OWML: QA/QC

Quality Assurance/Quality Control Manual: Ohio Water Microbiology Laboratory

Reagents and media for fecal-indicator analysis are prepared according to the methods and are labeled to indicate media, date prepared, and analyst.  Each lot of media is quality-control tested by using 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).  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).

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

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 when C. perfringens analysis is done to evaluate the test procedure and to ensure anaerobic culture conditions.
   
• 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 20^th sample to evaluate the test procedure and aid in interpretation of results.