USGS Ohio Water Science Center

Compliance and official methods

Fecal-Indicator Bacteria
The methods used for analysis of fecal-indicator bacteria are those of the USGS, USEPA, and APHA and others(table 5). All fecal-indicator bacteria methods used by the Ohio Water Microbiology Laboratory (OWML) are compliance or official methods.

Table 5. Methods for fecal-indicator bacteria used by the Ohio Water Microbiology Laboratory (OWML) [DW is drinking water, RW is recreational water]

BACTERIA	METHOD	TYPE OF METHOD	REFERENCE
Total coliforms	mENDO method	Compliance—DW Official—other waters	Britton and Greeson (1989) APHA (1998) Section 9222B
	MI method	Compliance—DW Official—other waters	USEPA (2000a and 2002) (Appendix C)
	Colilert method	Compliance—DW Official—other waters	Idexx Corp., Westbrook, ME APHA (1998) Section 9223 (Appendix D, E)
Fecal coliforms	mFC method	Compliance—DW Official—other waters	Britton and Greeson (1987) APHA (1998) Section 9222D

*Escherichia coli*	Modified mTEC	Compliance—RW, DW Official—other waters	USEPA (2000b and 2006c) (Appendix G)
	MI method	Compliance—DW Official—other waters	USEPA (2000a and 2002)
	Colilert method	Compliance—DW, RW Official—other waters	Idexx Corp., Westbrook, ME APHA (1998) Section 9223
	mTEC method (not recommended)	Compliance—DW, RW Official-other waters	USEPA (2000 and 2006a) (Appendix F)

Enterococci

mEI method

Compliance—RW, DW

Official—other waters

USEPA (2006b)

APHS (2005) Section 9230c

(Appendix H)

Clostridium perfringens

Modified mCP method

Official—all waters

USEPA (1996), modified by OWML (Appendix I)

Reagents and media for fecal-indicator analyses are prepared according to the methods cited above. Bottle and plates are labeled with the name of the reagent or media, the preparation date, and the initials of the person who prepared it. Each lot of media is quality-control tested with a pure culture of the target bacterium or a sewage sample as a positive control; negative controls are also required (Appendix J). Fresh sewage samples are obtained from the Olentangy Wastewater Treatment Plant as needed. Stock cultures of the positive and negative controls are kept on slants in the refrigerator and transferred once a month. Transfer dates are recorded in the LIMS. When preparing positive and negative controls to be sent to other Water Centers, stock cultures must be transferred within a week before use.

QC results are recorded in the “Media and Buffer” logbook on quality-control sheets (Appendix K); documentation of preparation procedures is also kept in this logbook. Media storage requirements and holding times are strictly followed (table 6). Target pH values for media and reagents are followed as described in table 7 and are recorded on the quality-control sheets in the “Media and Buffer” logbook. Requests for media, buffered-dilution water, and reagent preparation by project personnel are made using the “Expendable supplies request forms” (Appendix L).

The type of buffered-dilution water used by the OWML is phosphate buffer with magnesium chloride dilution water (U.S. EPA, 2000b). Instructions for preparation are listed in Appendix M.

Table 6. Information on media, buffered-dilution water, and reagents prepared and stored in the Ohio Water Microbiology Laboratory (OWML).

TYPE OF MEDIA/BUFFER	SOURCE	STORAGE	HOLDING TIME
mENDO agar	Difco, Detroit, MI	Dry agar – Cabinet Plates - refrigerator	As specified by manufacturer 3 days as plates
MI agar	OWML	Refrigerator	6 months in dilution bottles 2 weeks as plates
MI agar	Becton Dickinson, Cockeysville, MD	Dry agar – Cabinet Plates - refrigerator	As specified by manufacturer 2 weeks as plates
Colilert	Idexx Corp., Westbrook, ME	Cabinet	As specified by manufacturer
mFC agar	Difco, Detroit, MI	Dry agar – Cabinet Plates - refrigerator	As specified by manufacturer 3 days as plates
mTEC agar	OWML	Refrigerator	6 months in dilution bottles 2 weeks as plates
mTEC agar	Becton Dickinson, Cockeysville, MD	Dry agar – Cabinet Plates - refrigerator	As specified by manufacturer 2 weeks as plates
Modified mTEC	OWML	Refrigerator	6 months in dilution bottles 2 weeks as plates
Modified mTEC	Becton Dickinson, Cockeysville, MD	Dry agar – Cabinet Plates - refrigerator	As specified by manufacturer
mEI	OWML	Refrigerator	6 months in dilution bottles 3 days to 2 weeks as plates*
mCP agar	OWML	Refrigerator	6 months in dilution bottles 1 month as plates
Phosphate buffer with magnesium chloride (Appendix M)	OWML	Cabinet (unopened) Refrigerator (after opening)	1 year (unopened) 1 week (after opening)
Phosphate buffer with magnesium chloride	Hardy Diagnostics, CA	Cabinet (unopened) Refrigerator (after opening)	As specified by manufacturer
Urea-phenol solution (Appendix F)	OWML	Refrigerator	6 months or until it is no longer a straw-yellow color

* If reagents that are added after autoclaving are filter sterilized, the longer holding time is applied.

