The Man Behind Gram’s Stain

The Man Behind Gram’s Stain

Gram,s stain, the cornerstone of microbiology, bears the name of the man who first noticed that some bacteria are
permanently stained by gentian violet, while alcohol removes the dye from others’
Hans Christian Joachim Gram was born in Denmark in 1853 and educated in Copenhagen, but his famous discovery
took place in Berlin, Germany. His academic pursuits were varied, He studied botany before changing to medicine,
pursued research in hematology, and eventually became interested in microbiology. ln 1883, he moved to Berlin to
work in the pathology laboratory of Dr. Carl Friedlander, where he planned to help this scientist research lobar
pneumonia.
paul Ehrlich, Gram’s contemporary, lived in Berlin at the same time. Ehrlich is best known for his work on immunity. ln
190g, he shared the Nobel prize in physiology/medicine with Elie Metchnikoff. He wrote his doctoral thesis on staining
properties and later searched for useful drugs among the synthetic dyes. His work led to $reat advances in the area of
biological staining.
During his own experiments, Gram made good use of Ehrlich’s work. Gentian violet, one of the dyes that Ehrlich
investigated, formed the basis of Gram’s discovery. Gram’s objective was to develop a method for stainin$ kidney
tubules that would differentiate kidney tissue from urinary casts. He hoped to stain the tissue with gentian violet, then
selectivelyremove the stain so only cell nuclei remained cslored;Gram was disappointed to observe that the stain was
removed completely from the tissue when alcohol was applied. Still, he soon discovered that the bacteria in the tissue
were not decolorized, making them much easier to distinguish from surroundin$ tissue.
Friedlander mentioned Gram and his staining discovery in a paper on pneumonia late in 1883, noting, “One of our
collaborators Dr. Gram … succeeded in finding a method …. Even a few scattered cocci can be spotted with great
‘elegance.”, Meanwhile, Gram did further work on this method using Bismark brown as a counter stain and working
with various types of pathology specimens. By the time he published his staining method in 1884, he was able to
describe both Gram-positive and Gram-negative or$anisms.
Another important discovery was yet to come. Finding both Gram-positive and Gram-negative organisms in lun$ tissues
from pneumonia victims caused considerable confusion, eventually leading to the realization that lobar pneumonia
could be caused by more than one organism. The Gram-negative bacillus seen in lung tissues by Gram and Friedlander
came to be known as the Friedlander bacillus. Today we know it as Klebsiella pneumoniae’
Shortly thereafter, Gram studied pharmacologic properties of plant extracts briefly and then turned his attention to the
field of therapeutics. He died in 1938 at the age of 85. Though other investi$ators have made minor modifications to
Gram’s stain, the method remains much the same as when he left it.
A revolution in staining begins
christian Grart’s discovery of a staining method that wourd become a nrainstay of microbiology was fortuitous and unexpected’ lt would not have been possible, however, without a series of other events that were unfording from the
time of his birth’ The most significant was the accidental discovery of the first synthetic dye in 1g56.
The use of dyes to color textiles has been traditional in many cultures for thousands of years. tsefore the late 1g00s,
however’ dyes were obtained from naturat sources, particularly plants. ln the tgth century, advances in chemistry
resulted in the prociuction of synthetic dyes useful in industry. Many of them proved important in medicine, as well.
lrcnically’ a chemistry student who was attempting to synthesize quinine for the treatment of malariadiscovered the
first synthetic dye.
william H’ Perkin was just 18 years old, studying at the Royal coilege of chemistry in London, England, when a
chemistry experiment gone awry made him both famous ancl wealthy. His director, the German chemist August
Hofmann’ suggested that a synthetic form of quinine was urgenuy needed. while perkin believed this could be
achieved by oxidizing allyltoluidine, his experiment produced a coal-tar derivative with a deep purple color. This was not
the drug he was hoping for, yet he was quick to recognize its potential as a dye and, after some testing. patented the
substance’ iie sson went into biisiriess pi’otiucirrg iiie dye, which he iraiiied iriaijve, for the texiiie indusiry.
