From the outset, let’s admit that the current state of microbial identification is a little confusing. We can ID by traditional biochemical tests (API Strips) or by elegant refinements of the traditional methods (for example, the Vitek 2 Compact). We can identify microorganisms by carbohydrate utilization (Biolog systems) or by the GC pattern of the cell’s fatty acids (Sherlock System). If you want to go genotypic, then you currently have a choice between the Dupont RiboPrinter or Applied Biosystems MicroSeq systems. Your identification (genus and species) may well depend on which system you use as there is no objective standard, and each system is reliant on its proprietary database to assign an identification to the data.
Virtually all of these systems require a preliminary Gram stain to accurately identify the sample. The Gram stain requires a relatively fresh culture for best results (PMF Newsletter, Feb. 2006). This is the first reason to restreak for single colonies after isolation of a contaminant. However, each system also is dependent on the presentation of a monoclonal sample for accurate results. In fact, only one of these systems provides you with enough information to recognize if you have a contaminated (polyclonal) sample (no, I am not going to tell which one it is).
The basic fact is that acceptable microbiological practice (I am not even going to say “good lab practice” or “best lab practice” but “acceptable” or, if you prefer, “adequate”) requires streaking for well isolated, single colonies of good health for identification purposes. This is not difficult.
The best starting material is a relatively “clean looking” colony on your primary plate. This colony should not show obvious signs of being multiple pinprick colonies that merged into a single CFU. Using a sterile loop, sample from the center of the colony and begin a heavy streak onto a new plate of appropriate agar media. This is quadrant #1 (see accompanying figure).
Streaking in quadrant #1 (and all subsequent streaking events) should be in the same direction, with the same part of the loop in contact with the agar. After the completion of the streaking in this quadrant, the loop should be resterilized (thoroughly flamed or discarded for a new, sterile disposable loop) and the plate streaked into quadrant #2 by drawing the fresh loop across two or three lines in quadrant #1. This should be done once or twice, then subsequent streaks performed without touching any of the previous line in the agar surface. The loop is resterilized, and the process is repeated in quadrants #3 and #4, each time the loop becoming contaminated by drawing it across a few lines in the previous quadrant. The idea is a successive dilution of the level of CFU in each quadrant, on each successive line after the initial inoculation in the prior quadrant. The plate is then incubated overnight for colony growth. Single, well-isolated colonies should be evident in quadrant #4 or quadrant #3. Care should be taken not to accidentally contaminate the colony when harvesting.
One note of caution. This streaking for single colony isolates should be conducted a second time if the original plate was heavily contaminated, or if there are multiple colony morphologies evident on this initial streaked plate. The integrity of the microbial identification process requires a monoclonal colony (a colony that is from a single bacterial strain).
Remember – the only assurance you have of a correct identification is proper preparation of the monoclonal sample. To this concern, the final isolation plate should never be used as a storage device – the single well-isolated colony chosen should be restreaked on a separate plate or agar-slant tube for storage.
This seems like a lot of work, and requires an additional day (at least) to the turn-around time for identification of a contaminant when compared to the time required if single-colony isolation is omitted from the process. However, if microbial identification is attempted directly from the colony on the primary test plate (environmental monitoring or bioburden plate) any resultant microbial identification must be suspect as there is no assurance that you are working with a pure culture. When auditing your microbiology lab (or your contract lab), check to see if an SOP is in place requiring this step, and also check the refrigerators and incubators to see if you can find evidence that this is, in fact, occurring. It is an unfortunately common practice to omit this essential step in microbial identification in an ill-advised attempt to save time and money. However, as there are few quality controls possible on the microbial identification process, you have to build the quality into the process to avoid the GIGO phenomenon.
We are in a period of high regulatory interest in environmental monitoring identifications (as part of aseptic production controls), and in the demonstration of absence of objectionable microorganisms in nonsterile finished drug products. This is not the time (if there ever was one) to save a few pennies by omitting a step necessary to the accurate and confident identification of a microbial colony.
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