Table 7: Target pH values for media and reagent preparation

Media/Reagent	Final pH	+/-	Reference
Actinomycete Isolation Agar	8.10	0.2	Difco #212168 Bottle
Bile Esculin Agar	6.80	0.2	USEPA; Method 1600; EPA-821-R-06-009; 2006
Brain Heart Infusion Agar	7.40	0.2	USEPA; Method 1600; EPA-821-R-06-009; 2006
Brain Heart Infusion Broth	7.40	0.2	USEPA; Method 1600; EPA-821-R-06-009; 2006
EC Broth	6.90	0.2	USEPA; Method 1603; EPA-821-R-06-011; 2006
ISP Media 1	7.00	0.2	Difco #276910 Bottle
ISP Media 2	7.20	0.2	Difco #277010 Bottle
mCP Agar*	7.60	not stated	Bisson and Cabelli (1979)
mE Agar	7.10	0.2	Difco #233320 Bottle
mEI Agar**	7.10	0.2	USEPA; Method 1600; EPA-821-R-06-009; 2006
mEndo Agar	7.20	0.2	Difco #273620 Bottle
mEndo Broth MF	7.20	0.1	Difco #274930 Bottle
mFC Agar	7.40	0.2	Difco #267720 Bottle
MI Agar***	6.95	0.2	USEPA; Method 1604; EPA-821-R-02-024; 2002
MI Broth	7.05	0.2	USEPA; Method 1604; EPA-821-R-02-024; 2002
mod mTEC Agar	7.30	0.2	USEPA; Method 1603; EPA-821-R-06-011; 2006
mTEC Agar	7.30	0.2	USEPA; Method 1103.1; EPA-821-R-06-010; 2006
NA-MUG Agar	6.80	0.2	Standard Methods for the Examination of W & WW; (9222G); 2005
Nutrient Agar	6.80	0.2	USEPA; Method 1603; EPA-821-R-06-011; 2006
Phosphate Buffered Saline	7.40	0.2	USEPA; Method 1603; EPA-821-R-06-011; 2006
Simmons Citrate Agar	6.90	0.2	USEPA; Method 1603; EPA-821-R-06-011; 2006
Tryptic Soy Agar	7.30	0.2	USEPA; Method 1604; EPA-821-R-02-024; 2002
Tryptic Soy Broth	7.30	0.2	USEPA; Method 1603; EPA-821-R-06-011; 2006
Tryptone	7.30	0.2	USEPA; Method 1603; EPA-821-R-06-011; 2006
Working Phosphate Buffered Dilution Water	7.00	0.2	USEPA; Method 1603; EPA-821-R-06-011; 2006
*pH adjust before autoclaving
**Note: pH after nalidixic acid and TTC addition (aliquot a portion to check the pH)
***Note: pH after cefsulodin addition (aliquot a portion to check the pH)
Updated 10/1/08

Analytical quality-control samples for fecal-indicator bacteria by membrane filtration (mENDO, MI, mFC, mTEC, modified mTEC, mEI, and mCP agar methods) include the following:

Filter blank—a 50-100 mL aliquot of sterile buffered water is plated before the sample to confirm the sterility of equipment and supplies.
Procedure blank—a 50-100 mL aliquot of sterile buffered water is plated after every fifth sample to measure the effectiveness of the analyst’s rinsing technique or presence of incidental contamination of the buffered water.
A sewage sample is plated daily on mCP agar when C. perfringens analysis is done to evaluate the test procedure and to ensure anaerobic culture conditions.
For most media, positive and negative controls are plated for each batch of agar prepared or more often for some projects (Appendix J).
For MI, positive and negative controls are plated every 10 samples to ensure proficiency with the method and evaluate the integrity of the medium. Positive and negative controls include the following:
- Positive controls of E. coli and Serratia marcescens
- Negative controls of Pseudmonas ATCC 10145 (unable to grow on MI and ensures the selectively of the agar) and Providencia alcalifaciens (grows on MI but will not fluoresce and ensures target colonies are correctly identified).
For some projects, a sewage sample is plated with each batch of MI plates at the time of sample analysis to evaluate the effectiveness of cefsulodin (an antibiotic added at the time of plate preparation).

Analytical quality-control samples for Colilert include the following:

Positive (E. coli) and negative-control (Pseudomonas) cultures are included with every 20th sample to evaluate the test procedure and aid in interpretation of results.

OWML: QA/QC