This discovery inspired other nineteenth century chemists to search for additional coal-tar derivatives that courd be
used as dyes’ The cornpounds they produced soon replaced natural dyes in ilre textile industry, perkin,s mentor,
Hofmann’ became one of the leading researchers in this area, Returning to Germany in 1g65, his knowledge of organic
chemistry and his experience with coal-tar derivatives helped rnake Germany a leader in this field.
These events set the stage for investigators like Paul Ehrlich and christian Gram to discover new applications for
synthetic dyes in medicine’ By the time these men. and others like them, began working with biological staining
techniques’ investisators connected to, and funded by, the textile industry hacl already done much research. Gustav Mann’ a pioneer in histologic tee hniques’ recognized the contributions of this industry to his field. ln 1909, he wrote: “The method of staining “‘ grew and grew. till to be an histologist became practically synonymous with being a dyer, with this difference’ that the professional dyer knew what he was about, while the histologist, with few exceptions, did not know, nor does he to the present clay.,,
ln a roundabout fashion’ wiliiam Perkin achieved his original objective and much more. [n addition to discoveries made by medical researchers’ many medicinal uses for chemical dyestuffs were discovered by chemists in the textile industry during testing of the compounds they created. The discovery of a synthetic chemical substitute for quinine was achieved before the e,d of the century by someone else, but when perkin serenflipitousry revorutionized the dye and textile industry’ he set in motion a chain of events that ultimately resulted in medical advances far beyond his expectations.
Gram’s stain: The key to microbiology’
Gram,s stain remains one of the most valuabte methods we have for identifying isolates accurately and rapidly’ Despite
our long-standing familiarity with this method, it still warrants careful attention every step of the way-from preparation
and QC of reagents to stainin$ and interpretation.
The telephone rings in the microbiology lab. A stat specimen is on its way. A patient in the operating room has had
some tissue excised from an area of necrotizing fasciitis on the left leg. Time is of the essence; a limb, or a life, may be
lost.
clinical microbiology at the beginning of the 21st century still relies heavily on culturing patho$ens in the laboratory
prior to identifying them and performing susceptibility testing, and few results are available stat’ Even new molecular
methodologies typically take hours rather than minutes. Fortunately, Gram’s stain, devised by a Danish pathologist in
1gg4, exists (see ‘,The man behind Gram,s stain,” page 26, and “A revolution in staining beglins,” page 27). This simple
staining procedure remains the most useful test perfornred in the microbiology lab. Results from a Gram’s stain can tell
volumes about an infection within 15 minutes of a specimen’s arrival in the lab, while most other microbiolo$y results
require 24 hours or more. Nevertheless, Gram’s stain findings can be equivocal and, therefore, must be assessed
carefully in li$ht of the clinical picture.
A TELLTALE CLINICAL TOOL
organisms stained by Gram,s method can be divided into groups that have taxonomic si$nificance and that guide the
physician in choosing appropriate antibiotic treatment. Frequently, presumptive identification to a species level can be
made on Gram,s stain findings alone, For example, tissue from necrotizing fasciitis containing numerous Gram-positive
cocci in chains suggests streptococcus pyogenes infection. Many Gram-positive diplococci in sputum suggest the
presence of Streptococcus pneumoniae. And Gram-negative diplococci in a spinal tap sample stren$then a
presumptive diagnosis of menin$ococcal menin$itis’
A report of ,,many Gram-negative rods” or “moderate Grarn-positive cocci suggestive of staphylococci” helps determine
whether empiric antibiotics will be effective. Likewise, the absence of organisms strengthens a suspicion that an
inflammatory process is noninfectious. lnfections caused by mixed organisms do occur; however, a blend of organisms
in a Gram,s stain may indicate that bacteria unrelated to infection have colonized a site, or that a specimen is
contaminated with superficial material’
Human cells (particularly white blood cells and epithelial cells) can be stained as well, providing a means of assessing
whether culture results are likely to be useful. squamous epithelial cells suggest the presence of superficial material
and indicate the culture is likely to grow indigenous organisms of no ctinical relevance. Polymorphonuclear neutrophils
(PMNs), on the other hand, indicate an inflammatory process’
* organisms possess autolytic enzyme systems that can break down cell walls, causing a chewed-up or unevenly stained appearance. S. pneumoniae often appears this way.
