Culture media is a basic tool of microbiology, supporting most microbiological assays such as propagation, obtaining pure cultures, enumerating cells, preservation, and selecting microorganisms. For pharmaceutical quality control laboratories, media is frequently used for environmental monitoring, sterility testing, and microbial enumeration tests. As such, the quality of microbial-based tests greatly depends on the quality of the culture media; if the media is not the right quality, it undermines all of the tests that the media is used for. Therefore, safeguarding the quality of culture media through routine quality control testing can help ensure reliable, consistent microbiological test results.
Culture media are traditionally defined as substances that encourage the growth, support, and survival of microorganisms. This is achieved through the preparation of liquid broth or solid agar media comprising reagents required to support microbial growth, including basic nutrients, energy sources, growth factors, minerals, buffer salts, and metals1. To help minimize lot-to-lot variability, culture media manufacturers often attempt to standardize the preparation of media; however, there are frequently unavoidable differences in raw materials from natural sources, intra-lab skill level, or the storage and shipping conditions of the media. To help counter this, culture media should be physically inspected for color, clarity, damage, pH, and gel strength following all preparation steps. Moreover, media that has not been quality control tested or assessed should be quarantined to prevent premature use.
In addition to the physical inspection of culture media, it is important to analyze media for microbiological characteristics. As these media are frequently used for the analysis of sterility throughout the manufacturing process, or for the detection of objectionable microorganisms, it is imperative that the media is not only sterile, but able to promote the growth of any possible contaminants. For the analysis of media sterility, uninoculated media is commonly incubated. Here, the growth temperature and incubation period will depend on the type of media being analyzed. Media exhibiting the absence of growth following the recommended incubation period are considered sterile. In contrast, growth promotion testing is one of the most important quality control tests performed on media and is used to determine if the media in question is able to promote and sustain growth. The primary objective of the test is to detect if a new batch of media is functional and of the same standard as the most recent batch of media tested, as well as ensures the consistent use of standardized media between labs.
Currently, there are several approaches that can be taken for testing media for growth promotion. For agar-based media, a simple method of analysis is to serially dilute microbial strains and plate them on test media using the spread plate technique. These agar plates are then compared to the growth characteristics of a control plate, which is a batch of media that has been previously assessed for growth promotion and approved for use. Two other, more robust, approaches include the Miles-Misra and the ecometric techniques2. The Miles-Misra method is a quantitative technique used to determine the surface viable count. Here, droplets of titered microbial suspensions are dropped onto plates and allowed to spread naturally; the test plate is compared to a control plate following incubation. Colonies are counted in the sector where the highest number of discrete colonies can be enumerated. The results of the assay are examined using a productivity ratio, which is equivalent to the mean of the test plates divided by the mean of the comparative control plates. In this case, an acceptable productivity ratio should fall within 0.5-2, which is equivalent to 50%-200% recovery. In contrast, the ecometric method is a semi-quantitative approach that involves streaking a loopful of a microbial suspension onto four quadrants of an agar plate so that the inoculum is diluted with each streak. In this method, growth should occur in each of the quadrants.
For liquid broth culture media, growth promotion testing typically involves inoculating the media with an estimated number of microorganisms (< 100 colony forming units) and observing it for turbidity within a required time. For bacterial and fungal cultures, this is typically 3 and 5 days, respectively. These inoculated test cultures are compared to control batch media, and the inoculum level is verified via plate count. Both the test media and the control media must show turbidity for the analysis to be considered successful.
For each media type, an appropriate panel of microorganisms is required in order to demonstrate the suitability of the media for the required test. Depending on the required use of the media, a suitable microbial panel may differ in the microbial strains selected as well as how many strains comprise the panel. Generally, the pharmacopeia recommends a preselected list of specific microorganisms for each chapter or general test that must be traceable to a reputable culture collection, such as ATCC. For example, a standard set of cultures may include Staphylococcus aureus subsp. aureus (ATCC® 6538™), Bacillus subtilis subsp. spizizenii (ATCC® 6633™), Pseudomonas aeruginosa (ATCC® 9027™), Clostridium sporogenes (ATCC® 19404™), Candida albicans (ATCC® 10231™), Aspergillus brasiliensis (ATCC® 16404™), Escherichia coli (ATCC® 8739™), and Salmonella enterica subsp. enterica serovar Typhimurium (ATCC® 13311™). In addition to the recommended set of cultures, isolates that are commonly found in the manufacturing environment are commonly used for media testing as well. For example, media used for the analysis of clean room sterility are often tested for the growth promotion of microorganisms commonly found in clean rooms, such as Staphylococcus, Corynebacterium, Micrococcus, Bacillus species, and common skin microflora.
ATCC Genuine Cultures® are maintained using the seed lot system recommended by the United States Pharmacopeia (USP) General Chapter, Microbiological Best Laboratory Practices <1117>. Moreover, each ATCC Genuine Culture® has been thoroughly authenticated and characterized using a polyphasic approach comprising genotypic and phenotypic analyses. To conserve the characteristics of ATCC Genuine Cultures®, it is recommended that each laboratory has a seed lot system in place for preserving and maintaining reference cultures; these cultures must be handled carefully at all times to avoid genetic drift, phenotypic changes, contamination, and strain damage.
Overall, microbial culture media is an important tool used in the pharmaceutical quality control process. As the quality and functionality of the media directly affects its use in microbiological assays, it is imperative that it is thoroughly tested for quality, sterility, and growth promotion prior to its use. This can be analyzed through the use of fully characterized ATCC Genuine Cultures®. All ATCC Genuine Cultures® are maintained and authenticated in accordance with the USP Microbiological Best Laboratory practices, ensuring reliable results of microbial assays.
1. Bridson E, Brecker A. Design and Formulation of Microbiological Culture Media. In J.R. Noris and D.W. Ribbons (Editors). Methods in Microbiology, Volume 3A, London: Academic Press, 1970.
2. Mossel DAA, et al. Quality control of solid culture media: a comparison of the classic and the so-called ecometic technique. J Appl Bacteriol 49: 439-454, 1980.