CHAPTER 7

THERMOTOLERANT COLIFORMS (Formerly FECAL COLIFORMS) TEST

For Drinking Water and Wastewater
[Ref: Standard Methods (22nd Ed, 2012), 9221E; p. 9-74]
Learning Objectives
What we will learn in Chapter 7:
7.1 What is Thermotolerant Coliform (Fecal Coliform)?
7.2 Requirements for Thermotolerant Coliform Test in the Laboratory?
7.3 Sample collection &preservation
7.3.1 For Drinking Water
7.3.2 For Wastewater
7.4 Thermotolerant Coliform Test
7.4.1 Preparation of EC medium
7.4.2 Procedure
7.4.3 Interpretation
7.4. 4 Results
Two Figures (7.2 & 7.3), schematically describe -
• Figure 7.2: Simultaneous determination of Total Coliform and Fecal Coliform in
Drinking Water sample
• Figure 7.3: Simultaneous determination of Total Coliform and Fecal Coliform in
Wastewater (or Sewage) sample
7.5 Thermotolerant Coliform Direct Test (A-1 Medium)

Expected Learning Outcomes:

1. What is Fecal Coliform (Thermotolerant Coliform)?
2. Requirements for Fecal Coliform (FC) test – for water & Wastewater
3. How to Pre-pare EC medium?
4. How to conduct simultaneously Total Colifrom (TC) and Fecal Coliform (FC) test in the
laboratory for Drinking Water sample?
5. How to conduct simultaneously Total Colifrom (TC) and Fecal Coliform (FC) test in the
laboratory for Waste Water sample?

70 Dr Subodh Kumar Maiti, Professor, Dept of Env Sc & Engg, ISM Dhanbad 826 004
7.1 What are Thermotolerent Coliform (Fecal coliform)

This test is use to distinguish between Total Coliform(TC) and Fecal Coliform (FC)

Application: Fecal coliform test (using EC medium) is applicable to investigation of drinking

water, stream pollution, raw water source, wastewater treatment system, bathing water and general
water quality monitoring.

The term “Fecal Coliforms (now replaced with Thermotolerent coliform)” has been used in
water microbiology to denote coliform organisms that grow at 44.5oC and ferment lactose to
produce acid and gas. So, thermotolerant coliform is more correct term and is becoming more
commonly used. These thermotolerant coliforms also have been documented in organically rich
waters or tropical climates in the absence of recent fecal contamination.

So, testing for Escherichia coli – a

specific indicator of fecal contamination
is recommended (Standard Methods,
2012). In the multiple- tube fermentation
technique, this group of oraganisms is
identified by their ability to ferment
lactose to produce gas at 44.5oC.

Figure 7.1 explain the density of Total

Coliform, fecal Coliform and E. coli in
water sample.

Figure 7.1: Representative portion of Fecal Coliform

within Total Coliform

71 Dr Subodh Kumar Maiti, Professor, Dept of Env Sc & Engg, ISM Dhanbad 826 004
7.2 Requirements for Thermotolerant Coliform (Fecal Coliform) Test
1. Pre-sterilized Sample bottle (capacity 250 mL): As per nos water samples to be collected
2. Sample transport Box
3. Refrigerator/Freezer
4. Autoclave
5. Laminar flow chamber
6. Water bath or Bacteriological incubator
7. Sterile metal loops (3 to 3.5 mm diameter)
8. Fermentation tube – Capped: 10 nos/ drinking water sample; 15 nos/ wastewater sample
9. Durham Vials
10. Sprit lamp
11. Test-tube racks
12. Sterilized pipettes (10 mL, 1 mL Capacity); Pipettes containers
13. Wash bottle
14. pH meter
15. Dilution bottle [For Wastewater test]
16. Dilution water [For Wastewater test]
17. Reagent grade water
18. Sodium Thiosulfate solution (10%) – for dechlorination purposes
19. EC (E. coli)- medium - use dehydrated, commercially available medium or prepare
20. Lauryl Tryptose Broth (LTB) - use dehydrated, commercially available medium or prepare
21. Brilliant Green Lactose Bile Broth (BGLB) - use dehydrated, commercially available medium or
prepare
22. Cotton, analytical balance, Beaker etc
23. Chemicals
Sl No Name of Ingredient
1. Trpytose
2. Peptone
3. Lactose
4. Oxgall
5. Brilliant Green
6. Bile salts mixture or bile salt No.3
7. Dipotassium Hydrogen Phosphate, K2HPO4
8. Potassium dihydrogen Phosphate, KH2PO4
9. Sodium Chloride, NaCl
10. Sodium Lauryl Sulfate
11. Reagent grade water
Bile salts inhibit gram-positive bacteria especially bacilli and faecal Streptococci.

72 Dr Subodh Kumar Maiti, Professor, Dept of Env Sc & Engg, ISM Dhanbad 826 004
7.3 SAMPLE SIZE

Collect a sufficient volume of water for analysis, at least 100 mL of sample. World Health
Organization guidelines suggest 200 mL per sample, while Standard Methods (2012)
guidelines suggest 100 mL per sample.