when working with cultured organisms, the best results are obtained from colQnie s Lg-24hours old. culturing in liquid
media typically provides excellent morphology. Erroneous results are obtained when smears are nrade from very old or
very young cultures’ or when organisms are grown on culture media containing antibiotics. rncubation temperature and atmosphere can affect organism morphology, too.
other materials intrinsic to sBecimens are a potential source of problems, as well. For instance, mucus will pick up
counter stain’ sometimes to the degree that background staining interferes witfr the reading of slides. Additionaily, a
heavily stained background can obscure bacteria, particularly Gram negatives, and uneven background staining can mimic Gram negatives, making interpretation difficult.
Technical trouble spots
A reliable Gram’s stain result begins with a properly made smear. selecting purulent material whenever possible, the specimen should be rubbed or spread gently and evenly onto a crean grass sride. ldeaily, the preparation shourd have a
single layer of organisms and cells that do not overlap. smears that are too thick will be dark, dense, and impossible to reaci wiih accuracy. A smear of uneven thickness wiii resuii in uneven staining and Gram_vai.iabie organisms
some laboratories use a cytocentrifuge to deposit concentrated specimens (fluids in particular) on a marked area on the slide’ while the advantages of this procedure are obvious when bacteria and human celrs are rare, for many specimens’ especially those that are purulent or thick, this method is likely to produce a smear that is too heavy to read.
Methanol is the superior method of fixation, even though fiame fixing is used rnost freguengy. Because organisms fixed with methanol for a minimum of 1 minute are more resistant to decororization, fhis method provides greater contror
over the decolorization process’ ln addition, methanol fixation 1) prevents riquid specimens, such as urine and spinar fiLrid’ fronr washing off the slide during staining; 2) preserves red blood cells; and 3) results in a clearer background
over decolorization is probably the most persistent problem encountered in Gram,s staining. rt can resurt from ce, wair dama$e to an organism (inherent in some specimens), from decolorizing reagent being applied too zealously, or from a number of other variables (see Table 3, page 2S).
Excessive heat during fixatiorr’ Fixing with heat, as opposed tc merery drying the sride without further fixation, renders organisms more resistant to decolorization. Excessive heat, however, has the opposite effect and renders organisms more susceptible to decolorization’ lt also alters cell morphology. Frame-fixed srides shourd be passed through the flame severar times quickry, rather than herd in the frame for rong periods.
Generally, any specimen containin$ squamous epithelial cells in numbers greater than’ or equal to’ the number of
pMNs is of poor quality and should not be processed. ln addition, when squamous cells are present in lower numbers’
indigenous frora shourd be considered. To herp determine the significance of pathogens isorated from specimens,
raboratories use the Q (quarity) score, which provides an earry assessment of specimen quality and which allows
laboratorians to reject specimens unlikely to yield useful results. Most often used to assess sputum, the Q score is also
useful for screening specimens from external body sites (e’g’, superficial wounds) and to determine the suitability of
specimens for anaerobic culture. Note that in certain circumstances (e’$” leukopenia)’ the number of PMNs may be
deceivingly low in relation to squamous cells, and culturing may be warranted despite a low Q score’
Additionally, Gram’,s stain is a key identifier of isolates grown on culture media’ An organism’s Gram reaction and
morpholo$yoftenareusedtoplaceitwithinataxonomicgroup,providin$$uidanceintheselectionoffurther
identification tests. Frequenly this stain is one of few tests chosen to confirm presumptive identification’
AN ANALYSIS OF GRAM’S PROCEDURE
The conventional method for performing Gram’s stain begins with a thin, air-dried, heat-fixed preparation on a $lass
slide that is flooded with crystal violet and allowed to sit for at least 30 seconds (see Table 1, above)’ The slide is then
rinsed gently under running tap water and flooded with Gram’s iodine for an additional 30 seconds’ Following a second
tap water rinse, the preparation is decolorized. This involves rinsing the slide with an acetone-alcohol solution until all
