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Laboratory perspective of gram staining and its significance in investigations of

infectious diseases

Article · November 2014

DOI: 10.4103/2384-5147.144725

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Review Article

Laboratory Perspective of Gram Staining and its

Significance in Investigations of Infectious Diseases
Yunusa Thairu, Idris Abdullahi Nasir, Yahaya Usman1
Department of Medical Microbiology, University of Abuja Teaching Hospital, Gwagwalada, Abuja, 1Department of Medical Laboratory Science,
Faculty of Medicine, Ahmadu Bello University, Zaria, Kaduna, Nigeria

ABSTRACT
Clinical microbiology laboratory plays several important roles in the management of bacterial infections. Isolation,
identification of pathogenic microorganisms in cultures and subsequent antimicrobial susceptibility testing always
assists in selecting appropriate antimicrobial agent and prevention of unnecessary complications. The most
important and primary test to perform directly on some special samples such as cerebrospinal fluid and positive
cultures is Gram staining which serves as the most rapid and simplest test to characterize microorganisms. It
is therefore highly likely that the information provided by the Gram staining will help to assess the adequacy
of preliminary diagnosis and antimicrobial therapy selected after collecting culture specimens and before final
identification of the microorganism. In recent reports, the impact of Gram staining results on patient mortality has
been documented. On the other hand, there remains the possibility that Gram staining results do not match with
the final identification of microorganisms. This would carry a risk leading to inadequate antimicrobial therapy and
potentially affecting patients’ clinical course and mortality. The aim of this mini review is to analyze and discuss
the clinical significance and limitations of reporting Gram staining results for sample meant for bacteriological
investigations.

Keywords: Positive culture, Bacteriological, Limitations and special samples

INTRODUCTION The Gram stain easily divides bacteria into two groups,
Gram-positive and Gram-negative, on the basis of
Effective utilization and understanding of the clinical
their cell wall and cell membrane permeability. The
bacteriology laboratory can greatly aid in the diagnosis
mechanism further implies that solvent decolorization
of infectious diseases.[1,2] Although described more
causes significant damage to the cell surfaces of Gram-
than a century ago, the Gram stain remains the
negative bacteria and only limited damage to Gram-
most frequently used rapid diagnostic test, and in
positive bacteria. This suggests Gram-negative bacteria
conjunction with various biochemical tests is the
cornerstone of the clinical laboratory. First described are more “leaky,” causing these thin-walled lipid-rich
by Danish pathologist Christian Gram in 1884 and later cells to lose their crystal violet (CV) stain and appear
slightly modified.[3,15] red from the counterstain. Gram-positive cells, thick
walled and lipid-poor, appear blue from retaining the
original CV.[4] The Gram stain is not an infallible tool
for diagnosis, identification, or phylogeny, however. It
Access this article online
is of extremely limited use in some instances and has
Quick Response Code:
Website: been largely superseded by molecular techniques even
www.ssajm.org
in the medical microbiology laboratory. Given that some
organisms are Gram-variable (i.e., they may stain either
DOI:
negative or positive), and that some organisms are not
susceptible to either stain used by the Gram technique,
10.4103/2384-5147.144725
its true utility to researchers should be considered limited

Address for correspondence: Dr. Yunusa Thairu, Department of Medical Microbiology, University of Abuja Teaching Hospital, P.M.B. 228,
Gwagwalada, Abuja, Nigeria. E-mail: samhaamal200@gmail.com
Received: 03-06-14, Accepted: 11-09-14

