HACH METHOD SPECIFICATION SHEET

For Hach Microbiological Methods

Total Coliforms and E. coli

Membrane Filtration Method Sensitivity: 1 CFU/100mL

m-ColiBlue24® Broth

USEPA Approved for Drinking Water

Method No. 10029 Revision 2, 8/17/99

 

m-ColiBlue24 is a registered trademark of Hach Company

1.0 Scope and Application

1.1 This method determines the presence or

absence of total coliforms and E. coli in surface

and ground source waters and finished potable

water using a selective and differential

membrane filtration (MF) medium,

m-ColiBlue24 Broth.

1.2 This method can detect the presence or

absence of both total coliforms and E. coli

simultaneously within 24 hours and without the

need for a confirmation step.

1.3 The detection limit of the method is one

colony forming unit (CFU) of coliform bacteria

or E. coli per 100-mL of sample. See attachment

1.3.

2.0 Summary of Method

2.1 Coliform bacteria are identified in water

either as total coliforms or E. coli. Total

coliforms can be present in water without E. coli

being present; E. coli cannot be present in water

without total coliforms also being present.

2.2 A 100 mL volume of sample is filtered

through a 47-mm membrane filter using

standard techniques. The filter is then

transferred to a 50-mm petri plate containing an

absorbent pad saturated with m-ColiBlue24

Broth. The filter is then incubated at

35oC ± 0.5 oC for 24 hr. If coliform bacteria

are present in the sample, they will appear as

red colonies and if E. coli is present in the

sample, they will appear as blue colonies.

2.3 m-ColiBlue24 Broth is a nutritive, lactosebased medium, containing inhibitors to

selectively eliminate growth of non-coliforms.

It is analogous to an improved version of

m-Endo. Total coliform colonies growing on

the medium are highlighted by a non-selective

dye, 2,3,5-Triphenoltetrazolium Chloride

(TTC) which produces red colored colonies.

Among the total coliform colonies which grow

up on the medium, any E. coli colonies are

distinguishable by a selective blue color,

resulting from the action of β-glucuronidase

enzyme on 5-Bromo-4-Chloro-3-Indolyl-BetaD-glucuronide (BCIG).

3.0 Definitions

3.1 Material Safety Data Sheet (MSDS) -

Written information provided for each

chemical reagent or standard about a

chemical’s toxicity, health hazards, physical
properties, flammability, and reactivity. It also

includes storage, spill, and handling precautions.

3.2 Total Coliform Bacteria- Bacteria belonging

to the genera Klebsiella sp., Enterobacter sp.,

Citrobacter sp., or Escherichia sp.

3.3 Coliform Positive Colony - A red or blue

colony.

3.4 Coliform Negative Colony - A clear or white

colony.

3.5 Escherichia coli or E. coli Bacteria - A

genus within the total coliform group typified

by possession of the enzyme β-Glucuronidase,

ability to grow at 44.5oC, and form indole from

tryptophan.

3.6 E. coli Positive Colony - A blue colony.

3.7 E. coli Negative Colony - A non-blue

colony.

4.0 Interferences

4.1 No interferences to the colony color

development have been found in source waters

or finished potable water samples. Similarly,

particulates in water samples do not alter the

efficacy of the medium, although excess

particulates may cause colonies to grow together

on crowded filters or slow the sample filtration

process.

5.0 Safety

5.1 Standard safety practices appropriate to

microbiology laboratories should be followed.

Follow the test procedure carefully and observe

all precautionary measures.

5.2 Solid and liquid waste materials containing

or suspected to contain viable bacteria should be

decontaminated using an autoclave or by using

an appropriate disinfectant before discarding.

5.3 Refer to the appropriate Material Safety

Data Sheets supplied for each reagent for

comprehensive safety data essential for proper

use.

6.0 Equipment and Supplies

6.1 Equipment

6.1.1 Air Incubator -

Capable of operating at 35oC ± 0.5 oC 

6.1.2 Vacuum pump

6.1.3 Membrane filtration funnel unit

and flask.

6.1.4 Dissecting microscope, capable of

10-15X magnification. Microscope

should be equipped with a fluorescent

illuminator.

6.2 Supplies/Glassware - Cleanse all glassware

thoroughly with a suitable detergent and hot

water, rinse with hot water to remove traces of

detergent residual, and rinse again with

laboratory-pure water. Sterilize all glassware

by autoclaving 15 min. at 121oC or by heating

in an oven for at least 1 hour at 170 oC.

6.2.1 Presterilized 50-mm MF petri

plates with or without absorbent pads.

6.2.2 0.45 μm presterilized membrane

filters with or without absorbent pads.

6.2.3 Sterile Forceps

6.2.4 Sterile glass or plastic sample

collection containers.