color has been washed out. Finally, the slide is counterstained for 3o seconds with safranin, rinsed, and air dried’
Most bacteria, as well as many fungi and some parasites, are stained via this method’ White blood cells appear
uniformly red, and squamous epithelial cells exhibit a characteristic mixture of purple and red staining’ Background
material often takes on the color of the counter stain to some degree. Gram-positive organisms retain the crystal violet
and appear deep purple in color, while Gram negatives appear red’ Other or$anisms have a variable/mottled stain
result. The American society for Microbiology (ASM) provides a COmprehensive summary of expected Gram’s stain
results and the morpholo$ies of different organisms’
over the years, experts have tried to explain why some organisms stain Gram positive and others stain Gram negative’
The simple textboot< explanation evolved into variations of the followin$: A chemical reaction occurs between the
crystal violet and the iodine inside bacterial cells, forming a molecule, often referred to as the crystal violet-iodine
complex, that is too large to escape from the cell. The cell wall of a Gram-negative organism contains more lipid than
that of a Gram positive. During decolorization, this lipid is extracted by the sotvent, leaving holes in the Gram-negative
cell wall, and allowing the crystal violet-iodine complex to wash out. The cell is subsequently stained by the counter
stain.
while this explanation has served us well, it is only half the story. Gram-negative cells have an outer layer of
lipopolysaccharides and lipoproteins underlain by a thin peptidoglycan layer, while many Gram-positive bacteria
possess a thick peptidoglycan cell wall interspersed with lipoteichoic acids’ Electron microscopic studies have shown
Low concentration of crystal violet. Very low concentrations of crystal violet can be used successfully, but much greater
flexibility with decolorization time exists when higher csncentrations are used (up to 2%).specificaly, Gram positives
are more readily decolorized after being stained with low concentrations of crystal violet. Nonetheless, at the
concentration commonly used (0.3%), decolorization for 5-10 seconds generally is sufficient for good results.
Excessive washin$ between steps’ Excessive washing with water between steps is another source of over
decolorization’ The method relies on the crystal violet-iodine dye complex being removed differentially from bacterial
cells in alcohol and other solvents, but not in water. Therefore, it is surprising that crystal violet is more susceptible to
washing out than the dye complex. Researchers have tried to minimize this problem by omitting the water wash after
crystal violet and usin$ the iodine solution as a rinse. Unfortunately, this procedure increases risk of stain deposit on
the sride, A S-second wash usuaily is sufficient at any stage of the procedure.
lnsufficient iocline exposure’ The time of exposure to the iodine mordant is important, as is the amount of avairable
iodine in the solution. Like crystal violet, higher concentrations of iodine allow more flexibility with decolorization: the
lower the concentration, the more easily over decolorization will occur. concentrations of 0.33%-1olo commonly are
used.
Lack of available iodine. HiEher temperatures and exposure to air hasten the loss of Gram,s iodine. lnvestigators found
that a closed bottle of Gram’s iodine (o-33o/o)stored at room temperature lost more than half its available iodine in 3o
days’ while an open bottle retained only loo/o of its available iodine after the same period of time. At 37oc, an open
bottle of iodine had no detectable available iodine after just 15 days. ln this study, bacteria became more susceptible
to over decolorization when only 4oo/o of the available iodine was lost; they stained uniformly Gram negative when less
than 2oo/o remained’ To ensure reliable results, a fresh batch of Gram’s iodine, or polyvinylpyrrolidone iodine, shourd be
used.
Pro,onged decolorization. Sonre solvents decolorize bacteria more quickly than others do. For inexperienced
microbiologists, using a slower solvent such as 95% ethanol is recommended. ln more experienced hands, acetone
alcohol or plain acetone can be used. The decolorizer should flow over the stainqd area of the slide (not be applied
directly) until all color has washed out. This procedure can be difficult, particularly if the rnaterial on the slide is
unevenly distributed’ lf all the material on the slide is adequately decolorized, some of it wiil undoubtedly be over
decolorized.