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Yunusa, et al.: Laboratory perspective of Gram staining and its significance in investigations of infectious diseases
and specific. In molecular microbiology laboratory, most
identification is done using genetic sequences and other
molecular techniques which are far more specific and
information-rich than differential staining.[5]
The Gram stain has been used for several purposes that
include:
1. To directly examine specimens submitted for
microbiologic examination, e.g., body fluid or biopsy
when infection is suspected. It yields results much
more quickly than culture, and is especially important
when infection would make an important difference
in the patient’s treatment and prognosis; examples
are cerebrospinal fluid for meningitis and synovial
fluid for septic arthritis
2. To provide preliminary information to the clinician
regarding presumptive bacterial pathogens
3. To characterize the type of bacteria growing in culture
media (including blood cultures)
4. And to assess the quality of specimens submitted for
culture (i.e., sputum specimens) to determine whether
they are likely to yield clinically useful information
and whether they should be processed (cultured).
This specimen quality assessment is possible because the
leukocytes and epithelial cells of the human host also
stain with the Gram stain.[6]
As a general rule of thumb (which has exceptions), Gram-
negative bacteria are more pathogenic due to their outer
membrane structure. The presence of a capsule will often
increase the virulence of a pathogen. In addition, Gram-
negative bacteria have lipopolysaccharide (LPS) in their
outer membrane, an endotoxin that increases the severity
of inflammation. This inflammation may be so severe
that septic shock may occur. Gram-positive infections
are generally less severe because the human body does
not contain peptidoglycan; in fact, humans produce an
enzyme (lysozyme) that attacks the open peptidoglycan
layer of Gram-positive bacteria. Gram-positive bacteria
are also frequently much more susceptible to beta-lactam
antibiotics, such as penicillin.[7]
Exceptions to the rule include branching and filamentous
Gram-positive bacteria such as Mycobacterium tuberculosis
and other agents of tuberculosis, or Nocardia species, the
agents of nocardiosis and some types of actinomycetoma.[7]
These organisms present unique problems in diagnosis
and treatment, and special stains such as the Ziehl-
Neelsen stain and the Kinyoun stain are used in their
laboratory workup.
Like other laboratory tests, the Gram stain has certain
inherent limitations and is subject to technical variation
and misinterpretation — the subject of an article by Rand
and Tillan.[8] This retrospective study reviews major
169
errors, defined by the authors as errors in which the
original Gram stain of a positive blood culture reports
a single organism whose Gram stain morphology is
opposite (Gram-positive vs. Gram-negative or vice
versa) to the Gram stain morphology of the final culture
organism identification. The authors found that in
57 (0.7%) of 8,253 patients with positive blood cultures
the Gram stain was misread, resulting in major errors
that fell into three categories:
1. Six cases where the original Gram stain report was
Gram-positive cocci and the culture yielded Gram-
negative bacilli, 3 of which were Acinetobacter species.
2. Twenty-five cases where the original Gram stain
report was Gram-negative bacilli and the culture
yielded Gram-positive organisms; of these were
Bacillus species and 2 were Clostridium species (this
type error is probably due to overdecolorization, a
common technical problem in Gram stains).
Twenty-eight cases where the original Gram stain
showed a single organism but the culture yielded
multiple organisms; most often in this group the original
Gram stain showed Gram-positive cocci. When the
medical charts of these patients were reviewed, there
were four patients in whom delays of 14 h to 3 days
occurred in starting appropriate antibiotics, with two
deaths, although the authors believed that the erroneous
Gram stain report was probably not contributory.
Although the study by Rand and Tillan has limitations
(e.g., the reasons for the errors in the Gram stain
reports — technical artifact, interpretation error, or
clerical error — could not be specifically determined in
this retrospective study), it is still a useful contribution to
the field of clinical microbiology for several reasons. First,
the data presented show that the overall performance
of the clinical laboratory scientists in interpreting Gram
stains (99.31% of the Gram stains were interpreted
accurately over 23 months was very good, especially
when one considers that the Gram stain morphology
of bacteria can be affected by antibiotic therapy,
technical variables including overdecolorization and
underdecolorization, and misinterpretation by those
reading the Gram stains. Although 100% accuracy
in reporting is always the goal of the laboratory, the
accuracy of >99% for reporting of positive blood cultures,
which are critical laboratory values, is to be commended.
Second, the authors provide important information to
laboratory directors by identifying the most common
types of major errors in blood culture Gram stains
so that the directors can emphasize these problems
in training and continuing education of the bench
microbiologists responsible for interpreting Gram stains.
Third, the finding that a frequent error was reporting a
single organism when 2 or more were present prompts
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Yunusa, et al.: Laboratory perspective of Gram staining and its significance in investigations of infectious diseases

