There are some significant concerns as to the need for Growth Promotion testing of standard media. It can be argued that since all preparation conditions are under control and the physical parameters of the finished media are checked, there is little additional information gathered by the labor-intensive and time-consuming procedure of checking the growth promoting capabilities of the media. This topic has been debated not only among workers in QC laboratories but also in the clinical microbiological industry.
Clinical microbiology laboratories in the United States are not required to test most common media under NCCLS standard M22-A2 Quality Assurance for Commercially Prepared Microbiological Culture Media although this stance has come under question by the relevant NCCLS committee and is being re-evaluated (Krishner 1999). The current understanding of which clinical media to test was based on a survey performed in the early 1980's of 1,164 laboratories. From their reported experiences it was determined that most media could be accepted safely on the manufacturer's data (Krishner 1999). This result confirmed an earlier study (Nagel and Kunz 1973) that called into question the need for excessive growth-promotion testing of commercially prepared media. They examined 900 lots of 46 different media representing 350,000 units of purchased culture media and found only 17 lots to be unsatisfactory. These lots were of specialized media containing labile components.
There has been no convincing scientific evidence published that would argue for the need to test Trypticase Soy media, for example, for growth promotion. However, both the Sterility Test and the Microbial Limit Test require such testing. Given the compendial requirement to test, the first decision may reasonably be to determine the challenge organism. In addition to the compendial organisms required in the tests, addition of specific microorganisms of interest could be useful if they have been recovered from past tests (e.g. a Sterility Test contaminant or a frequent environmental monitoring isolate).
The next concern is test design. There are two types of media commonly used in the microbiological lab - broth and agar. These two types must be considered separately as they show growth by completely different means. The fundamental question of GP testing can be expressed as: Is the new batch of media as good as a previously qualified batch? This question cannot be answered adequately except by statistical comparison, given the variability of microbiological data. The statistical design of GP studies will be developed in the following discussion which has been influenced by the excellent review by Weenk (1992).
Growth Promotion Testing of Agar Media
A singular advantage of agar media tests is that they provide numbers - colony forming units (CFU). To analyze CFU you must use statistical tools designed for the Poisson distribution (Ilstrup 1990) or else convert the data to approximate the normal distribution. This data conversion can be done by using its log10 values or by taking the square root of (n+1) (Ilstrup 1990). Once this is done, plate counts can be directly compared using the Student's T-Test or other tests of normally distributed data.
There are, of course, several less demanding tests for demonstration of equivalency between two agars:
Spread Plates or Pour Plates
The compendia assume a Growth Promotion test by comparison of CFU, with the cells plated in the normal fashion for the lab. The compendia generally require that the colony counts derived from growth on the current batch of media be no less than 50% (USP 2003b) or 70% (USP 2004) of a previously qualified batch. This approach provides the advantages of colony counts and a large area for the colonies to grow, but it is somewhat laborious and expensive in terms of material.
Miles-Misra (Drop Count) Technique
This technique involves dropping the cells in a 10 �L aliquot onto the surface of an agar plate (Miles and Misra 1938). When used carefully, an entire 6-fold dilution scheme can be plated in a single Petri dish and if read early, the individual drops can be used to yield estimates of the number of CFU/mL in the challenge suspension. This method offers significant advantages in terms of labor and material resources.
Growth Promotion Testing of Broth Media
Copious Growth
This is the current compendial method of choice. In this method, the challenge organism is inoculated at a very low level (< 100 CFU per unit) and incubated at the prescribed temperature for the prescribed period of time (3 days or 5 days). Growth in the batch of media is then compared to a parallel sample from a previously qualified batch of the same media. The growth is to be comparable between the two and copious. The advantage of this method is that it does not require a great deal of labor, but the quality of the data for the comparison between the growth promoting characteristics of the media is exceptionally poor. This can be described as a crude end-point test with an “n†of 1.
End-point Methods
In this approach to growth promotion testing, very low levels of inoculum are added to multiple tubes of the two media being examined. Then the resultant growth frequency is compared between the two media to determine equivalency. For example, comparing an old and a new batch of Trypticase Soy Broth (Soy Bean Casein Digest Broth) might be performed by taking 100 tubes of each media, and then inoculating all 200 tubes with <5 CFU of the challenge organism Staphylococcus aureus. After incubation, the number of turbid tubes would be compared - say 30/100 of the new media turbid vs. 46/100 of the old media. The statistical comparison could be performed using the Chi Square Test or Fisher's Exact Test. This evaluation would be performed separately for each challenge organism. The number of tubes used can be decreased (or increased) at the expense of the statistical power of the method.
End-point methods to growth promotion of broth media are obviously very laborious and technically demanding. It is not difficult to envision a design that would require more than a thousand tubes and the need to accurately create an inoculum of <5 CFU of a variety of challenge microorganisms.
MPN
The Most Probable Number method of enumerating microorganisms is most commonly used in the QC lab as part of the Microbial Limits Test (USP 2003b) or in other situations where the sample cannot be put into an appropriate suspension or be filtered (Aspinall and Kilsby 1979). In this technique, the unknown sample is prepared in a ten-fold dilution series and added to nutrient broth in replicate tubes (normally either 3, 5 or 10 replicates are used). The tubes will then either turn turbid (growth) or remain clear, and allow for an estimate of the most probable number of microorganisms.
Ankur Choudhary is an experienced pharmaceutical blogger.