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APG has been providing microbiological samples now for over two years. Last year was the first time that USEPA included microbiological samples in the DMRQA. This caused problems for many laboratories who were running micro samples for the first time in a proficiency testing format. The experience DMRQA also lead APG to redesign our microbiological samples. We received comments from customers on our micro samples and those of our competitors. These included: the container size of the sample, the type of pipette provided, and most importantly the matrix of the samples. We began development of our microbiological product in the spring of 2005. Although, it was a long arduous journey, it resulted in accreditation as a NELAC approved Microbiological Proficiency Testing (PT) Provider in November 2006. This article addresses the Non-Potable Water (WP) PT product development and product development which took place after the 2006 DMRQA Study to improve our samples.
Our development process included addressing the comments of our customers on the size of the vial used to contain the freeze-dried organisms. The previous vial was a 1.5 mL size and was difficult to make the buffer transfer required to reconstitute the sample. We now provide the sample in a 5 mL vial to allow for easier transfer of buffer to the vial and then to transfer the reconstituted organisms back to the buffer bottle. The pipette used to make the transfer was also changed from a plastic disposable pipette with no volume indicators to a serological graduated pipette.
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Customers complainted that several PT providers used a sample matrix that was difficult to dissolve and would tend to foam up or generate “dirt” in the vial that affected the ability to transfer the organism to the buffer bottle. Our new matrix quickly dissolves without incubation and generates a clear sample. |
Finally, and probably most important is the performance of the product. We had received comments of no growth from a small percentage of our customers. As we collected the information over time a pattern became evident. The most common occurrence of no growth came from customers using m-FC media, with or without rosolic acid. However, at the same time, we have had a large percentage of customers report using m-FC media successfully. This prompted a research project as to why this happening. We had used m-FC media with and without rosolic acid as part of our internal testing to verify the performance of our product and had not experienced no growth results as reported by these customers. Through many hours of literature research, we discovered that m-FC with rosolic acid provides a harsh growth environment for bacteria especially for stressed organisms. Research performed on wastewater effluents revealed low recoveries for E. coli in wastewaters treated with chlorination on m-FC with rosolic acid media when compared to other E. coli selective medias. Recoveries were enhanced by using a stressed organism recovery technique on the m-FC with rosolic acid. One of our initial theories was that the freeze-drying process might mimic the chlorination treatment and stress the organisms. We began testing of a different matrix. We worked with our customers to provide external testing as part of our verification of performance. The results were still mixed as to acceptable performance and no growth. We next began research into the strain of E. coli being used for the PT samples. We had selected a strain of E. coli based on many factors but mostly because this strain is used by many media manufacturers to demonstrate the effectiveness of their products, including manufacturers of m-FC media. After more literature research and talking to various media manufacturers, we learned that certain strains tends to tolerate the m-FC with rosolic acid growth environment better. The new strain being used is described is a more thermo-tolerant strain of E. coli. After performing side by side testing of various medias which are incubated at temperatures ranging from 35-44.5˚C, we found that m-FC media performed better with the new strain. We sent trial QC samples using the new strain to customers who had previously experienced no growth using the m-FC media. The results from these customers were excellent and all reported highly consistent growth.
The three most important factors that affect bacteria growth when performing analysis are media, incubation temperature and incubation time. The most common problem has been incubation temperatures being too high. The 44.5±0.2˚C incubation temperature when using m-FC media is very critical to the accurate identification of fecal coliforms, which includes E. coli. The very tight tolerance of 0.2˚C indicates the need for very accurate incubation temperature controls, because temperature variations of as little as 1˚C too high may impair or totally inhibit the growth of E. coli while allowing other fecal coliforms to grow. This could lead to a false sense of security that your analytical procedure is working correctly because you experience normal growth on your routine samples. Then you analyze a PT sample which contains E. coli as the representative organism for fecal coliforms and you get low counts or no growth. It is important to verify the temperature of incubation with a NIST traceable thermometer or a thermometer calibrated with a NIST traceable thermometer.
Media quality is another very important item. Media should be tested using known strains of bacteria for proper response. While most media manufactures provide this testing prior to shipment, you may want to include your own testing of media at some routine frequency during the time you are using a given lot. Appropriate positive and/ or negative controls are important. The media may perform well when you receive it, but you should verify it’s effectiveness throughout the time you will use it, since all media tends to deteriorate over time. The quality of the media may also be affected by storage conditions so it is important to follow manufacturers instructions. At a minimum the media quality should be verified when you receive it or before its first use as described in Standard Methods 9020B Intralaboratory Quality Control Guidelines. Standard Methods 9222D states unused broth should be discarded after 96 hours if stored in sealed plastic container at 4-8˚C. This shelf-life is much shorter than the expiration dates used by most commercially prepared media manufacturers.
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