microbiologists to continue to vigorously search for recovered from a five-year old child with cerebrospinal
other organisms, even after identifying 1 organism in a meningitis [Figure 4].
blood culture Gram stain, especially since Gram-positive
organisms are generally more easily visualized than
Gram-negative organisms.

Clinical Significance of Gram Staining

The Gram stain is a very important preliminary step in
the initial characterization and classification of bacteria.
It is also a key procedure in the identification of bacteria
based on staining characteristics, enabling the bacteria
to be examined using a light microscope. The bacteria
present in an unstained smear is invisible when viewed
using a light microscope. Once stained, the morphology
and arrangement of the bacteria may be observed as well.
Furthermore, it is also an important step in the screening
of infectious agents in clinical specimens such as direct
smears from a patient. Although the Gram staining is
used for detection and differentiation of bacteria, other Figure 1a: Gram positive stained colonies of Candida spps showing
microorganisms, most frequently yeasts and fungi, can typical large oval budding cells, source: www.candida-albicans.co.uk
be seen on a Gram-stained smear.[9] Like Clostridium
organisms, yeasts can appear Gram-positive or Gram-
negative. Yeasts are generally at least 10-20 times the size
of bacteria and hence differentiation from bacteria is not a
problem. However, yeasts are the size of CV precipitate,
which is occasionally present.

This large purple structure can even appear to be

budding. CV precipitate can be differentiated from
yeasts because:
1. The precipitate may be present in an area with several
other purple artifacts of various size and shape,
2. The precipitate has a homogenous deep purple color,
while the yeast is often mottled, and
3. The precipitate may be perfectly round, while Candida
species, the yeast most commonly encountered, are
generally oval.[9] Figure 1b: Candida albicans showing extensive pseudohypae, source:
http://pathmicro.med.sc.edu

Below are list of commonly encounter pathogens on

Gram stain smears.

Gram-positive bacteria
Stain dark purple due to retaining the primary dye
called CV in the cell wall. Example Gram positive
stained colonies of Candida spps showing typical
large oval budding cells and extensive pseudohypae
[Figure 1a and 1b respectively]; Gram stained colonies
of Staphylococus aureus recovered from Blood culture of
a baby with sepsis [Figure 2].

Gram-negative bacteria
Stain red or pink due to retaining the counter staining
dye called Safranin or neutral red. Example: Gram
Negative Bacilli of Escherichia coli from a woman Figure 2: Gram stained colonies of Staphylococus aureus recovered
suffering from Urinary tract infections [Figure 3] and from Blood culture of a baby with sepsis, source: www.cdc.org/ncidod/
Gram negative Coccobacilli of Haemophilus infleunzae diseases/submenu/sub_staphylococcus

