The U.S. Pharmacopeia (USP) created quite the challenge when it designed the growth promotion test (GPT) for selective media. Laboratories not only need to test new batches of media with less than 100 colony-forming units (CFU), the colonies must also grow on agars such as MacConkey within 18 hours. We’ve compiled nine best practices to help you become a selective media GPT expert.
1. Don’t expect a microorganism to grow as well on selective agar as on non-selective agar (even if the non-selective agar was designed for the microorganism species)
For example, the crystal violet and bile salts in MacConkey Agar inhibit Gram-positive microorganisms while allowing many types of Gram-negative microorganisms to grow. Just because the MacConkey Agar allows Gram-negative strains to grow, it doesn’t mean they will flourish.
For instance, if Tryptic Soy Agar (TSA) and MacConkey Agar are tested in parallel from an Escherichia coli suspension containing ≤100 CFU per inoculum, the E. coli will usually recover more colonies the nutrient-rich TSA than on MacConkey. From the E. coli’s viewpoint, growing on TSA is like eating a well-balanced diet containing plenty of fruits and vegetables, whereas growing on MacConkey is like eating nothing but potato chips.
Keep in mind there is no requirement for what percent recovery there must be on selective agar versus non-selective agar, so there is no need to fret if you don’t get even 50% recovery.
2. Don’t worry about the factor of two
The factor of two shouldn’t be on your mind when testing the growth-promoting properties of selective media because it isn’t a requirement according to the USP. Instead, the USP states growth on the new batch of selective media should be “comparable” to growth on the previously approved batch of selective media.
3. Confirm the number of CFU in your inoculum on non-selective agar
What if you recover no colonies when you inoculate MacConkey Agar with E. coli? Does this mean the MacConkey Agar is unacceptable or that you have no E. coli in your inoculum? If you test a non-selective agar such as TSA in parallel with the selective agar, you can confirm whether there were viable E. coli cells in the inoculum. Remember, as mentioned above, there is no requirement for what percent recovery must be achieved when comparing non-selective to selective recovery.
4. Incubate plates in stacks of four or less
The USP puts a time limit on how many hours you can incubate your new batch of selective media before seeing growth. For example, colonies of E. coli should appear on VRBG agar within 18 hours of placing the plates in the incubator.
Eighteen hours is not much time! One way to ensure the bacteria can meet this strict deadline is to stack agar plates only four plates high. An incubator full of tall stacks of agar plates takes longer to warm up than an incubator with small stacks, and the plates in the middle of the stacks will also take longer to warm up. The agar plates need to reach 30˚C to 35˚C quickly to give the bacteria enough time to grow.
5. Use more CFU
If you inoculate your agar with <10 CFU, it is possible you will get no growth when using media that is very selective.
If the mean assay value of your inoculum is less than 50 CFU per 0.1 ml on non-selective media, you can try doubling the inoculum to improve your chances of recovery on selective media. For example, if the mean assay value is 30 CFU per 0.1 ml on TSA, you can inoculate a new batch of MacConkey agar with 0.2 ml and still be under the USP limit of ≤100 CFU.
6. Use the environmental conditions required by the species
- Remember fungus prefers cooler temperatures. USP <62> recommends growing Candida albicans and Aspergillus brasiliensis at 20-25˚.
- Use an anaerobic indicator when growing anaerobes such as Clostridium sporogenes.
- Check and record incubator temperatures twice a day.
- Validate incubators and calibrate thermometers on a routine basis.
- Validate incubators to ensure they stay in correct temperature range. Beware of hot spots in your incubator.
7. Use high-quality media
Results can vary with the type of media used. We have seen this when testing Pseudomonas aeruginosa on TSA. Below are our results when we inoculated six brands of media with 0.1 ml from the same suspension of P. aeruginosa.
|Tryptic Soy Agar||CFU per ml|
8. Give pour plates more time to grow
Colonies often grow more slowly on pour plates compared to spread plates. For instance, you may need to incubate pour plates an extra 24 hours before you can see tiny Staphylococcus aureus colonies. A background light can help you spot them.
9. Use a standardized inoculum
By using a standardized inoculum of 10-100 CFU, you can avoid the unpleasant surprise of finding 120 colonies on your agar plate the day after you inoculated it with the suspension prepared with a turbidimeter.
Using standardized suspensions also saves time. Michael Sinclair from the Microcheck Microbial Analysis Laboratory performed a time study that compared the time it takes to perform the growth promotion test using commercially-prepared microorganisms versus traditionally-prepared microorganisms. The study found that the total hands-on- time (HOT) for traditional spectrophotometric and turbidimetric methods was 115 minutes for five microorganisms, whereas the total HOT time for methods using commercially prepared microorganisms was only 30 minutes for five microorganisms.
Microbiologics offers a broad collection of QC microorganism products for performing the growth promotion test on selective media. EZ-Accu Shot™, EZ-Accu Shot™ Select, EZ-CFU™ and EZ-CFU™ One Step are designed to make the test hassle-free. All four products deliver 10-100 CFU per 0.1 ml.
About the Authors
Karla I. Fjeld is the Research and Development Scientist at Microbiologics. Karla received a Bachelor of Arts in biology and chemistry at the College of St. Benedict, St. Joseph, Minnesota in 2001, and a PhD in biochemistry and molecular biology at Michigan State University, East Lansing, Michigan in 2007. As the R&D Scientist, she works on both new products and product and process improvements.
Laurie Kundrat, MT (ASCP) has over 25 years of experience as a Microbiologist and a Clinical Technologist. Laurie is an active member of the Personal Care Products Council (PCPC) and serves as a member of the Microbiology Committee. She graduated from Case Western Reserve University with a degree in Biology. She also earned a Medical Technology degree from Fairview General Hospital.