14 Jul 2025
Introduction
Accurate recovery of mycobacteria is important for high-level (HLD) and intermediate-level disinfection (ILD) validations of reusable medical devices. The use of Middlebrook agar often results in overgrowth of fast-growing bacteria and fungi, leading to underestimation of mycobacterial counts. Rapidly growing mycobacterium (RGM) medium is a selective formulation that simplifies enumeration by suppressing competing flora, improving the efficiency of these types of validations.
What is RGM medium?
Based on Middlebrook 7H10 agar, the RGM medium is supplemented with an antibiotic cocktail that selectively inhibits non-mycobacterial organisms. The medium was originally developed for the detection of non-tuberculous mycobacteria in sputum samples but has been repurposed for environmental and disinfection studies due to its ability to:
- Support Mycobacterium terrae (M. terrae) enumeration, equivalent to standard Middlebrook agar (Log₁₀ CFU/mL difference ≤ 0.1)
- Suppress Gram‐negative and Gram‐positive bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae, in mixed suspensions
- Inhibit water‐acclimated contaminants without affecting the recovery reliability of M. terrae from filtered samples
Performance comparison
| Middlebrook Agar | RGM Medium | |
|---|---|---|
| Incubation time | 14–21 days | 14–21 days |
| Enumeration* | Frequent overgrowth by non-mycobacterial contaminants, including P. aeruginosa, E. coli, S. aureus, K. pneumoniae and fungi, often obscuring or outcompeting mycobacterial colonies, making accurate enumeration challenging | Clear, distinct and countable colonies of M. terrae with suppression of most non-mycobacterial contaminants |
| Quantitative recovery (Log₁₀ CFU/mL) | 7.2 ± 0.2 | 7.1 ± 0.3 |
| Co-culture with P. aeruginosa | 24 CFU P. aeruginosa, 52 CFU M. terrae | 0 CFU P. aeruginosa, 47 CFU M. terrae |
| Mixed-species suspension (10⁻⁵) | Too numerous to count (mixed flora) | 9 CFU (all M. terrae) |
| Environmental water sample | Mixed growth of water microorganisms | Only M. terrae colonies |
CFU = colony forming unit
*The comparison between Middlebrook Agar and RGM medium utilized a mixed bacterial culture to evaluate the performance of the two media in suppressing non-mycobacterial contaminants and supporting M. terrae enumeration.
Streamlined workflow
By preventing non-mycobacterial overgrowth, RGM medium eliminates the need for exhaustive colony differentiation, thus:
- Reducing hands-on time needed for plate reading and data verification
- Minimizing false negatives due to obscured mycobacterial colonies
- Maintaining high accuracy in HLD/ILD validation without sacrificing turnaround time
Regulatory compliance
When properly validated, RGM medium used for selective recovery of M. terrae in bioburden and disinfection efficacy testing aligns with key industry standards, including AAMI TIR12:2020/(R)2023, USP <1229.12> and ISO 11737-1.
Conclusion
RGM medium offers performance equivalent to that of traditional Middlebrook agar for M. terrae enumeration while improving selectivity against non-mycobacterial contaminants. The use of the RGM medium streamlines workflows, improves enumeration, and when validated, complies with regulatory standards for reusable medical device disinfection studies.
References
- Scohy A, Gohy S, Mathys V, et al. Comparison of the RGM medium and the mycobacterial growth indicator tube automated system for isolation of non-tuberculous mycobacteria from sputum samples of cystic fibrosis patients in Belgium. Journal of clinical tuberculosis and other mycobacterial diseases. J Clin Tuberc Other Mycobact Dis. 2018;13:1-4. doi:10.1016/j.jctube.2018.07.003
- Preece CL, Perry A, Gray B, et al. A novel culture medium for isolation of rapidly-growing mycobacteria from the sputum of patients with cystic fibrosis. J Cyst Fibros. 2016;15(2):186-191. doi:10.1016/j.jcf.2015.05.002
- Application of rapidly growing mycobacteria (RGM) media for disinfection efficacy testing. Labcorp. 2024. https://content.labcorp.com/sites/default/files/biopharma_knowledge_library/whitepapers/application-of-rapidly-growing-mycobacteria-media-for-disinfection-efficacy-testing.pdf