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Yunusa, et al.: Laboratory perspective of Gram staining and its significance in investigations of infectious diseases
Figure 3: Gram Negative Bacilli of Escherichia coli from a woman
suffering from Urinary tract infections, www.cdc.org/ecoli
GRAM STAIN MECHANISM
Gram-Positive Cell Wall
Gram-positive bacteria have a thick mesh-like cell wall
which is made up of peptidoglycan (50-90% of cell
wall), which stains purple. Peptidoglycan is mainly a
polysaccharide composed of two subunits called N-acetyl
glucosamine and N-acetyl muramic acid. As adjacent
layers of peptidoglycan are formed, they are cross-linked
by short chains of peptides by means of a transpeptidase
enzyme, resulting in the shape and rigidity of the cell
wall. The thick peptidoglycan layer of Gram-positive
organisms allows these organisms to retain the CV-iodine
complex and stains the cells as purple.[4]
Lipoteichoic acid is another major constituent of the
cell wall of Gram-positive bacteria that is embedded
in the peptidoglycan layer. It consists of teichoic acids
that are long chains of ribitol phosphate anchored to the
lipid bilayer through a glyceride. It acts as a regulator of
autolytic wall enzymes (muramidases: Bacterial enzymes
located in the cell wall that causes disintegration of the
cell following injury or death).
Lipoteichoic acid also has antigenic properties that
stimulate specific immune responses when it is released
from the cell wall after cell death. Cell death is mainly
due to lysis induced by lysozymal activities, cationic
peptides from leucocytes, or beta-lactam antibiotics.[1]
Gram-Negative Cell Wall
Gram-negative bacteria have a thinner layer of
peptidoglycan (10% of the cell wall) and lose the CV-
iodine complex during decolorization with the alcohol
rinse, but retain the counter stain Safranin, thus appearing
reddish or pink. They also have an additional outer
membrane, which contains lipids, which is separated
from the cell wall by means of the periplasmic space.[10]
171
Figure 4: Gram negative Coccobacilli of Haemophilus infleunzae
recovered from a five-year old child with cerebrospinal meningitis,
source: www.tommytoy.typepad.com
Relevance of Gram-Negative Cell Wall
The cell wall of Gram-negative bacteria is often a virulence
factor that enables pathogenic bacteria to cause disease.
The virulence of Gram-negative bacteria is often associated
with certain components of the cell wall, in particular, the
LPS (otherwise known as LPS or endotoxin). In humans,
LPS elicits an innate immune response characterized by
cytokine production and activation of the immune system.
Inflammation occurs as a result of cytokine production,
which can also produce host toxicity.[11]
STAIN REACTION
The four basic steps of the Gram stain (as described by
WHO, 2013) are.[12]
Application of the Primary Stain to a Heat-Fixed
Smear of Bacterial Culture
CV dissociates in aqueous solutions into CV+ and Cl–
ions. These two ions then penetrate through the cell
wall and cell membrane of both Gram-positive and
Gram-negative cells. The CV + ions later interact with
negatively charged bacterial components and stains the
bacterial cells purple.
Addition of Gram’s Iodine
Iodine (I− or I3−) acts as a mordant and as a trapping
agent. A mordant is a substance that increases the affinity
of the cell wall for a stain by binding to the primary stain,
thus forming an insoluble complex, which gets trapped
in the cell wall. In the Gram stain reaction, the CV and
iodine form an insoluble complex (CV-I), which serves
to turn the smear a dark purple color. At this stage, all
cells will turn purple.
Decolorization with 95% Ethyl Alcohol
Alcohol or acetone dissolves the lipid outer membrane of
Gram-negative bacteria, thus leaving the peptidoglycan layer
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Yunusa, et al.: Laboratory perspective of Gram staining and its significance in investigations of infectious diseases
exposed and increases the porosity of the cell wall. The CV-I
complex is then washed away from the thin peptidoglycan
layer, leaving Gram-negative bacteria colorless.
On the other hand, alcohol has a dehydrating effect on
the cell walls of Gram-positive bacteria that causes the
pores of the cell wall to shrink. The CV-I complex gets
tightly bound into the multi-layered, highly cross-linked
Gram-positive cell wall thus staining the cells purple.
The decolorization step must be performed carefully.
Otherwise over-decolorization may occur. This step is
critical and must be timed correctly otherwise the CV
