, your test results

would read 5–1–0. Table 46.1 indicates that the

MPN for this reading would be 33 coliforms per

100 ml of water sample.



Second Period

Confirmed Test



1. Record your results of the presumptive test in the

report for exercise 46.

2. Using an inoculating loop, from the tube that has

the highest dilution of water sample and shows

gas production transfer one loopful of culture to

the brilliant-green lactose bile broth tube.

Incubate for 48 ± 3 hours at 35°C. The formation

of gas at any time within 48 hours constitutes a

positive confirmed test.



Third Period

Completed Test



1. Record your results of the confirmed test in the

report for exercise 46.

2. From the positive brilliant green lactose bile broth

tube, streak a LES Endo or Levine’s EMB plate.

3. Incubate the plate inverted for 24 hours at 35°C.

4. If coliforms are present, select a well-isolated

colony and inoculate a single-strength, brilliant



288



Environmental and Food Microbiology



© The McGraw−Hill

Companies, 2002



green lactose bile broth tube and streak a nutrient

agar slant.

5. Gram stain any bacteria found on the slant.

6. The formation of gas in the lactose broth and the

demonstration of gram-negative, nonsporing rods

in the agar culture is a satisfactorily completed

test revealing the presence of coliforms and

indicating that the water sample was polluted.

This is a positive completed test.



Procedure for the P-A Coliform Test

First Period

1. Inoculate 100 ml of the water sample into a 250ml P-A culture bottle containing 50 ml of triplestrength P-A broth. Mix thoroughly by inverting

the bottle five times to achieve even distribution of

the triple-strength medium throughout the sample.

2. Incubate at 35°C.



Second Period

1. Inspect the P-A culture bottle after 24 and 48

hours for acid production. A distinct yellow color

forms in the medium when acid conditions exist

following lactose fermentation. If gas also is

being produced, gently shaking the bottle will

result in a foaming reaction.

2. With an inoculating loop, transfer any culture that

shows acid production or acid and gas (a positive

presumptive test) to a tube of brilliant green

lactose bile (BGLB) broth containing a Durham

tube for incubation at 35°C.



Third Period

1. Turbidity in the BGLB broth and gas in the Durham

tube within 48 hours confirm the presence of

coliform bacteria (e.g., Escherichia coli).

2. Record results as presence-absence test positive

or negative for coliforms in 100 ml of water

sample in the report for exercise 46.

HINTS AND PRECAUTIONS

(1) Do not confuse the appearance of an air bubble in a

clear Durham tube with actual gas production. If gas is

formed as a result of fermentation, the broth medium

will become cloudy. Active fermentation may be shown

by the continued appearance of small bubbles of gas

throughout the medium outside the inner vial when the

fermentation tube is gently shaken. (2) When taking

your water sample, the upper 38 cm of most waters usually contains the greatest numbers of live bacteria.

(3) Use sterile containers to collect water samples.



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



VIII. Environmental and

Food Microbiology



Laboratory Report



© The McGraw−Hill

Companies, 2002



46. Stand. Coliform Most

Prob. Num. (MPN) Test &

Presence Absence

Coliform Test



46



Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————



Standard Coliform Most Probable Number

(MPN) Test and Presence-Absence Coliform Test

1. Results of presumptive test.

Number of Positive Tubes

5 tubes DSLB

10 ml H2O added



5 tubes SSLB

1.0 ml H2O added



5 tubes SSLB

0.1 ml H2O added



24 hours



48 hours



24 hours



48 hours



24 hours



48 hours



_______



_______



_______



______



_______



_______



MPN



_____________________________



2. Results of confirmed test.

Positive

24 Hours



48 Hours



_________



Negative

24 Hours



_________



________



48 Hours

_________



EMB or LES Endo Agar Results ___________________________________________________________________________________________



3. Results of completed test.

Lactose Fermentation Results



Morphology



____________________________________



_______________________________________________________________________________



Presence-Absence (P-A) Coliform Test

1. Results in P-A culture bottle. ________________________________________________________________

2. Results from brilliant green lactose bile (BGLB) broth.

Acid: ___________________________________



Gas: _______________________________________



289



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



VIII. Environmental and

Food Microbiology



46. Stand. Coliform Most

Prob. Num. (MPN) Test &

Presence Absence

Coliform Test



© The McGraw−Hill

Companies, 2002



Review Questions

1. Why are coliforms selected as the indicator of water potability?



2. Does a positive presumptive test indicate that water is potable?



3. Why is the MPN test qualitative rather than quantitative?



4. What is the function of the following in the MPN test?

a. lactose broth

b. Levine’s EMB or LES Endo agar

c. nutrient agar slant

d. Gram stain

5. What does a metallic green sheen indicate on an EMB plate? Pink to dark red colonies with a metallic surface

sheen on LES Endo agar?



6. What bacterial diseases can be transmitted by polluted water?



7. What does a positive presence-absence test indicate? A negative presence-absence test?



290



Environmental and Food Microbiology



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



VIII. Environmental and

Food Microbiology



© The McGraw−Hill

Companies, 2002



47. Memb. Filter Tech. for

Coliforms & Fecal Strept.;

Test for Fecal Coliforms



E X E RC I S E



47



Membrane Filter Technique for Coliforms and Fecal

Streptococci; KONFIRM Test for Fecal Coliforms

SAFETY CONSIDERATIONS

In this experiment, students will be taking unknown

samples and growing them to large concentrations. Any

of these samples could contain human pathogens; thus,

extreme caution should be taken when working with and

disposing of the final products. Be careful with the Bunsen burner flame. The 95% ethyl alcohol is flammable—

do not use around an open flame. No mouth pipetting.

