Semiloka Mutu 2017

Jakarta, 5-7 Mei 2017

STANDARD MICROBIOLOGY REPORT FOR GRAM STAIN

Adhi Kristianto Sugianli

Department of Clinical Pathology, Faculty of Medicine Universitas Padjadjaran,
Dr. Hasan Sadikin General Hospital, Bandung - Indonesia

Abstract

Effective utilization and understanding of the clinical bacteriology laboratory can greatly
aid in the diagnosis of infectious diseases. The Gram stain is the most frequently used
rapid diagnostic test, and in conjunction with various biochemical tests as the cornerstone
of the clinical laboratory. The implication of Gram stain smears guides the physician on
the initial choice of antibiotics (empirical). Therefore, it is clinically important to have a
standardized schema for assigning quantities of cells and bacteria. Several methods
have been developed to quantify the Gram stain. The criterion included is number of
microorganism, PMNs and epithelial cells. Those of parameter are quantified using grade
system (1+, 2+, 3+, 4+). Although international standard has not yet well developed and
implemented for Gram stain report, few study has successfully showed the important of
standard report. Therefore, this will enhance the accurate result of Gram stain, as well as
gives impact to the quality of patient care.

Keywords: Gram stain, standard report, quantitative grade

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Semiloka Mutu 2017
Jakarta, 5-7 Mei 2017

STANDARD MICROBIOLOGY REPORT FOR GRAM STAIN

Adhi Kristianto Sugianli

Department of Clinical Pathology, Faculty of Medicine Universitas
Padjadjaran, Dr. Hasan Sadikin General Hospital, Bandung - Indonesia

1. INTRODUCTION
Effective utilization and understanding of the clinical bacteriology
laboratory can greatly aid in the diagnosis of infectious diseases. The
Gram stain remains the most frequently used rapid diagnostic test as the
cornerstone in the clinical bacteriology and most useful in conjunction with
various biochemical tests. Although it was described more than a century
ago as first described by Danish pathologist Christian Gram in 1884 and
later slightly modified, the Gram stain easily divides bacteria into two
groups, Gram-positive and Gram-negative, on the basis of their cell wall
and cell membrane permeability (1).
The useful of Gram stain smears is it guides the physician on the initial
choice of antibiotics (empirical treatment); judges the quality of specimen
in conjunction with culture result; 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 (1, 2).
Despite the clinical importance of the Gram stain, there are few available
standards for the reading and interpretation of this test. The assignment of
semi-quantitative and quantitative values to the number of cells and
bacteria seen is clearly arbitrary, since published criteria for general use
vary dramatically. Although all aspects of Gram stain interpretive criteria
need to be standardized for all specimen types, the lack of standard semi-
quantitative and quantitative criteria for the analysis of cells and bacteria is
particularly problematic (3).
Therefore, it is clinically important to have a standardized schema for
assigning quantities of cells and bacteria to individual semi-quantitative

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scores. Since several clinical studies have shown that the presence of
moderate to heavy amounts of pus and/or the presence of bacteria on
Gram stains correlates with the presence of infection, information reported
from specimen Gram stains needs to be accurate (1, 3).

2. QUANTITATIVE GRADE AS STANDARD REPORT

Several grading or classification systems for the Gram stain have been
derived to aid the technologist in arriving at decisions relating to culturing
the specimen or interpretation of growth from culture of a specimen. Most
evaluations are aimed especially at specimens such as sputum, for which
collection is complicated by contamination with throat and mouth oral
because culture alone can be misleading. The objective is to separate the
representative sample from the contaminated sample before culture or
culture evaluation. The Bartlett’s method and Murray-Washington method
was the first easiest method for screening the quality of specimen. The
Bartlett’s method for scoring sputum and the Murray-Washington method
for contamination assessment document the association of 10 to 20 squa-
mous epithelial cells (SECs) per 10x microscopic field with unacceptable
specimens and 10 to 25 PMNs per 10x microscopic field with significant
specimens [Figure 1] (2, 4).
Many diagnostic microbiology laboratories use the documented
observations related to SECs and PMNs. The proven document for
observation related to SECs and PMNs was the Clinical Microbiology
Proficiency-Testing [CMPT] program, University of British Columbia,
Vancouver, British Columbia, Canada that encourages laboratories to
report both semi-quantitative and quantitative criteria on Gram stain
surveys for cells and bacteria. This proficiency-testing program surveyed
all microbiology laboratories regarding the interpretative criteria being
routinely used to read Gram-stained smears. Participating laboratories
were requested to provide a copy of their laboratory’s criteria for Gram
stain interpretation of various elements which included definitions for both
semi-quantitative (e.g. grades 1, 2, 3, and 4) and quantitative reporting of