stain will be removed from the Gram-positive cells. If the
decolorizing agent is applied on the cell for too long time,
the Gram-positive organisms to appear Gram-negative.
Under-decolorization occurs when the alcohol is not left
on long enough to wash out the CV-I complex from the
Gram-negative cells, resulting in Gram-negative bacteria
to appear Gram-positive.
Counterstain with Safranin
The decolorized Gram-negative cells can be rendered
visible with a suitable counterstain, which is
usually positively charged safranin, which stains them
pink. Pink color that adheres to the Gram-positive
bacteria is masked by the purple of the CV (basic fuschin
is sometimes used instead of safranin in rare situations).
It is a prudent practice to always include a positive and
negative control on the staining procedure to confirm
the accuracy of the results[13] and to perform proficiency
testing on the ability of the Laboratory scientist to
correctly interpret the stains.[14]
ERRORS DURING GRAM STAINING
Excessive Decolorization
It is clear that the decolorization step is the one most
likely to cause problems in the Gram stain. The particular
concerns in this step are listed below.[6]
Excessive heat during fixation
Heat fixing the cells, when done to excess, alters the cell
morphology and makes the cells more easily decolorized.
Low concentration of crystal violet
Concentrations of CV up to 2% can be used successfully,
however low concentrations result in stained cells that
are easily decolorized. The standard 0.3% solution is
good, if decolorization does not generally exceed 10 s.
Excessive washing between steps
The CV stain is susceptible to wash-out with water (but
not the CV-iodine complex). Do not use more than a 5 s
water rinse at any stage of the procedure.
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Insufficient iodine exposure
The amount of the mordant available is important to
the formation of the CV-iodine complex. The lower
the concentration, the easier to decolorize (0.33–1%
commonly used). Furthermore, QC of the reagent is
important as exposure to air and elevated temperatures
hasten the loss of Gram’s iodine from solution. A closed
bottle (0.33% starting concentration) at room temperature
will lose >50% of available iodine in 30 days, an open
bottle >90%. Loss of 60% iodine results in erratic results.
Prolonged decolorization
95% ethanol decolorizes more slowly, and may be
recommended for inexperienced technicians while
experienced workers can use the acetone-alcohol mix.
Skill is needed to gauge when decolorization is complete.
Excessive counterstaining
As the counterstain is also a basic dye, it is possible to
replace the CViodine complex in Gram-positive cells with
an over-exposure to the counterstain. The counterstain
should not be left on the slide for more than 30 s.
REPORTING RESULTS
Results and Interpretation (as Described by WHO,
2003)
a. Evaluate the general nature of the smear under low-
power magnification (10X).
i. Determine if smear has been properly decolorized.
Depending on the source of the specimen, the
background should be generally clear or gram
negative. If WBCs are present, they should appear
completely gram negative. Do not mistake thin
crystal/gentian violet precipitate needles for
gram-positive bacillus-shaped bacteria.
ii. Determine if thickness of smear is appropriate.
For proper interpretation, areas must be no more
than one cell thick with no overlapping of cells.
b. Examine smears prepared from clinical specimens
under low power for evidence of inflammation. If
appropriate for culture source, note the following:
i. Relative amounts of PMNs, mononuclear cells,
and RBCs
ii. Relative amounts of squamous epithelial cells,
bacteria consistent with normal microbiota,
which may indicate an improperly collected
specimen
iii. L o c a t i o n , s h a p e a n d a r r a n g e m e n t s o f
microorganisms
c. Examine several fields a of the smear under oil
immersion for the presence of microorganisms.
i. If no microorganisms are seen in a smear of a clinical
specimen, report “No microorganisms seen.”
ii. If microorganisms are seen, report relative
numbers and describe morphology.
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Yunusa, et al.: Laboratory perspective of Gram staining and its significance in investigations of infectious diseases
d. Observe predominant shapes of microorganisms
overall shape: coccus, coccoid, coccobacillary, bacilli,
filamentous, yeast-like
i. Appearance of ends; rounded, tapered, flattened,
clubbed (swollen), concave, swelling of sides can
suggest the presence of spores but can also be
