Streaking for Single Colonies: An Essential First Step in
Microbial Identification
Scott Sutton,
Ph.D.
Vectech
Pharmaceutical Consultants
This article first appeared in the
PMF Newsletter
of December, 2006 and is protected by copyright to PMF.
It appears here with permission.
The computer programming guys have a useful
acronym: GIGO (Garbage In, Garbage Out). This is useful in
pretty much all aspects of life, and I am willing to defend that
statement with pithy arguments at a later time. Right now, I
want to take a brief look at how it applies to microbial
identification.
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.
Consulting with Scott Sutton |
