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Reference:
Sandle, T. ‘Gram’s Stain: History and Explanation of the Fundamental
Technique of Determinative Bacteriology’, IST Science and Technology Journal,
April 2004 (No. 54), pp3-4
Gram’s Stain: History and Explanation of the Fundamental Technique of
Determinative Bacteriology
By Tim Sandle
For those not familiar with Microbiology, the Gram stain (or more accurately
Gram’s stain) is a laboratory staining technique that distinguishes between two
groups of bacteria by the identification of differences in the structure of their cell
walls. It is an important tool in the process of bacterial identification by diving
bacteria into two groups (the so called Gram-positives and Gram-negatives) and
in allowing their morphological types (coccid or rod shaped) to be clearly seen.
The staining technique involves a number of steps:
Staining bacterial cells with crystal violet.
Fixing the stain.
Using a solvent to remove the stain from some types of bacteria.
Using a counter stain.
The first step involves taking single (pure) colonies from an agar plate of still
growing bacteria (often 18 – 24 hours old) and heat-fixing the cells (which kills
them) onto a microscope slide. Following this the cells are then stained with a
basic dye, crystal violet, which stains all bacterial cells
blue. The second step involves adding an iodine-
potassium iodide solution. The iodine solution enters the
cells and forms a water-insoluble complex with the
crystal violet dye. For the third step, cells are treated
with alcohol or acetone solvent in which the iodine-
crystal violet complex is soluble. Following solvent
treatment, only Gram-positive cells remain stained.
After the staining
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{picture: stained slide
ready for microscopic
examination}
procedure, cells are treated with a counterstain, i.e., a red acidic dye such as
safranin, in order to make Gram-negative (decolorized) cells visible.
Counterstained gram-negative cells appear red, and gram-positive cells remain
blue. The slide is then examined microscopically using a x 100 objective.
In order to understand how this relatively simple stain works, the origins of the
technique and of its ‘discoverer’ provide some of the answers. The discoverer of
the stain was Hans Christian Joachim Gram, who was born in Denmark in 1853.
He was born to Frederik Terkel Julius Gram, a professor of jurisprudence, and
Louise Christiane Roulund into the relative comfort of middle-class Danish
society. The young Christian Gram (as most text books call him) studied natural
sciences and graduated from the Copenhagen Metropolitan
School in 1871 and gained a medical degree in 1878 from the
University of Copenhagen, and became a physician.
{picture of
Christian
Gram}
Gram spent a great deal of his life travelling Europe, lecturing and pursuing
studies in pharmacology and bacteriology. It was said that his lecturers, although
very detailed, were not the most inspiring and tended to stretch on for some time.
However, it was during his study of red corpuscles in 1884 that he developed his
method for staining bacteria. Gram was working in the rather grisly setting of a
Berlin morgue, under the leadership of a Dr. Friedlander, where he was trying to
isolate and view the causative agent of bacterial pneumonia. It was whilst he was
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examining lung tissue from patients, that Gram noticed that some stains were
taken up by some bacteria but not by others.
In view of this phenomenon, Gram dried a smear from a lung sample and poured
Gentian (crystal) violet over it (or more precisely “anilinegentain violet solution
of Ehrlich”). Following a water wash, Gram added Lugol’s solution (of
potassium tri-iodide in water) to act as a mordant. Following this step Gram used
ethanol to wash the dye away. Through a number of observations, Gram
observed that certain bacteria (such as the pneumococci) retained the purple
colour (which he termed a positive reaction) whereas other species did not (the
negative reaction).
The reaction, as it was later theorised, was based on the differences in the cell
wall composition for the two ‘groups’. The bacteria that retained the stain (the
Gram-positive bacteria) had a higher peptidoglycan and lower lipid content than
the Gram-negative bacteria. The effect of the solvent was to dissolve the lipid
layer in the cell wall for the Gram-negative bacteria, thereby causing the crystal
violet to leach out whereas for Gram-positive the solvent dehydrates the thicker
cell walls, blocking any diffusion of the violet-iodine complex, which closes the
pores of the cell and retains the stain.
Gram ran a series of experiments, which demonstrated that the new staining
technique was selective for a different
range of bacteria. The key to the technique
proved to the length of time that the solvent
is applied during the ‘decolourisation’ step,
for too long an exposure removed the stain
from both groups of bacteria. The
phenomenon of ‘Gram-variability’, also
{picture: Gram-stained rod shaped cells
examined microscopically at x 100}
shown by ageing cells where logarithmic growth has been suspended, remains a
problem to this day.
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The idea for a counter-stain was developed some years later by a German
pathologist named Carl Weigert (1845 – 1904) who used safranin as a counter
stain, which stained the cells red. Gram himself never used a counter stain.
Gram was modest about his discovery, and in his publication stated:
“I am aware that as yet it is [the stain] very defective and imperfect; but it
is hoped that also in the hands of other investigations it will turn out to
be useful.”
1
Gram’s polychromatic stain went onto be modified several times during the
twentieth century. With the use of the counter-stain, crystal violet became termed
the ‘primary stain’. Occasionally methylene blue is substituted for crystal violet;
sometimes basic fuchsin is substantiated for safranin (fucshin, generally, stains
bacteria more intensely than safranin). The test remains the first test to be used
for the identification of bacteria and without the discovery of basic staining it is
arguable that bacteriology would not have advanced as quickly in the
identification, and hence recognition, of many medically and industrially
important species.
References
Cowan, S. T. and Steel, K. J. (1965): ‘Manual for the Identification of Medical
Bacteria’, Cambridge University Press
Gram, H. C. J. (1884): Über die isolirte Färbung der Schizomyceten in Schnitt-
und Trockenpräparaten, Fortschritte der Medizin, Berlin, 2: 185-189.
Web-sites:
http://www.whonamedit.com/doctor.cfm/696.html
http://www.arches.uga.edu/~emilyd/history.html
http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/Gram1.htm
1
Über die isolirte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten.
Fortschritte der Medizin, 1884, 2: 185.
