Introduction:

Growth promotion testing is a requirement put forth by Pharmacopeia

documents to ensure that the media used for detecting microbial

contamination in products or processes is effective in detecting target

organisms. Testing specifically for Mycoplasma species is critical for

processes that include the use of cell culture as these organisms can affect

metabolism pathways of the host cells, altering the cells’ normal growth and

phenotype. Standardized methods for culture based testing for

Mycoplasma requires detection of low concentrations of cells

(approximately 100 CFU present in the inoculum).

Use of Enumerated Mycoplasma Controls to Assess Media Quality for Growth Promotion Testing Karla I. Fjeld, PhD

Department of Research and Development, Microbiologics, Inc., St. Cloud, MN

Results:

• Each result shown is the average of multiple lots of organisms and media.

• The viable, countable recovery for each organism, on each media in relation

to the reference media is different for each individual organism. The relative

recovery for SP4 glucose agar MFG B ranges from 7% - 263%, and the

Mycoplasma agar ranges from 7% - 329% from the reference media.

• Figure 1 graphically illustrates the differences between the three types of

media for each individual organism

• Some organisms showed inconsistent morphology when plated on the various

medias.

• Figures 2-4 show the morphological differences that occur when three

representative organisms are grown on the different medias.

Conclusion:

Recovery and morphology of Mycoplasma species was generally inconsistent

between suppliers and types of media. The patterns for each organism were

consistent across different lots of media from the same manufacturer. Given the

importance and requirement of Mycoplasma testing, careful evaluation of media

with qualified control materials is critical for understanding the performance of

media used for growth promotion testing. Variability in the growth media

performance is a concern given the increase in the number of products requiring

Mycoplasma testing.

This study highlights the fact that consistent performance of a media cannot be

assumed across multiple species of Mycoplasma. It is recommended that a

study of each organism and medias of interest is conducted before commencing

routine testing to determine the best media for recovery of each organism from

the product to be tested. The use of a commercial, enumerated suspension can

help with consistency of recovery. Microbiologics will be launching a line of

enumerated Mycoplasma suspensions in 2020.

Materials and Methods:

A comparison of commercially prepared media was performed using internally

prepared, frozen, standardized concentrations of six Mycoplasma species

obtained from NCTC. The organisms are specified by USP <63> for GPT.

The media used were Mycoplasma Agar, and two brands of SP4 Glucose Agar.

The differences in the SP4 agars are as follows: MFG A contains thalium

acetate (excluded in MFG B); MFG B contains polymyxin B and amphotericin B

(excluded in MFG A).

The agar plates were inoculated with 0.2 ml from the same microorganism

suspension, or dilutions in the same dilution series, to obtain easily countable

plates. The plates were counted using a stereoscope.

The testing was done in triplicate utilizing different lots of media and three

different lots of microorganism suspensions for each organism. All plates from

each suspension were inoculated at the same time and incubated in the same

special atmosphere jar in microaerophilic conditions at 34-38°C.

Enumerated Mycoplasma

Figure 1: Relative recovery differences among three medias inoculated from the same organism suspension or dilution series

Figure 2: Mycoplasma fermentans: SP4 Glucose Agar - MFG A (left), SP4 Glucose Agar—

MFG B (middle), Mycoplasma Agar (right)

Figure 3: Acholeplasma laidlawii SP4 Glucose Agar - MFG A (left), SP4 Glucose Agar—

MFG B (middle), Mycoplasma Agar (right)

Figure 4: Mycoplasma gallisepticum SP4 Glucose Agar - MFG A (left), SP4 Glucose Agar—

MFG B (middle), Mycoplasma Agar (right)