Colony age for susceptibility testing.

Q The Clinical and Laboratory Standards Institute (CLSI) says that susceptibility testing in particular (and probably ID panels as well) should be done using 18 to 24 hours' growth. In real life, this is not altogether practical. We are really good at not doing ID/susceptibility panels on growth that is more than 24 hours old. Instead, we subculture and do it on fresh growth--although this does delay results by 18 hours (Gram negative bacilli) to 24 hours (staph and enterococcus). When we err, it is by setting up the panels on growth that is only 10 to 14 hours old.

Is it better to subculture the organism and delay results by a day, or do an ID/susceptibility panel on growth that is between 24 and 48 hours old? My understanding is that the problem with "old" organisms is that some may be dead but still give turbidity when preparing the inoculum. Therefore, the panel would be under-inoculated and results might be falsely susceptible. That is why we are strict about only using 18-to 24-hour growth in this scenario.

What is the downside of using growth that is "too young"? Can we get erroneous results by using growth that is less than 18 hours old? We use conventional panels (not rapid). Since some rapid panels rely on enzymatic reactions, I can see how too young a growth could affect those results. I am not as clear on what erroneous results we could get in an overnight (conventional) panel.

A CLSI (formerly NCCLS) states that a suspension of an 18- to 24-hour-old culture can be used for direct broth or saline preparation for testing of antimicrobial susceptibility of bacterial isolates. (1,2) This is the recommended method for testing fastidious organisms, Haemophilus spp., Neisseria spp., and Streptococcus spp., and for staphylococci when testing for potential oxacillin resistance.

Alternatively to this, the CLSI standards also indicate that at least three to five well-isolated colonies of the same morphological type can be selected from an agar plate culture (age not specified). The top of each colony is touched with a wire loop, and the growth is transferred to a tube containing four mL to five mL of a suitable broth medium, such as tryptic soy both. The broth culture is incubated at 35[degrees]C until it achieves or exceeds the turbidity of the 0.5 McFarland standard (usually two to six hours). This suspension can then be used to prepare your 0.5 McFarland standard for use in antimicrobial-susceptibility testing.

Thus, either the direct colony suspension method or the growth method can be used with confidence to prepare bacterial isolates for susceptibility testing. There is an exception to this, however. The manufacturer of your panels says in its package insert that for Staphylococcus aureus only the direct colony suspension method should be used if looking for methicillin-resistant S aureus, or MRSA. Always follow the manufacturer's directions, and discuss any concerns you have with appropriate representatives from the company concerning their instructions.

I would not recommend extensive subculturing of organisms in order to prepare susceptibility testing, which would delay patient-result reporting but, instead, incorporate one or both of the two methods outlined above and standardized by the CLSI into your routine workflow. I would look at your workflow and try to redesign it so that you are not delaying your patient reports, especially on critical specimens (e.g., positive blood cultures, body fluids), but working on them first thing in the morning so that you can get them on your susceptibility-testing instrument the same day. Work towards this with all of your clinically significant isolates.

--Susan E. Sharp, PhD, (DABMM)

Director of Microbiology

Kaiser Permanente Pathology

Regional Laboratory; Associate Professor

Oregon Health and Science University

Portland, OR

References

1. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard--7th ed. CLSI Document M7-A7. Wayne, PA: Clinical and Laboratory Standards Institute. 2006.

2. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests; Approved Standard--9th ed. CLSI Document M2-A9. Wayne, PA: Clinical and Laboratory Standards Institute. 2006.

Daniel M. Baer, MD, is professor emeritus of laboratory medicine at Oregon Health and Science University in Portland, OR, and a member of MLO's editorial advisory board.

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