• Therefore, collect at least 100 mL of sample in pre-sterilized bottles (in sterile glass
or plastic sample bottles).
• Sample containers should not be filled completely.
• Maintain at least 2.5 cm (approximately 1") of air space to allow adequate space for
mixing the sample prior to analysis.

7.3.1 Drinking Water

Potable water should contain no coliforms per 100 mL, so testing should be done on
undiluted samples. Use 5-tube or 10-Tube Most Probable Number test or
Presence/Absence test for potable water.

7.3.2 Waste Water

Non-potable water testing generally requires dilution of the original sample, based on
probable coliform concentration. For MPN testing, three different dilutions should be
tested, using 5 tubes for each dilution, for a total of 15 tubes. Sample dilutions procedure
and preparation of dilution water were discussed in Chapter 6 section 6.6 in Figure 6.2 &
6.3.

7.4 Thermotolerant Coliform Test

Do not use EC medium for the direct isolation of Thermotolerant Coliform from water.
Prior enrichment in a presumptive medium is required for optimum recovery of
Thermotolerant Coliform.

73 Dr Subodh Kumar Maiti, Professor, Dept of Env Sc & Engg, ISM Dhanbad 826 004
7.4.1 Preparation of EC medium
Preferably use a dehydrated, commercially available medium.
The EC- medium can also be prepared in the laboratory (Table 7.1).
Table –7.1: Composition of EC medium
Sl No Ingredients Quantity / L
1 Trpytose 20 g
2 Lactose 5g
3 Bile salts mixture or bile salt No.3 1.5 g
4 Dipotassium Hydrogen Phosphate, K2HPO4 4 g
5 Potassium dihydrogen Phosphate, KH2PO4 1.5 g
6 Sodium Chloride, NaCl 5g
7 Reagent grade water 1000 mL
1. Add dehydrated ingredients to water, mix thoroughly, and heat to dissolve.
2. Before sterilization, dispense sufficient medium in fermentation tubes with an
inverted vial, to cover the inverted vial at least one-half to two-third after
sterilization.
3. Close tubes with metal or heat-resistant plastic caps.
4. Autoclave medium at 121 oC for 12 to 15 minutes.
5. Ensure that inverted vials are free of air bubbles.
6. Medium pH should be 6.9 ± 0.2 after sterilisation.
7.4.2 Procedure
1. Gently shake or rotate the positive fermentation tubes of total coliform MPN test (i.e.
presumptive test) to EC medium. This examination may be performed simultaneously
with the confirmatory procedure using brilliant green lactose broth.
2. Use a sterile metal loop with a minimum 3 to 3.5-mm diameter to transfer growth from
each positive presumptive or confirmed fermentation tube to EC broth.
3. Place all EC tubes in a water bath within 30 minutes after inoculation. Incubate
inoculated EC broth tubes in a water bath at 44.5 ± 0.2 oC for 24 ± 2 h. Maintain a
sufficient water depth in the water bath incubator to immerse tubes to the upper level of
the medium.

74 Dr Subodh Kumar Maiti, Professor, Dept of Env Sc & Engg, ISM Dhanbad 826 004
7.4.3 Interpretation

1. Gas production and growth in an EC broth culture within 24 ± 2h or less is

considered a positive reaction indicating fecal origin.
2. Failure to produce gas (with little or no growth) constitutes a negative reaction
indicating a source other than the intestinal tract of warm-blooded animals.

3. If multiple tubes are used, calculate MPN of Thermotolerant Coliform from the
number of positive EC broth tubes.

7.4. 4 Results
Record the number of positive tubes and refer Table-6.2 for drinking water

Table – 6.2: MPN index and 95% confidence limits for various combinations of positive
and negative results when ten 10-mL portions are used

No. of tubes MPN 95% confidence limits

giving positive index/ (Approximate)
reaction out of 10 100 mL Lower Upper
(10-mL each)
0 <1.1 - 3.4
1 1.1 0.051 5.9
2 2.2 0.37 8.2
3 3.6 0.91 9.7
4 5.1 1.6 13
5 6.9 2.5 15
6 9.2 3.3 19
7 12 4.8 24
8 16 5.8 34
9 23 8.1 53
10 >23 13 --
Source: Standard Methods (2012)

75 Dr Subodh Kumar Maiti, Professor, Dept of Env Sc & Engg, ISM Dhanbad 826 004
 Collect Drinking water sample in
a Pre-sterilized sampling Bottle

Inoculate 10 mL of Drinking
water sample in each Tube

Sterilized fermentation
tube filled with Lauryl
Tryptose Broth (10 mL)
with inverted Durham Vial
Presumptive test
Incubate at 35 ± 0.5 oC for 24 ± 2 h

Select all (+) tubes (gas produced)

Transfer inocula with a sterile metal loop

simultaneously to BGLB and EC media
Sterilized Sterilized
fermentation tube fermentation tube
filled with BGLB filled with EC
Medium (10 mL)
Broth (10 mL) with with inverted
inverted Durham Vial Durham Vial