6.2.5 Sterile graduated cylinders.

6.2.6 Sterile pipets.

6.2.7 Sterile MF filtration unit
6.2.8 Side-arm flask

6.2.9 Biohazard bag

6.3.0 150 mL of sterile rinse water

7.0 Reagents and Standards

7.1 Growth Medium

7.1.1 The membrane filtration method

for Total Coliforms and E. coli utilizes

m-ColiBlue24 Broth, catalog no.

26084-20. The broth is provided in

ready to use 2-mL ampules. Under

proper storage conditions (2-8oC) the

broth has a shelf life of one year. The

broth contains a nutritive medium and

colorimetric indicators.

7.2 Dechlorinating Reagent

7.2.1 Decholorinating Reagent Powder

Pillow (14363-69). Contains Sodium

Thiosulfate (Na2S2O3 ) and Sodium

Sulfate (Na2SO4).

7.2.2 To make a 3% Sodium Thiosulfate

(Na2S2O3 ) Solution, add 48.18 g of

Sodium Thiosulfate, Pentahydrate (460-

01) to approximately 500 mL of

deionized water, and dilute to 1 L with

deionized water.

8.0 Sample Collection, Dechlorination,

Preservation, Shipment and Storage.

8.1 Water Sample Collection

8.1.1 Sample Collection Containers -

Samples should be collected in sterile,

clean glass or heat-resistant bottles.

Presterilized Whirl-Pak Bags, catalog

no. 20733-53, may also be used.

8.1.2 Sampling Procedure - Potable

water samples are taken by first

flushing the tap 2-3 minutes to clear

service line. Collect samples using

aseptic techniques to avoid

contamination. For other water

samples, aseptically collect water

representative of the source.

8.3 Dechlorination - Water containing chlorine

or other halogens must be treated with sodium

thiosulfate to allow accurate evaluation of

microbial content. Add one Decholorinating

Reagent Powder Pillow (cat no. 14363-69) by

aseptically cutting off the tip of an alcoholrinsed pillow and pouring the contents into 100

mL of chlorinated water sample. Alternatively,

pipet 0.1 mL of a 3% Sodium Thiosulfate

(Na2S2O3) Solution into 100 mL of a

chlorinated water sample. Presterilized WhirlPak Bags, catalog no. 20733-53, contain

sufficient Sodium Thiosulfate powder to

neutralize a 100 mL chlorinated water sample.

8.4 Preservation, Shipment, Storage - Samples

should be tested as soon as possible. If

analysis cannot be done within 1 hr of

collection, potable water samples should be

held on ice or at 2-8oC in a refrigerator for a

maximum holding time of 30 hours (16.1).

Non-potable samples should be held at <10oC

for a maximum of 8 hours from sampling to

analysis.

9.0 Quality Control

9.1 m-ColiBlue24 Broth undergoes quality

control (QC) testing at the time of manufacture.

A Certificate of Analysis is included with every

box of 20 ampules stating that m-ColiBlue24

Broth, as received by the analyst, is ready for

use in analyzing water samples by the

membrane filtration procedure. Shelf life data

is available on request. Each laboratory should

test quality control using the following

procedure. Three ampules will be required, as

well as membrane filtration equipment. The
three ampules will be used as an uninoculated

blank, a positive control for both total coliforms

and E. coli, and a negative control.

9.2 The day before the QC procedure, inoculate

three 10-mL Tryptic Soy Broth tubes with E.

coli (ATCC 25922), Enterobacter cloacae

(ATCC 23355), and Pseudomonas aeruginosa

(ATCC 27853). Incubate at 35oC ± 0.5 oC for

24±4hr.

9.3 Aseptically open 3 ampules of

m-ColiBlue24 Broth and saturate the pads in

three MF petri dishes.

9.4 Filter 100 mL of sterile water and place

filter on the first pad. This is the uninoculated

blank.

9.5 Prepare serial dilutions as described in

Standard Methods 9222B (16.2). Prepare a

serial dilution of the 24 hr P. aeruginosa culture

so that filtration of a 100-mL volume would

theoretically yield 20-80 colonies. Filter the

bacteria-containing diluent. After filtration,

rinse the funnel with several 20-30 mL volumes

of sterile rinse water. Place membrane filter

into the second plate of m-ColiBlue24 Broth.

This is the negative control. Prepare appropriate

serial dilutions of the E. coli culture and filter.

Rinse the funnel as previously indicated. Using

this same membrane filter, serially dilute the E.

cloacae culture, filter and rinse. This will

introduce both E. coli cells and E. cloacae cells

onto the same filter for a dual positive control.

Incubate all three MF petri plates 24 hr at 35oC

± 0.5 oC. The uninoculated control should

remain blank. The P. aeruginosa control should

have no colonies. The positive control should

have both blue (E. coli) and red (E. cloacae)

colonies.