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Jakarta, 5-7 Mei 2017

cells (e.g. numbers of PMN and epithelial cells per oil immersion field) and
bacteria when 10 or more oil immersion fields are read (oil immersion field
of 1,000). Nevertheless, the data finding showed several participant do not
consistent with the quantitative criteria specified by the CMPT program
with vary explanation. Therefore, diagnostic microbiology laboratories
should ensure that the Gram stain smears from clinical specimens is being
consistently interpreted by using common interpretive criteria in-house for
grading the presence of cells and bacteria. In fact, many physicians
clinically rely in many cases on an initial Gram stain report to confirm the
presence of infection by showing the presence of pus and bacteria in
submitted clinical specimens. Furthermore, the results of the initial Gram
stain should also be correlated to the culture results from individual clinical
specimens to ensure that all of the bacteria of interest found in the smear
are recovered. Accurate interpretation of Gram stains is therefore a critical
VOL. 38, 2000
performance parameter that should be monitored by the laboratory’s
internal and external quality assurance programs (2, 3)
TABLE 1. CMPT program’s recommended Gram stain performance
reporting criteriaa oratory’s inte
No. per oil immersion field
However, t
($1000) dardized Gra
Grade Description
cepted and w
Cells Bacteria
our province
1! Rare "1 "1 particular sch
2! Few 1–5 2–10 creasing tran
3! Moderate 6–10 11–50 has made it
4! Many #10 #50 dures across
a
Based on the reading of more than 10 fields.
the province
located in dif
Table 1. CMPT program’s recommended Gram stain reporting criteria
lem for medi
recommendations of our external proficiency program. Data transferred fr
Adoptedinto
were entered from aChruch (3).
spreadsheet program, Microsoft Excel used to educa
version 6.0, and analyzed using standard descriptive methods. not using sta
A total of 28 of 32 (87.5%) laboratories completed and addition, an
Another method attempted to coordinate observations related to
returned the Gram stain survey. Two laboratories that do not nologists to e
routinely materials,
background perform Gramwhich stains
consistdid
of not
localparticipate
materials, in the sur-
contaminating terpretation
vey. There was a wide variability in the Gram stain
materials, and purulence, and to describe the relationship of reporting Despite th
criteria used to grade both cells and bacteria. Many laborato- currently on
microorganisms to these background materials. The body site of the
ries use semiquantitative criteria (grades 1!, 2!, 3!, and 4!) numbers of c
ratherand
sample than
the quantitative
classification criteria for reporting
of the smear Gram stain
together determine the re-
extent ated for inte
sults. Gram stain s
Table 2 outlines the interpretive criteria for reporting cells nosis have b
and bacteria used by all of the laboratories
4 that completed the scoring criter
survey. There was better agreement among laboratories in criteria for t
interpreting the number of cells than the number of bacteria gonorrhoeae
 direct Gram-stained sputum samples. (Box 1-4). Using this
system, negative numbers are assigned to a smear when squa-
mous epithelial cells are observed. indicating contamination photomicrographs of Gram-stained sputum preparations il-
with oropharyngeal secretions (saliva). Positive numbers are lustrating this grading system are shown in Image Plate 1-1.
Semiloka
assigned for the presenceMutu 2017of segmented neutrophlls. indicat- A similar grading system has been proposed by Murray
86
Jakarta,of
ing the presence 5-7 Mei 2017
active inflammation. The magnitude of and Washin.gton (Box1-5). The large number of epithelial
these negative and positive determinations depends on the cells in groups 1 to 4 of this system indicates contamination
relative numbers of epithelial cells and segmented neutro- with oropharyngeal secretions and invalidates the samples
(i.e., the specimen should be rejected). Only group 5 speci-
phils as shown in the outline of Bartlett's grading system. mens are considered clinicallyand
relevant. In atoclinical study,
of culture
A final score of o or less evaluation.
indicates either This lackmethod
of inflam-alsoVan seems 15
complicated lead
matory response or presence of significant salivary con- Scoyl recommended that sputum samples contain-
inconsistent
tamination. thus invalidating on the reporting
specimen.(2, 4).
Representative ing more than 25 neutrophils be accepted for culture even if
more than 10 epithelial cells are present (group 4). These sug-
gested criteria for evaluation of sputum have been evaluated
by applying them to matc.hed pairs of respiratory seaetion
obtained by expectoration and by transtracheal a
Bartlen•s Grading System for technique that bypasses the oropharyngeal flora.
Assessing the Quality of Sputum The sputum grading system cannot be employed if pul-
Samples monary infections caused by mycobacteria, fungi. Legionella
spp.• and viruses are suspected. Additionally, the signitlcance
No. of Ne•raphils per 1DX of PMNs is altered in a few situations: (1) when the patient
Law-Power Field Grade is neutropenic from disease or therapy. (2) when the patient
<10 0 does not mount an effective inflammatory response. and
(3) where a foreign body causes irritation to a mucosal sur-
11H5 +1
face. Neutropenia can result from an inherited deficiency or
>25 +2 because the Phases of the Diagnostic
inflammatory Cycle
cells or their 17
precursors are de-
P111sence of mucus +1 stroyed, either by a disease process or by chemotherapy for a
disease. In some conditions. the ability of neutrophil.s to mi-
No. ofthese
bacteria are present. With Epilhalial
cluesCalls .-r 10X
in hand. the cli- grate to the site of an infection is impaired. From the infor-
nician can make moreLaw-Power Field
rational decisions about initial mation ordinarily supplied by clinicians, it is rarely possible
Murray
for theand Washington's
clinical microbiologist to Grading
