Historically, microbiological methods to enumerate bacteria, yeast or mold include conducting decimal dilutions of a sample, mixing the decimal dilutions with molten agar (semi-solid) into Petri Plates and allowing for microorganisms to replicate and appear as colonies. The Colony Forming Units (CFU) can then be enumerated and the amount of the microorganisms in the sample can be estimated.
The plate count method is based upon two major assumptions: (i) that every cell forms one colony and (II) that every colony originates from one cell. Neither assumption is accurate.
- Each cell form a colony: The ability of a given cell to form a colony depends upon a myriad of factors, including the physiological state of the cell, the medium used, the incubation temperature, oxygen, etc. as well as previous treatment of the cells (cell injury).
- Each colony originates from a single cell: this may not be true if pairs, chains, or clumps of cells are not completely broken apart before plating. Mixing, shaking or other procedures cannot assure that all cells are separated.
“With all the errors and assumptions, the plate count may represent somewhere between 10% and 90% of the real number of bacteria present in the sample” (Banwart, 1981). The main value of a plate count is the ability to compare the results.
After incubation, plates with 30-300 (USP) colonies per standard-sized plate are counted (Bacteriological Analytical Manual (BAM) uses 25-250 colonies to be counted on a plate). This number of colonies (30-300) was chosen because the number counted is high enough to have statistical accuracy, yet low enough to avoid nutrient competition among the developing colonies. This theoretically mean that any specified level below 250 cfu/g (1:10 dilution x 25 colonies = 250) cannot be accurately counted by the plate count method. Otherwise, recording a CFU of “less than,” or too numerous to count (TNTC) (Yousef, 2003 p. 7-11) must be followed. Typically, plate methods take 2 – 5 days to incubate before the results are available.
Dilute to spec is a different approach to enumeration of bacteria in a sample. The Dilute-to-Specification protocols require diluting the sample to the specification limit required for product action or release. If there is growth, the sample fails; if there is no detection, the sample passes since the counts are below the specification limit. If we use an example of a specification level of <10 cfu/g, and assume that one organisms can detect in the vial. If there is a detection after adding one ml of a 1:10 dilution then there are counts >10cfu/g, and if there is no DT it means <10cfu/g. For a spec. level of <100 cfu/g, 0.1 ml needs to be added to the vial. For higher spec levels we just add more dilutions. The following equation can be used to calculate the required dilution:
1/spec level=dilution * amount to vial
For example if we use a specified level of 500 cfu/g the required dilution is 1/500=0.002:
– Dilute 1:100
– Add 0.2 ml to a vial
– 1:100=0.01; 0.01*0.2=0.002
After the appropriate dilution if the sample does not have a DT it means that the count is <500 cfu/g. If there is a DT the count is >500 cfu/g.
The dilute to spec. approach correlates well with the plate count methodology while yielding a clear cut distinction between samples above and below spec.
Protocols were developed for raw materials, finished products, and environmental swabs as well as processed water. The system automated reporting system collates the data from all these assays to generate a single report.
Multi Assay Protocol
The figure below shows an example of a flow chart for performing multiple assays. A streamlined protocol was developed allowing the analysis of tablets, capsules, suppositories, and liquid material.
In this example the specified level for yeast and mold is <1,000 cfu/g. A 1:10 dilution of the product in TSB is followed by a 1:100 dilution in buffer. 1.0 ml is transferred to a yeast and mold vial (shown in yellow in the figure). The total dilution is 1:1,000, therefore if there is growth in the vial resulting in a DT, the vial contains yeast and mold >1,000 cfu/ml. A flat curve without a DT indicates that the sample has <1,000 cfu/g.
Absence of specific organisms, such as E. coli, S. aureus, and Salmonella is determined by pre-incubating 10.0 grams of sample in TSB for 18 hours followed by the distribution of the pre-incubated broth into the appropriate vials.
Four hundred thirty-seven dietary supplement and OTC samples were analyzed for total aerobic count at various specification levels. Therefore, there was 99.8% agreement between the USP plate count method and the BioLumix dilute-to-spec method. This indicates the ability of the BioLumix system to yield equivalent results to the plate count method over the range of 100 cfu/g to 10,000,000 cfu/g. Similar results were obtained for yeast and mold counts and Gram Negative Bile Tolerant bacteria.
Why Use it?
The dilute to spec approach allow automating the assays, streamlining and simplifying the procedures, save labor and create a paperless laboratory, while generating results that correlate well with the plate count methodology.
Banwart, G. 1981. Basic Food Microbiology. AVI Publishing Company Westport, CT.
Yousef, A., and Carlstrom, C (2003). Food Microbiology: A Laboratory Manual. Hoboken, NJ: John Wiley & Sons, Inc.
USP <2021> Microbial Enumeration Tests-Nutritional and Dietary Supplements
USP <61> Microbial Examination of Nonsterile Products: Enumeration Tests
Bacteriological Analytical Manual (BAM) 8th Edition (1981)