How to overcome low-population assay resultsMost users of biological indicators test incoming BIs for viability and population as part of their acceptance criteria for use. This reasonable or mandated examination prior to routine use or the beginning of a validation is also stressed in U.S. Pharmacopeia:
“Upon initial receipt of the biological indicator from a commercial supplier, the user should verify the purity and morphology of the purchased biological indicator microorganisms. … Also, a microbial count to determine the mean count per biological indicator unit should be conducted.”The USP provides information explaining how to perform a microbial count of BIs in the section titled “Microbiological Tests - Total Viable Spore Count.” According to the USP, after the assay procedure is performed, the total viable CFU count needs to be within minus 50% and 300% of the labeled BI population. The process of performing a total viable spore count on the large variety of BIs being used with differing carrier materials and BI size can be more difficult than it may appear.
Any difficulty encountered in obtaining acceptable results to qualify a BI should in most cases be offset by the wide range of acceptability that the USP allows, minus 50% to 300% of the labeled population. Successful population verification should be easily obtainable; however, even with this wide range, many end users are unable to obtain acceptable assay verification. Nearly all assay result failures are due to low population results, below the acceptable minimum of minus 50%.
Some of the causes for low testing results are easier to locate than others. One challenging part of a population failure investigation is just getting the end user who is actually performing the test to look at what was done. Most lab techs consider the assay procedure to be pretty straightforward and are not aware of the procedure problem areas that can occur to make a test result falsely low. Some find it much easier to just accept the fact that the BI is unacceptable for population and request a replacement than to take a look at the possibility of user error.
During the nearly 19 years I have been addressing complaints of low population on BIs, I have found it is extremely unusual to find a BI complaint where the BI was indeed low in population. Most of those who reported low populations have been able to achieve success by making a few procedural changes and then repeating the assay.
One of the first questions that I ask when investigating a low population complaint is, “Are you following USP procedure?” In most cases the answer is yes. However, many who say they follow the USP have made several unintentional deviations from USP procedure that could account for the low population result.
- You must either follow USP or not. Unless you are using a validated excursion from USP, do not deviate from the procedure as written. Most BI manufacturers follow USP Total Viable Count procedure to make the label claims for the BIs. If you are using a different method from that of the manufacturer, difficulties in population verification can occur.
- USP procedure for paper strips states that the initial step for BIs to be tested is to “dispense the paper into component fibers by placing the test specimens in a sterile 250 ml cup of a suitable blender containing 100 ml of chilled, sterilized purified water and blend for three to five minutes to achieve a homogeneous suspension.” If you are using something else, you are neither following USP nor following the assay procedure used by this BI manufacturer. The use of a 250 ml cup is not the same as a large blending vessel that is usually used for mixed or blended drinks. It is a 250 ml jar. Such jars are common half pint jars used for canning and can be found in most hardware stores. The threaded lip on the jar fits the same plastic collar and blade setup used with a typical blender. Using this initial 250 ml container is an important part of the procedure and is also required according to USP. The photos below illustrate how easy it is to use these jars. Because they are canning jars, they are also heat treated and autoclavable. They are usually purchased by the dozen and are very reasonably priced.
Case Study: Three Strikes and You Are OutAfter failing to accept three different, consecutive lots of spore strips for use due to low population testing results at a particular company, I was asked to audit the USP assay procedure being used there, because these particular lots of strips had already passed verification efforts at several other sites using the USP procedure. An investigation was requested to look into the assay procedure being used to determine whether there was an error in the procedure or if the strips truly had a low population.
The facility in question followed USP procedures, and lab personnel said that they were following the Total Viable Spore Count procedure as published in USP. When I arrived at the facility, the lab tech had all the necessary tools ready to perform a population assay. The first step was to remove the paper strips from the glassine envelopes and prepare them to be assayed.
STRIKE ONE: In order to macerate the paper, the tech put the strips into a blending vessel that looked much like those used to prepare mixed drinks. The vessel must have been able to hold at least a liter of water, not as specified in USP. The strips were put into the container, and 100 ml of water was added. This large container was put on top of the blender, and it was turned on. Soon the strip/water mixture was churning in the vessel, high enough on the sides of the vessel that liquid could easily spill out.
STRIKE TWO: After the strips were blended in the container, a glass pipette was used to remove a 10 ml aliquot to start the dilution series. There was so much fiber content present that the tip of the pipette drawing up the aliquot got clogged. The lab’s remedy for this problem was to use a large-bore pipette tip that would not clog as easily. This drew even more paper pulp into the 10 ml sample.
STRIKE THREE: Near the end of the dilution series, the last two dilutions were plated out in triplicate petri dishes, with a 1 ml aliquot put into each dish. Approximately 20 ml of TSA was added to each dish, and the dishes were allowed to cool and were inverted as they were placed into the incubator. The TSA used had a high content of dipotassium phosphate and other buffers.
The final assay results for all three lots of strips were over one log low in population and thus failed the verification. On a subsequent assay using a different medium on the same lots of spore strips, and with the above deviations corrected, all three lots passed verification.
Even in what would seem to be a pretty straightforward procedure, you either follow USP or you don’t.—RN
- Blend to a homogenous solution and, while blending, look at the paper material through the glass jar to see when the strips are thoroughly blended. You should be able to draw up a 10 ml aliquot that does not have paper pulp in the sample.
- Make sure, while vortexing between dilutions, that a vortex actually occurs in the dilution tube and that the solution is being mixed well.
In the full study, six groups of log 6 spore strips were processed using the USP Total Viable Spore Count procedure. All strips were treated the same except for the TSA used in final plating. The groups were blended and went through the usual dilution series. Six sterile petri dishes were inoculated with 1 ml aliquots from the last dilution tube. On three of the plates, Brand X TSA was used, and on the other three plates from the same tube, Brand Y TSA was used in making the final pour plates. Colony-forming units recovered with the different brands of TSA are given in Table 1.
The CFU data from above shows that anyone using Brand X TSA in their verification would never confirm or meet USP acceptance criteria even if all the previous steps had been done correctly. Just changing to a different TSA brand can make a huge difference in your CFU recovery.
Based upon the testing results, a recovery medium that is free from additives such as phosphates, diphosphates, or dipotassium phosphate buffers is recommended. In the above study, Brand Y media did not contain phosphate buffers, while Brand X did. Media that contain phosphates and/or buffers are very suitable for a large amount of typical laboratory or micro work, but this study shows that these additives can be inhibitive to the accuracy of a quantitative recovery of injured spores.
Russ Nyberg has worked at the Omaha Biological Indicator manufacturing facility for Mesa Labs (formerly Raven Biological Laboratories) for the past 19 years. He has held the positions of director of manufacturing and production and now works in technical support.
- Sutton S. Counting colonies. Microbiology Network website. Available at: www.microbiol.org/resources/monographswhite-papers/counting-colonies. Accessed Feb. 5, 2012.
- U.S. Pharmacopeia. General chapter 55: Biological indicators — resistance performance tests. U.S. Pharmacopeia. Available at: www.usp.org/usp-nf/notices/retired-compendial-notices/second-supplement-usp-31-nf-26-online-and-cd-includes-incorrect-version-usp-general. Accessed Feb. 5, 2012.
- Shintani H, Sakudo A, McDonnel GE. Methods of rapid microbiological assay and their application to pharmaceutical and medical device fabrication. Biocontrol Sci. 2011;16(1)13-21.