Excessive counter staining’ over decolorization is stiil possible once Ure counterstainingstep is reached. Basic dyes
applied in sequence will replace each other in bacterial celrs if left on too long. lq other words, Gram-positive bacteria
that retain the crystal violet iodine dye complex until the counter stain is applied may still stain Gram negative if
excessive counterstaining occurs; the second stain simply replaces the first. Researchers found that basic dyes do this
consistently regardless of the order in which they are applied, though the time required for complete reptacement
varies’ while the use of a mordant’ such as iodine, significantly srows this process, over time, replacement occurs
nevertheless’ The counter stain should not be left on the slide for prolonged periods (3o seconds is adequate).
that solvents such as alcohol and acetone damage the cell walls of both Gram-positive and -negative bacteria, althouBh
damage to the latter is more extensive. Additionally, the chemical composition of the walls of many cells (e’g’, yeast’
some species of Bacillus, and animal cells other than bacteria) fail to conform to this standard explanation’
Recently, it has been suggested that an organism’s reaction in Gram’s stain is not strictly a result of the chemical
composition of its cell wall, but rather is directly related to cell wall thickness. Experts note that “only thick walled
organisms can be Gram-positive because the wail acts as a permeability barrier restrictin$ diffusion of the crystal
violet:mordant complex.” The peptidoglycan layer in Gram positives is, on avera$e, 1O-15 times thicker than that in
Gram negatives. Gram-positive organisms, such as yeast, have a thick cell wall as well, even though the chemical
composition is significanfly different. The crystal violet-iodine complex, therefore, is retained because the cells are not
exposed to the decolorizer lon$ enou$h for sufficient damage to occur.
This explanation is supported by the observation that dama$e to the cell wall caused by prolonged exposure to
solvents, in addition to the age of the organism and use of antibiotics, will cause Gram positives to stain Gram
negative. cells with variable wall thickness (e.g., dividing cells and those undergoing autolysis) tend to yield a Gramvariable
stain result.
MODIFYING GRAM’S STAIN
The deep purple dye used by Gram was g;entian violet, a mixture of crystal violet and other related dyes. Since then,
gentian violet has been replaced by crystal violet, a pure chemical that is, therefore, less variable. Though other basic
dyes have been investi$ated, crystal violet remains the preferred primary stain’
several other modifications have been made to improve Gram’s stain results For instance, Gram’s $entian violet
solution was found to be unstable (i.e., dye precipitated out of the solution within a few weeks). To correct this problem,
one scientist recommended the addition of aniline sulfate to make the dye stable indefinitely. Today, many people add
ammonium oxalate to the alcoholic dye solution.
Gram,s iodine (iodine, potassium iodide, and water) is still the standard mordant, or trappin$ agent, used today’ Studies
have shown, however, that the available iodine in Gram’s iodine solution is rapidly lost, resulting in over decolorization
of organisms. Atkins created a more stable solution by substituting sodium hydroxide for potassium iodide’ Hucker’s
modification incorporates sodium bicarbonate, which prevents the development of an acid pH as iodine oxidizes’ This
alteration does not prevent loss of available iodine, however. Since then, polyvinylpyrrolidone iodine has proven to be a
stable solution that leads to reliable results’
Several alternate decolorizers have been used since Grain’s original ethanol solvent. One investi$ator experimented
with mixtures of alcohol, aniline oil, xylol, and acetone. Other researchers recommended a solution of equal parts of
acetone and alcohol. Bartholomew supports the use of n-propyl alcbhol. Different solvents act at different speeds:
ethanol and n-propyl alcohol decolorize relatively slowly, while acetone and methanol decolorize quickly’ Acetone’
Quality control
QC slides for Gram’s stain can be made usingi stock strains of staphylococcus spp. and Escherichia coli. check stains
and reagents regularly; the ASM recommends stairling Qc slides daily, whenever a new stain or reagent is put into use,
and when results are poor’ Additionaliy, ail stains and reagents used in the Gram,s stain process shsuld be visually
examined for precipitate or other changes in appearance. ll a precipitate is present, the stain should be filtered.
occasionally, it may be useful ts use smears made from mouth swabs, rvhie h yield a variety of Gram-positive and –
negative or$anisms’ as \tell as squamous epithelial cells. These cells are a good inclicator of the success of staining as
they usually exhibit a typical speckled Gram-variable pattern. lf squamous cells in any smear are uniformly Gram
negative’ the smear is over decolorized’ similarly, squamous eells or PMNs that appear Gram positive indicate an uncler
decolorized smear.