9.6 Colonies may be picked from

membrane filters and inoculated into Lauryl

Tryptose Broth (LTB), Brilliant Green Lactose

Bile (BGLB), EC+MUG or other media for

further QC testing if desired.

10.0 Calibration and Standardization

10.1 No calibration or standardization of

m-ColiBlue24 Broth is required.

10.2 All equipment used in Section 6 should

be calibrated and maintained according to

manufacturer's instructions.

11.0 Procedure

11.1 Test Procedure

11.1.1 Aseptically open an ampule

containing m-ColiBlue24 Broth and

pour the broth onto the pad in a 50-mm

MF petri plate.

11.1.2 Place a sterile filter onto a

sterile filter holder. Using sterile

graduated cylinders and pipets, measure

an appropriate sample aliquot. Pour

water sample into reservoir funnel on

filter holder and draw the water through

the filter using a vacuum pump. Rinse

the funnel with several 20-30 mL

volumes of sterile rinse water. With

sterile forceps, transfer the filter to a

petri plate containing a m-ColiBlue24-

saturated pad. Invert plate and incubate

at 35oC ± 0.5 oC for 24 hr.

11.2 Interpretation

11.2.1 Examine filters for colony

growth. Colonies are typically readily

visible, but a dissecting microscope

may be useful.

11.2.2 A red or blue colony is a Total

Coliform Positive result. A clear or

white colony is a Total Coliform

Negative result. A blue colony is

specifically an E. coli Positive result. A
non-blue colony is an E. coli Negative

result:

 Positive Negative

 Result Result

Total Coliform Red or Blue

Colony

Clear or White

Colony

E. coli Blue Colony

only

Non-Blue Colony

11.2.3 The incubation time is 24 hours.

If no colonies are visible after 24 hours,

the sample is free of total coliforms and

E. coli.

12.0 Data Analysis, Calculations,

Interpretation and Reporting

12.1 Presence-Absence

12.1.1 The presence of at least one red

or blue colony indicates the sample is

total coliform positive. The presence of

at least one blue colony also indicates

the sample contains E. coli. Absence of

red or blue colonies indicates the sample

contains no total coliforms or E. coli.

No further analysis or calculation is

required.

12.2 Quantitation

12.2.1 This method is approved by the

USEPA for the purposes of determining

the presence or absence of total

coliforms and E. coli in drinking water.

Since a protocol for evaluating the use of

a new method for the enumeration of

total coliforms and/or E. coli in drinking

water does not exist, new methods, such

as m-ColiBlue24, cannot be approved by

the USEPA as quantitative methods for

USEPA reporting purposes. However,

we have included the enumeration

protocol for other (non-USEPA

reporting) purposes.

12.2.2 Coliform density determinations

can often provide additional data for

problem solving within the water plant

or for analysis of source water samples.

m-Coliblue24 Broth can be used to

make density determinations.

12.2.3 If enumeration of total coliform

and/or E. coli populations is desired,

refer to Standard Methods 9222B for

appropriate dilutions of the sample to

filter so that 20-80 coliform colonies

are present after incubation. For

analysis of potable water samples, a

standard sample of 100 mL is always

required. Calculate the number of

microorganisms per 100-mL sample

also as described in Standard Methods

9222B.

13.0 Method Performance Characteristics

13.1 Specificity - The specificity of

m-ColiBlue24 Broth for recovery of total

coliforms and E. coli was evaluated. These

experiments were conducted according to the

EPA protocol of June 30, 1992 (16.3). For

E. coli, the false positive error was 2.5% and

the undetected target error was 0% (Section

17.0, Table 17.1). Overall agreement between

m-ColiBlue24 and E. coli reference methods

was 98.8%. For total coliforms, the false

positive error was 26.8% and the undetected

target error was 1.6% (Table 17.2). Using

M-Endo as a comparison for total coliform

recovery to m-ColiBlue24, m-Endo total

coliform false positive error was 29.6% and the

undetected target error was 3.4%. (Table 17.3).

Overall agreement for total coliform recovery

was 86.2% for m-ColiBlue24 and 85.7% with

m-Endo. Although the total coliform false

positive errors seemed large initially, review of

the literature revealed that many existing media

for total coliform recovery have similar false

positive error rates (16.4-16.10).
13.2 Precision and Bias: Not applicable, as this

protocol was conducted on a presence-absence

basis. If individuals wish to use this medium for

quantitative determinations they may use

precision and bias calculations specified by

ASTM (16.11).

14.0 Pollution Prevention

14.1 Base the quantity of chemicals purchased

on expected usage during its shelf life and

disposal cost of unused material. Actual reagent

preparation volume should reflect anticipated

usage and reagent stability.

14.2 For information about pollution prevention

that may be applicable to laboratories and

research institutions, consult Less is better:

Laboratory Chemical Management for Waste

Reduction, available from the American

Chemical Society’s department of Government

Regulations and Science Policy, 1155 16th

Street N.W., Washington D.C., 20036. Phone:

(202) 872-4477.