caused by vacuoles, marked pleomorphism, or
irregular staining.
ii. Appearance of sides: parallel, ovoid (bulging),
concave, irregular
iii. Nature of axis: straight, curved, spiral
iv. Pleomorphism (variation in shape): the
descriptive term “diptheroid” or “coryneform”
is used to describe gram positive bacilli that
are pleomorphic, club shaped, or irregularly
staining or that have palisading and/or angular
arrangements (V and L shapes).
v. Branching or cellular extensions
Recording Observations
Each laboratory must have SOPs (Standard operating
procedures) for reporting results and for all
procedures in the Microbiology laboratory. Clinically
significant findings should be communicated to the
pathologist or called to the attention of the attending
physician.
a. Smears of clinical specimens: for urine cultures,
examine 20 or more fields. Report as positive if an
average of one or more organisms are seen per oil
immersion field. This correlates with a colony count
of = 105 CFU/ml.
i. Record relative amounts of observed cells and
microorganisms. Commonly used quantitation
systems include the following.
1. Numerical
a. 1+ (<1 per oil immersion field, 100x)
b. 2+ (1 per oil immersion field)
c. 3+ (2 to 10 per oil immersion field)
d. 4+ (predominant or > 10 per oil immersion field)
2. Descriptive
a. Rare (<1 per oil immersion field)
b. Few (1 to 5 per oil immersion field)
c. Moderate (5 to 10 per oil immersion field)
d. Many (>10 per oil immersion field)
ii. Record the morphology of observed bacteria
VALUE OF DIRECT SMEARS
Direct Gram stain smears guides the physician on
the initial choice of antibiotics, pending the results
of culture and sensitivity, Judges Specimen quality,
Contributes to the selection of culture media, especially
with mixed flora and provides internal quality control
when direct smear results are compared to culture
results.[15]
173
FURTHER CONSIDERATIONS IN GRAM STAIN
A. Use results of Gram stains in conjunction with other
clinical and laboratory findings. Use additional
procedures (e.g., special stains, direct antigen tests,
inclusion of selective media, etc.) to confirm findings
suggested by Gram-stained smears.
B. Careful adherence to procedure and interpretative
criteria is required for accurate results. Accuracy is highly
dependent on the training and skill of microscopists.
C. Additional staining procedures are recommended for
purulent clinical specimens in which no organisms
are observed by the Gram stain method.
D. Gram stain-positive, culture-negative specimens
may be the result of contamination of specimen
with normal commensals, or artefacts from staining
reagents, Prior use of antimicrobial agents, or failure
of organisms to grow under usual culture conditions
(media, atmosphere, etc.)
SUMMARY
The differentiation of bacteria into either the gram-
positive or the gram-negative group is fundamental
to most bacterial identification systems. This task is
usually accomplished through the use of Gram’s staining
method. Incidentally, the Gram stain is a deceptively
simple procedure. Staining can be performed quickly and
easily but preparation and interpretation of the smear
requires considerable experience and training and can
therefore be prone to errors. The use of SOPs and Quality
controls are important in the performance of the test.
Reporting Gram stain results of direct smears are more
reliable and useful when samples are from sterile sites
and may guide the selection of the appropriate initial
antibiotic before the results of culture identification and
susceptibility testing become available especially for
infections like sepsis and meningitis where a delay in the
institution of appropriate initial antibiotic results in high
mortality. Identification of bacteria such as Acinetobacter
spp, Nocardia spp and Actinomycetes spp which can be
Gram variable remains a challenging problem The
quality of the results and confidence in the reports will
improve with the much needed interaction between the
Laboratory scientists who generates the results and the
Pathologist who interpretes the results in the light of the
patients clinical condition with the selection of the most
appropriate antibiotic for the management of the patient.
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How to cite this article: Thairu Y, Nasir IA, Usman Y. Laboratory perspective
of gram staining and its significance in investigations of infectious diseases.
Sub-Saharan Afr J Med 2014;1:168-74.
Source of Support: Nil, Conflict of Interest: None declared.
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