Dispose of all water samples properly. Do not look directly at the ultraviolet light.



Materials per Group of Students

vacuum pump or water faucet aspirator

sterile membrane filter apparatus

9 sterile plastic 50-mm diameter petri plates

Millipore (No. PD10 047 00 or Gelman plates)

sterile membrane filter disks, Millipore

(No. HAWG 047 AO) or Gelman

sterile absorbent disk pads (packed with filters)

5-ml pipettes with pipettor

M-Endo Broth MF

M-FC broth

KF streptococcus agar

LES Endo agar

HACH m-ColiBlue 24 broth

waterproof tape

Whirlpak bag

44.5 ±2°C water bath

300-ml water sample that each group of students

brings in from different sources

95% ethyl alcohol for sterilizing forceps

sterile forceps

Bunsen burner

sterile distilled water

35°C incubator

wax pencil

KONFIRM test tablets (for 100-ml samples)

1 comparator, 2 mls of developer (KEY Scientific

Products)



1 sterile cotton swab

1 nonfluorescent container (150 ml)

long wavelength ultraviolet lamp (Wood’s lamp),

KS 1699 or equal



Learning Objectives

Each student should be able to

1. Understand the principles of the membrane filter

technique

2. Determine the quality of a water sample using the

membrane filter technique and/or the KONFIRM

test

3. Quantitate the number of coliforms in a water

sample using the membrane filter technique

4. Perform a KONFIRM water test for fecal

coliforms



Suggested Reading in Textbook

1. Microbial growth in natural environments,

section 6.5.

2. Sanitary Analysis of Waters, section 29.5.



Medical Application

Historically, contaminated water has always been a serious

public health concern and remains so today. In the United

States, public drinking water is tested and treated daily to

maintain potability. This assures the safety of the public

water consumers. The membrane filter technique is one test

commonly used in combination with other tests to test for the

presence of fecal coliforms. Fecal contamination of drinking

water can lead to the bacterial transmission of Salmonella

typhi (typhoid fever), S. paratyphi (paratyphoid fever),

Shigella dysenteriae (bacillary dysentery), and Vibrio

cholerae (cholera) as well as viruses (poliomyelitis and hepatitis A) and parasites (Cryptosporidium).



291



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



VIII. Environmental and

Food Microbiology



© The McGraw−Hill

Companies, 2002



47. Memb. Filter Tech. for

Coliforms & Fecal Strept.;

Test for Fecal Coliforms



Figure 47.1 The Membrane Filter Technique for the Direct Recovery of Coliform Bacteria from Water Samples.



Membrane filter

on a filter support



Water sample filtered

through membrane

filter (0.45 µm)



Principles for Membrane Filter Technique

The second standard test for measuring coliform

numbers (quantity) in water is the membrane filter

technique (figure 47.1).a This technique involves filtering a known volume (100 ml for drinking water

samples) of water through a special sterile filter.

These filters are made of nitrocellulose acetate or

polycarbonate, are 150 µm thick, and have 0.45 µm

diameter pores. A grid pattern is typically printed on

these filter disks in order to facilitate colony counting. When the water sample is filtered, bacteria

(larger than 0.45 µm) in the sample are trapped on

the surface of the filter. The filter is then carefully removed, placed in a sterile petri plate on a pad saturated with a liquid or agar-based medium, and incubated for 20 to 22 hours at 35°C. One assumes that

each bacterium trapped on the filter will then grow

into a separate colony. By counting the colonies one

can directly determine the number of bacteria in the

water sample that was filtered.

The broth medium usually employed in detecting

total coliforms is M-Endo broth MF. Other media,

such as M-FC broth and KF streptococcus agar, are

available for the detection of both fecal coliforms and

fecal streptococci, respectively. Fecal streptococci are

the Lancefield Group D streptococci that occur in the

feces of humans and other warm-blooded mammals.

Total coliform colonies will be pink to dark red in



a As applied to the membrane filter technique, the coliform group may

be defined as comprising all facultatively anaerobic, gram-negative,

nonspore-forming, rod-shaped bacteria that produce a red colony with a

metallic sheen within 24 hours at 35°C on an Endo-type medium

containing lactose.



292



Environmental and Food Microbiology



Membrane filter

removed and

placed in plate

containing the

appropriate

medium



Incubation

for 20–22

hours



Typical

coliform

colonies



color and will appear to have a golden green metallic

sheen or luster (figure 47.2a,d). Fecal coliform

colonies will appear blue (figure 47.2b), and fecal

streptococci (figure 47.2c) colonies will appear light

pink and flat, or dark red.

In determining total coliforms, the amount of

water filtered should be enough to result in the

growth of about 20 to 80 colonies and no more than

a total of 200 bacterial colonies of all types. About

50 to 200 ml of unpolluted water is often adequate

for such bacterial counts. Polluted water may contain

so many coliforms that it will be necessary to dilute

1 ml or less of sample with about 50 ml of sterile

water. This is done in order to provide enough volume for uniform bacterial dispersion across the filter

surface, in addition to providing an appropriately

low coliform count.

Coliform density is expressed in terms of the

number of coliforms per 100 ml of water and is calculated according to the following formula:

Coliform colonies/100 ml =

coliform colonies counted × 100

ml of water sample filtered

The number of coliforms should be given to two significant figures per 100 ml.

The standard set for potable (drinking) water is a

limit of 1 coliform per 100 ml and an action limit of 4

coliforms per 100 ml. An action limit means that the

water company or other provider must take immediate

action to remedy the problem(s) that is/are responsible

for the presence of coliforms.

From positive fecal coliform and fecal streptococci test results, one can be fairly certain that the

water pollution is from a fecal source. However, in