determine whether a patient
antimicrobial therapy.11H5 -1
System
is neutropenic. One of the e:u::eptions isof
for Assessing the Quality the incompletely un-
In the past, the examination of >25 wet mounts of unstained ma- -2 Sputum
derstood Samples
deficiency in mobilization of neutrophils that is
terials by phase contrast or darkfield
Tot!I' microscopy was used found in infancy.:w The exact age at which a full neutrophilic
for demonstrating motility. spirochetes and endospores. but response can Epithelial
be mounted is not Leukourtes
clearly defined, but in the
this in uncommon performed "Average for the number of epithelial cella and neutrophila in
today. Giemsas. Wright's. or very youngestPower Cells Per
children Luw-
field
Per
(less than 2Law-
Power months
field of age) decisions
about 20 or 30 separate lOX microscopic fields is determined: to reject specimens or evaluate isolates incompletely should
acridine orange (AO) stains then
maya be totalhelpful in observing
is calculated. A final scorebac-
of 0 or less indicates Group
not 1be based on 25 the absence of10 neutrophils in the spec.imen.
terial forms that stain poorlylackorofthat have little contrast with
active inflammation or Cllltamination with saliva. Unsat-
background material isfactory specimens should be rejected, the provider should be Group2 The two situations
25 in whic.h foreign bodies modify the
notified and
Direct Grams stains of clinical a repeat sputum
material may alsospecimen should be reqUNted, if
be used interpretations associated with the presence of neutrophils.
Group3
These are when 25an endotrac.heal 25catheter is in place the lower
clinically indicated. The denotation
to determine whether a spec.imen is representative of the site of "if clinically indicated• is
important because if a message such as ·Repeat sruum respiratory tract and when there
Group4 25 is an indwelling catheter in
of infection. This techniqueture•
hasisbeen applied
given, then this mavtobethe evalua-
performed, even if the clinicallv the lower urinary tract, such as a Foley catheter. In each of
tion of sputum samples. Bartlett
findings has
of thedevised a grading
patient do not wanant repeat sys-testing. Group5 <10 25
these situations, the presence of neutrophils in a specimen
tem for evaluating sputum samples for the relative number may reflect irritation from the catheter rather than from an
of squamous epithelial cells and segmented
Figure 1. Bartlett’sneutrophilsGrading in System and Murray & Washington’s Grading
direct Gram-stained sputum samples. (Box 1-4).6 Using this
system, negative numbers are assigned toSystem.
a smear when squa- from Koneman (4).
Adopted
mous epithelial cells are observed. indicating contamination photomicrographs of Gram-stained sputum preparations il-
with oropharyngeal secretions (saliva). Positive numbers are lustrating this grading system are shown in Image Plate 1-1.
assigned for the presence of segmented neutrophlls. indicat- A similar grading system has been proposed by Murray
86
ing the presence of active 2.1 GRAM STAIN
inflammation. REPORT
The magnitude and3,
of (1, Washin.gton
5-7) (Box1-5). The large number of epithelial
these negative and positive determinations depends on the cells in groups 1 to 4 of this system indicates contamination
Despite the clinical with
importance
relative numbers of epithelial cells and segmented neutro- of oropharyngeal
the Gram secretions
stain, and are
there invalidates the samples
currently only
(i.e., the specimen should be rejected). Only group 5 speci-
phils as shown in the outline of Bartlett's grading system.
A final score of o or lessaindicates
few published
either lackquantitative criteria
mens are for the numbers
considered of cellsInand
clinically relevant. bacteria
a clinical study,
of inflam- Van Scoyl 15
recommended that sputum samples contain-
matory response or presence of significant salivary con-
that have been clinically evaluated
tamination. thus invalidating the specimen. Representative ing more thanfor interpretation of specific
25 neutrophils be accepted types
for culture ofif
even
more than 10 epithelial cells are present (group 4). These sug-
clinical specimens. International standards
gested criteria haveof sputum
for evaluation not developed and
have been evaluated
by applying them to matc.hed pairs of respiratory seaetion
implemented yet for Gram obtained stain interpretation
by expectoration and of by alltranstracheal
types of clinical a
Bartlen•s Grading System for technique thatdescriptive
bypasses the oropharyngeal
Assessing thespecimens. No standards exist
Quality of Sputum for the naming of flora. cellular and
The sputum grading system cannot be employed if pul-
Samples bacterial morphotypes from monary Gram infections
stains. caused
Several by mycobacteria,
other aspects fungi. Legionella
of the
spp.• and viruses are suspected. Additionally, the signitlcance
No. of Ne•raphils per 1DX
interpretation and reporting of Gramis stains
of PMNs altered inalso
a fewneed to (1)
situations: be when
addressed.
the patient
Law-Power Field Grade is neutropenic from disease or therapy. (2) when the patient
<10 Therefore, article 0 and literature
does summaries
not mount an theeffective
important step forresponse.
inflammatory standard and
(3) where a foreign body causes irritation to a mucosal sur-
11H5 report Gram stain +1 mentioned as bellow.
face. Neutropenia can result from an inherited deficiency or
>25 +2 because the inflammatory cells or their precursors are de-
P111sence of mucus +1 stroyed, either by a disease process or by chemotherapy for a
disease. In some conditions. the ability of neutrophil.s to mi-
No. of Epilhalial Calls .-r 10X grate to the site of an infection is impaired. From the infor-
Law-Power Field 5
mation ordinarily supplied by clinicians, it is rarely possible
11H5 -1 for the clinical microbiologist to determine whether a patient
is neutropenic. One of the e:u::eptions is the incompletely un-
>25 -2 derstood deficiency in mobilization of neutrophils that is
Semiloka Mutu 2017
Jakarta, 5-7 Mei 2017

