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OWML: QA/QC

Quality Assurance/Quality Control Manual: Ohio Water Microbiology Laboratory
 

Compliance and official methods

Culture methods for indicator bacteria
The methods used for analysis of indicator bacteria are those of the USGS, USEPA, and APHA and others (table 5). All indicator bacteria methods used by the OWML are compliance or official methods.

Table 5.  Culture methods for 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
Appendix C1

Compliance—DW

Official—other waters

Britton and Greeson (1989)

APHA (2006, Section 9222B)

MI method
Appendix C2

Compliance—DW

Official—other waters

USEPA (2002)

Colilert method
Appendix C3

Compliance—DW, RW

Official—other waters

Idexx Corp., Westbrook, ME

APHA (2004, Section 9223)

Fecal coliforms

mFC method
Appendix C4

Compliance—DW

Official—other waters

Britton and Greeson (1987)

APHA (2006, Section 9222D)

Escherichia coli

Modified mTEC
Appendix C5

Compliance—RW, DW Official—other waters

USEPA (2006a)

MI method
Appendix C2

Compliance—DW

Official—other waters

USEPA (2002)

Colilert method
Appendix C3 (water),

Appendix C6 (sediment)

Compliance—DW, RW

Official—other waters
Official - sediment

Idexx Corp., Westbrook, ME

APHA (2004, Section 9223)

Myers and others (2007)

Enterococci

mEI method
Appendix C7

Appendix C8

Compliance—RW, DW

Official—other waters

USEPA (2006b)

APHA (2007, Section 9230C)

Enterococci Enterolert
Appendix C9

Compliance—DW, RW

Official—other waters

Idexx Corp., Westbrook, ME

APHA (2007, Section 9230D)

Clostridium perfringens

Modified mCP method
Appendix C10

Official—all waters

USEPA (1996), modified by OWML

 

Reagents and media for indicator bacterial 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 C11). 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 B4); 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 B5).

The type of buffered-dilution water used by the OWML is phosphate buffer with magnesium chloride dilution water (U.S. EPA, 2000a). Instructions for preparation are listed in Appendix A2.

 

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

Fisher Scientific or Hardy Diagnostics

Dry agar – Cabinet

Plates - refrigerator

As specified by manufacturer

5 days as plates

MI agar

OWML

Refrigerator

6 months in dilution bottles

2 weeks as plates

MI agar

Govern. Scientific Source or Fisher Scientific

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

Fisher Scientific or Hardy Diagnostics

Dry agar – Cabinet

Plates - refrigerator

As specified by manufacturer

3 days as plates

Modified mTEC

OWML

Refrigerator

6 months in dilution bottles

2 weeks as plates

Modified mTEC

Govern. Scientific Source or Fisher Scientific

Dry agar – Cabinet

Plates - refrigerator

As specified by manufacturer

 

mEI

OWML

 

Refrigerator

 

6 months in dilution bottles

3 days to 2 weeks as plates*

mEI Govern. Scientific Source or Fisher Scientific Dry agar - Cabinet Plates - Refrigerator As specified by manufacturer
2 weeks as plates
Enterlolert Idexx Corp., Westbrook, ME Cabinet As specified by manufacturer

mCP agar

OWML

Refrigerator

6 months in dilution bottles

1 month as plates

mCP agar Oxoid Dry agar - Cabinet As specified by manufacturer

Phosphate buffer with magnesium chloride (Appendix A2)

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

 

 

* 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

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, 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 C11).
  • 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 and Enterolert are purchased directly from IDEXX. For Colilert, E. coli and Klebsiella pneumoniae are the positive cultures and Pseudomonas aeruginosa is the negative culture. For Enterolert, Enterococcus faecalis is the positive culture and E. coli and Streptococcus bovis are the negative cultures. The acceptable ranges for each lot number of controls are provided in the manufacturer’s “Certificate of Analysis” available at http://www.idexx.com/view/xhtml/en_us/water/certificates.jsf. Control sets are run every week during busy periods or after every 20 samples to evaluate the test procedure and aid in interpretation of results.

Strain maintenance for positive and negative bacterial control cultures is described in Appendix A3. Strain information and culture maintenance are documented in the OWML LIMS.

   

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