Incubate in Water bath at

Incubate at 35-37 oC for 48± 3h
44.5 oC ± 0.2 oC for 24 ± 2h

Gas production anytime within 48± 3h

Gas production anytime within 24± 3h
Positive Reaction
or less [Positive Reaction]
Calculate MPN Index from Table Calculate MPN Index from Table

Results of Total Coliform Results of Fecal Coliform

Figure 7.2: Diagram showing simultaneous determination of Total

Coliform and Fecal Coliform in Drinking Water sample

76 Dr Subodh Kumar Maiti, Professor, Dept of Env Sc & Engg, ISM Dhanbad 826 004
 Collect Wastewater sample in a
Pre-sterilized sampling Bottle

Follow Decimal dilution

Presumptive test
1 mL 0.1 mL 0.01 mL Sterilized tubes
filled with Lauryl
Tryptose Broth
(10 mL)& inverted
Durham Vial

Incubate at 35 ± 0.5 oC for 24 ± 2 h & Select all (+) tubes (gas produced) Total tubes = Nos
of (+) tubes in
Presumptive test
Transfer inocula with a sterile metal loop
simultaneously to BGLB and EC media
Sterilized tube
filled with EC
Sterilized tube filled with Medium (10 mL)
BGLB Broth (10 mL) & & inverted
inverted Durham Vial Durham Vial

Incubate in Water bath at

Incubate at 35-37 oC for 48± 3h
44.5 oC ± 0.2 oC for 24 ± 2h

Gas production anytime within 48± 3h

Gas production anytime within 24± 3h
Positive Reaction
or less [Positive Reaction]
Calculate MPN Index from Table Calculate MPN Index from Table

Results of Total Coliform Results of Fecal Coliform

Figure 7.3: Flow diagram showing estimation of Total Coliform & Fecal Coliform
in a Wastewater sample

77 Dr Subodh Kumar Maiti, Professor, Dept of Env Sc & Engg, ISM Dhanbad 826 004
7.5 Thermotolerant Coliform Direct Test (A-1 Medium)
The A-1 medium may be used for the isolation of thermotolerant coliforms from source water, treated
wastewater, but not drinking water. Unlike EC medium, A-1 medium does not require prior enrichment in
a presumptive medium for optimum recovery of thermotolerant (fecal) Coliform.
Preferably use a dehydrated, commercially available medium.
Table: Composition of A-1 medium
Sl No Ingredients Quantity/ L
1 Lactose 5g
2 Tryptone 20 g
3 Sodium chloride, NaCl 5g
4 Salicin 0.5 g
5 Polyethylene glycol p-isooctylphenyl ether 1 mL
Total ingredients 30.5 g + 1 mL
6 Reagent-grade water 1000 mL
1. Heat and dissolve solid ingredients, add polyethylene glycol p-isooctylphenyl ether, and adjust to
pH 6.9 ± 0.1. For 10-mL samples, prepare double-strength medium (i.e., dissolve 30.5g +1mL in
500mL of Reagent grade water), so the final concentration of ingredients after sample addition is
correct.
2. Before sterilization dispense, in fermentation tubes with an inverted vial, sufficient medium to
cover the inverted vial at least one-half to two-thirds after sterilization.
3. Close with metal or heat-resistant plastic caps.
4. Sterilize by autoclaving at 121 0C for 10 minutes. Ensure that inverted vials are free of air bubbles.
5. Store in the dark at room temperature for not longer than 7 days.
6. Ignore formation of precipitate during storage.
7.5.1 Procedure
Inoculate tubes of A-1 broth as follows:
For non-potable water/ effluent /sewage/ wastewater sample
1. For wastewater use five replicates tubes having decimal dilution of 1.0, 0.1, 0.01 mL or more.
2. Take 3 nos. of beaker and mark the beaker as 1mL, 0.1 mL and 0.01mL and keep 5 pre-sterilized
fermentation tube filled with A-1 broth and inverted Durham’s tubes.
For source water:
• Five replicate tubes each containing 20mL; or
• Ten replicate tubes each containing 10mL, or
• One single bottle containing 100 mL of water.
Incubate for 3h at 35 ± 0.5oC.
Transfer tubes to 1a water bath at 44.5 ± 0.2oC and incubate for another 21 ± 2h.
7.5.2 Interpretation
Gas production in any A-1 broth culture within 24h or less is a positive reaction indicating the presence of
Thermotolerant coliforms. Calculate the MPN of Thermotolerant coliforms from the number of positive A-
1 broth MPN-index table.

78 Dr Subodh Kumar Maiti, Professor, Dept of Env Sc & Engg, ISM Dhanbad 826 004
 NOTES

79 Dr Subodh Kumar Maiti, Professor, Dept of Env Sc & Engg, ISM Dhanbad 826 004
 NOTES

80 Dr Subodh Kumar Maiti, Professor, Dept of Env Sc & Engg, ISM Dhanbad 826 004