15.0 Waste Management

15.1 It is the laboratory’s responsibility to

comply with all federal, state, and local

regulations governing waste management,

particularly the hazardous waste identification

rules and land disposal restrictions, and to

protect the air, water, and land by minimizing

and controlling all releases from fume hoods

and bench operations. Compliance with all

sewage discharge permits and regulations is also

required.

15.2 See the MSDS for product composition

information and further guidance on waste

disposal.

15.3 For more information on laboratory waste

management, consult Waste Management

Manual for Laboratory Personnel, available

from the American Chemical Society's

department of Government Regulations and

Science Policy, 1155 16th Street N.W.,

Washington D.C., 20036. Phone: (202) 872-

4477.

16.0 References

16.1 Federal Register, Title 40, Part 141,

Section 141.21 Coliform sampling. December

5, 1994.

16.2 “ American Public Health Association,

American Water Works Association, Water

Environment Federation. Microbiological

Examination, Part 9000” in: Standard

Methods for the Examination of Water and

Wastewater, 18th ed., A.E. Greenberg, L.S.

Clesceri, A.D. Eaton, eds. Washington, D.C.,

American Public HealthAssociation, 1993.

16.3 U. S. Environmental Protection Agency.

Requirements for the nationwide approval of

new or optionally revised methods for total

coliforms,fecal coliforms, and/or E. coli, in

national drinking water monitoring. Revision

1.2, June 30, 1992. Environmental Monitoring

Systems Laboratory, Cincinnati, OH.

16.4 Brenner, K.P., C. C. Rankin, Y.R.

Roybal, G.N. Stelma, P.V.Scarpino, and A.P.

Dufour. 1993. New medium for the

simultaneous detection of total coliforms and

Escherichia coli in water. Appl. Environ.

Microbiol. 59: 3534-3544.

16.5 Cenci, G., A. De Bartolomeo, and G.

Caldini. 1993. Comparison of fluorogenic and

conventional membrane filter media for

enumerating coliform bacteria. Microbios

76:47-54.

16.6 Covert T.C., L.C. Shadix, E.W. Rice, J.R.

Haines, and R.W. Freyberg. 1989. Evaluation

of the autoanalysis colilert test for detection

and enumeration of total coliforms. Appl.

Environ. Microbiol.55: 2443-2447.

16.7 Edberg, S.C., M.J. Allen, D.B. Smith, and

the national collaborative study. 1988.
National field evaluation of a defined substrate

method for the simultaneous enumeration of

total coliforms and Escherichia coli from

drinking water: comparison with the standard

multiple tube fermentationmethod. Appl.

Environ. Microbiol. 54: 1595-1601.

16.8 Jacobs, N.J., W. L. Zeigler, F.C. Reed,

T.A. Stukel, and E.W. Rice.1986. Comparison

of membrane filter, multiple-fermentation-tube,

and presence-absence techniques for detecting

total coliforms in small community water

systems. Appl. Environ. Microbiol. 51: 1007-

1012.

16.9 Lupo, L., E. Stickland, A. Dufour, and V.

Cabelli. 1977. The effect of oxidase positive

bacteria on total coliform density estimates.

Health Lab. Sci. 14: 117-121.

16.10 Sartory, D.P. and L. Howard. 1992. A

medium detecting β-glucuronidase for the

simultaneous membrane filtration enumeration

of Escherichia coli and coliforms from

drinking water. Lett. Appl.Microbiol. 15:273-

276.

16.11 American Society for Testing and

Materials. Determination of precision and bias

applicable methods of committee D-19 on

water. D 2777-86. Annual Book of ASTM

Standards, Vol. 11.01. ASTM, Philadelphia,

1993.
17.0 Tables

17.1 E. coli recovery on m-ColiBlue24

Reference

Positive Negative Total

m-ColiBlue24 Positive 234 6 240

Negative 0 250 250

Total 234 256 490

Sensitivity 100.0%

Specificity 97.7%

False Positive Error 2.5%

Undetected Target Error 0%

Overall Agreement 98.8%

17.2 Total Coliform Recovery on mColiBlue24

Reference

Positive Negative Total

m-ColiBlue24 Positive 183 67 250

Negative 2 248 250

Total 185 315 500

Sensitivity 98.9%

Specificity 78.7%

False Positive Error 26.8%

Undetected Target Error 1.1%

Overall Agreement 86.2%

Table 17.3 Total Coliform Recovery on m-Endo

Reference

Positive Negative Total

m-Endo Positive 149 61 210

Negative 5 245 250

Total 154 306 460

Sensitivity 96.8%

Specificity 80.1%

False Positive Error 29.0%

Undetected Target Error 3.2%

Overall Agreement 85.7%