Gram
Variable Bacteria
When
performing Gram staining, be aware that bacteria may not always exhibit the
same gram reaction. For instance,
gram-positive cells that should stain purple, end up
staining pink. And, gram-negative cells
that should stain pink, stain purple.
When this occurs, bacteria are said to be gram-variable. Unfortunately, such erroneous gram reactions
could lead to the misidentification of bacteria. Below are some of the factors that can contribute
to these discrepancies:
- Dirty slides: Poor Gram stain results might occur if
smears are prepared on dirty slides. Oils and other grime on the slide
might alter the effectiveness of the reagents. Slides should always be thoroughly
pre-cleaned.
- Uneven smears: Cells should always be spread into a
thin, single layer of cells. If a
smear exhibits uneven thickness, overlapping cells may not get the proper
exposure to the reagents. This
results in uneven or mottled staining.
For example, in the thicker areas of the smear, gram-negative cells
may not decolorize sufficiently and end up staining purple.
- Overheating smears: Excessive heat while heat-fixing a smear
is a potential problem. This heat
may alter or damage cell wall, thereby making them decolorize more
readily. Under this circumstance,
gram-positive cells tend to stain pink.
- Faulty
reagents: The use of faulty reagents
might lead to erroneous gram reactions.
For example, Gram’s iodine loses its effectiveness over time due to
exposure to air and higher temperatures.
Insufficient iodine exposure could cause cells to decolorize more
readily. Similarly, cells tend to
decolorize too easily if crystal violet is prepared at a concentration
that is too low. In either case,
gram-positive cells end up staining pink.
- Excessive
decolorizing: When ethyl alcohol is used
too abundantly or too long, a smear can be overdecolorized. As a result, gram-positive cells lose
all the crystal violet and end up staining pink.
- Excessive
rinsing: Another source of overdecolorization
can occur with excessive washing between steps. Water could remove the crystal violet
from gram-positive cells
before the Gram’s iodine is applied, thus causing them to
stain pink.
-
Excessive
counterstaining: If safranin is applied for a prolonged period, this
excessive counterstaining will replace the crystal violet in gram-positive
cells and make them stain pink.
- Old
cultures: For the most reliable
results, it is recommended that cultures used for Gram staining should be
18-24 hours old. Smears prepared
from old cultures tend to stain inconsistently. The older cells in the smear have
damaged cell walls that decolorize more readily, making gram-positive
cells stain pink.
- Broth
cultures: Smears prepared from broth
cultures may lead to more gram-variable reactions. Because this fluid medium ends up being
transferred to slide, the ingredients found in broths are thought to
interfere with the staining process.
To avoid this complication, smears can be made from the surface
growth of bacteria cultured on solid media.
- Non-conforming
bacteria: Because of the unique
chemical composition of some bacteria, these organisms simply do not
conform to the typical gram reactions observed with most bacteria. In addition to gram-variable species,
some bacteria may simply stain very poorly or not at all. These bacteria (e.g., Mycobacterium spp.) are then termed Gram neutral.
Bibliography
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Diagnostics. 2002. Nov. 6, 2003
http://www.hardydiagnostics.com/catalog/hugo/LanaGram.htm
Kelly,
S.G., and F.J. Post. “Gram Stain.” Basic Microbiology
Techniques (4th ed.) 85-88 (2002).
Star Publishing Company, Belmont, CA.
Kruczak-Filipov, P. and R. Shively. “Gram stain procedure.” In:
Isenberg, H.D. (ed.). Clinical
Microbiology Procedures Handbook.
Vol. 1, 1.5.1-1.5.18 (1992). American Society for
Microbiology, Washington, D.C.
McClelland, R. “Gram’s stain: The key to microbiology.” Medical Laboratory Observer 20-28 (April
2001). Nov. 6, 2003
http://www.mlo-online.com