James Zellner, Microbiologist and Technical Sales Representative
There are dozens of different types of microbial growth media available to Microbiologists, developed for a wide variety of purposes. The United States Pharmacopeia (USP) references many of these to demonstrate the quality of both compounded drug products and the control of compounding environments. The various types of media serve specific purposes based on the goal of the test. This might include recovery of the broadest range possible of organisms in a sterility test, screening for a specific organism if it would be particularly dangerous in a certain dosage form, or capturing anything in the compounding environment that might find its way into a finished product. Microbial growth media can be prepared in both liquid and solid forms, depending on the application. Tryptic Soy Agar (TSA) and Tryptic Soy Broth (TSB), for example, have the same nutrient profile and similar ingredients, but TSA is a solid and TSB is a liquid. For conducting passive air sampling, a solid TSA plate is easier to handle and leave out in the hood during compounding. Liquid media, like TSB, is generally better at growing organisms, and should be used where possible for recovery of microorganism in drug products. Liquid media is more conducive to growth, since nutrients, oxygen, and waste products move around more freely, and temperature is more uniform and constant in a fluid.
If the goal is to recover or grow the widest variety of organisms, a general growth media that provides a wide range of nutrients is the best choice. General growth medias mentioned in USP include TSB and TSA (aerobic), Fluid Thioglycolate Media (FTM, anaerobic), and Sabouraud Dextrose Broth and Agar (SDB/A, fungal). Tryptic Soy-based media provide a range of common nutrients for bacterial and fungal growth, and is referenced throughout the USP. If an anaerobic media is needed, FTM develops an oxygen gradient when left stationary, generating anaerobic conditions at the bottom and increasing oxygen levels at the top. Because of this property, FTM can grow both anaerobic (like Clostridium and Propionibacterium) and aerobic bacteria. SDB/A has a lower pH to encourage fungal and limit bacterial growth, making it an excellent general purpose fungal media.
General purpose medias are used to conduct USP <51>, USP <61>, USP <62> and USP <71> testing. In the case of USP <71> sterility testing, where any recovery is a potential Out-of-Specification result (simple pass/ fail criteria), general purpose media (TSB and FTM) may be the only type necessary. Other tests, like USP <62>, may start with TSB as an enrichment step, then move to more specialized media for the test to work properly.
Outside of testing product, TSA is referenced where environmental monitoring is discussed, since the goal is recovery of the broad range of organisms. TSA can also be incubated anaerobically, via Bio-Bag or specialized incubator, to recover anaerobic organisms.
In some cases, a general purpose media is not ideal for the test being conducted. A selective media might be used to exclude organisms if there is a specific species or group that is of interest. A USP example of this is Cetrimide Agar, used to screen for Pseudomonas aeruginosa in USP <62>. This agar contains a germicidal compound, Cetrimide, that P. aeruginosa and few other microorganisms can tolerate. If growth is observed on a Cetrimide Agar plate, it is almost certainly P. aeruginosa (though a microbial ID is recommended to confirm). Differential, sometimes referred to as indicative, media has some type of visual indicator added to quickly distinguish if a target organism is present. An example for this also comes from USP <62>, where MacConkey Agar is used to screen for Escherichia coli. MacConkey Agar contains lactose, which E. coli can ferment for energy, creating acid as a byproduct. A chemical additive in the media changes to a pink color as the pH lowers, providing the Microbiologist a visual cue that E. coli is likely present. Salmonella can also grow on MacConkey Agar, but cannot ferment lactose, so no color change will be observed. Finally, there are enriched growth media that contain additional nutrients, very specific combinations and amounts of nutrients, or additives to culture fastidious organisms (those with special growth requirements). USP <63> Mycoplasma refers to the use of enriched media for testing. Mycoplasmas are the smallest self-replicating bacterial organisms, and do not possess cell walls. They are of clinical concern because many are pathogenic, and can be a serious issue in laboratory and pharmacological cell cultures. The ability to screen for Mycoplasma is therefore extremely important. Their parasitic nature, very small genome, and lack of a cell wall mean they need very specific nutrient and growth conditions to be recovered in the lab. Hayflick Media, described in USP <63>, has additional nutrient sources added and antibiotics that act on organisms with cell walls (Penicillin) to meet the nutritional needs of Mycobacteria and exclude other bacterial organisms.
With the enormous range of microbiological media available, there is a “right tool” for many of the bacterial and fungal jobs in the lab. The USP provides a wealth of information and appropriate uses for the broths and agars encountered. An experienced Microbiologist, with the appropriate resources in equipment and media, is a powerful ally to ensure the highest quality and safest compounded drug products reach the patient.
For more information on growth media, contact ARL at info@arlok.com or 800-393-1595.