KEEP A CRITICAL EYE
No procedure in nricrobiology is nrore familiar to the lab professional than Gram,s stain. our long-standing familiarity
with this test encourages us to regard it as a “faiFme-never” method. Unfortunately, this perception can lead to a false
sense of security’ Bartholomew expressed the true situation well when he wrote: “obviously, more is involved in Gram
differentiation than merely the color of the organism, which results from a personal interpretation of what constitutes a
Gram staining procedure.”
when all $oes well, our beloved Gram’s stain supplies crucial information leading to correct identification of isolates
and provides rapid results to physicians waiting to treat seriously ill patients. To ensure the most accurate Gram,s stain
results, however, microbiologists must always use a critical eye when peering through the microscope.
Causes of over decolorization
* cell wall damage of or$anism due to a host inflammatory response, age of organism, use of antibiotics, and/or
production of autolytic euzymes
* Excessive heat during fixation
* Low concerrtration of crystal violet
* Excessive washing between steps
* lnsufficient iodine exposure
* Lack of available iodine
* Prolonged decolorization
* Excesslve counterstaining
alcohol is moderately rapid, depending on the amounts of each solvent used’ For routine work, an acetone-alcohol
mixture tends to be the most practical choice. ASM recommends a ratio of 3:7, acetone:alcohol’
Although Gram found Bismarck brown to be an effective counter stain, safranin is used in most microbiology labs
today, as it provides good contrast. Nevertheless, some workers prefer a O.Lo/o-O’2Yo solution of fuchsin’ Basic fuchsin
(0.gyo) and carbol fuchsin are recommended primarily for anaerobes and other weakly staining Gram-negative rods
that are poorly clemonstrated with safranin. While this modification atlows Gram negatives to stain more intensely,
background material tends to stain more intensely as well, making the slide difficult to read’
yet another interesting modification to Gram’,s stain involves the addition of a fast green and tartrazine step before
counterstaining with safranin. Though the final color of both Gram positives and Gram negatives is altered slightly, this
step results in better contrast between Gram negatives and back$round material, makin$ or$anisms easier to detect’
WHEN THINGS GO AWRY
As with any test, Gram’s stain has its flaws, so laboratorians must be mindful of potential problems. Some of these
problems are inherent to specimens, while others are the result of technical difficulties.
lnherent pitfalls
Some organisms cannot be demonstrated by Gram’s stain (e.g., Mycoplasma spp., Chlamydia spp’, and Rickettsia
spp.). Others typically stain poorly. A few Gram negatives tend to stain faintly with safranin and are more effectively
demonstrated using an alternate counter-stain, such as basic fuchsin or carbol fuchsin’ Among these organisms are
Campylobacter spp., Legionella spp., Bacteroides spp., Fusobacterium sPP., and Brucella spp’ Mycobacteria, in $eneral,
are not stained by Gram’s method, and Legionella spp. only stain when taken from a culture’
Some organisms fail to yield a typical Gram’s stain reaction due to cell wall damagle, which can cause Gram positives
to appear Gram negative or Gram variable. Possible reasons for loss of cell wall integrity include the followin$:
* Organisms that have been ingested by phagocytic cells often are visible within the cells but have peculiar morpholo$y
and variable staining.
* When a significant inflammatory response is present, organisms harvested for culture may already be so damaged
that visualization via Gram’s stain is impaired or even impossible.
* Organisms taken from a culture more than 48 hours old are more likely to have cell wall dama$e and, as a result,
falsely stain Gram negative. This may be the case with fresh swabs of infected sites, as well. According to researchers,
it takes about 4 days for an inflammatory response to appear, at which point swabbed or$anisms may be too old to
yield a reliable result.
* Administering antibiotic therapy to a patient before specimen collection can damage bacterial cell walls and prevent
organism growth.

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