A. Direct Smear
1. Evaluate the general nature of the smear under low-power
magnification (10X)
a. Determine if smear has been properly decolorized. Depending on
the source of the specimen, the background should be generally
clear or gram negative.
b. Examine the slide for cells including epithelial, red blood cells and
white blood cells. Red blood cells may stain faintly. White blood
cells should appear as light pink cells with a dark pink or red
nucleus (appear completely gram negative). White blood cells may
be differentiated into PMNs and mononuclear cells. No further
differentiation of white blood cells should be attempted using the
Gram stain.
c. Determine if thickness of smear is appropriate. For proper
interpretation, areas must be no more than one cell thick with no
overlapping of cells.
d. Examine smears prepared from clinical specimens under low power
for evidence of inflammation. If appropriate for culture source, note
the following:
a. Relative amounts of PMNs, mononuclear cells, and RBCs
b. Relative amounts of squamous epithelial cells, bacteria
consistent with normal microorganism, which may indicate
an improperly collected specimen
c. Location, shape and arrangements of microorganisms (in
general observation)

2. Examine several fields of the smear under oil immersion for the
presence of microorganisms (suggested 20 – 40 field)
a. If no microorganisms are seen in a smear of a clinical specimen,
report “No microorganisms seen.”
b. If microorganisms are seen, report relative numbers and describe

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morphology.

3. Examine the slide for microorganism characteristic morphologies and

arrangements including gram-positive versus gram-negative, cocci,
bacilli, spirochetes, curved-rods, large or small in singlets, pairs,
clusters, chains, or diplococci. Indicate pleomorphic, coccobacillary or
diphtheroids if applicable. In other word, the detail as bellow:
a. Observe predominant shapes of microorganisms overall shape:
coccus, coccoid, coccobacillary, bacilli, filamentous, yeast-like
b. 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.
c. Appearance of sides: parallel, ovoid (bulging), concave, irregular
d. Nature of axis: straight, curved, spiral
e. 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).
f. Branching or cellular extensions

4. If bacterial spores are present, indicate cellular location such as

terminal or subterminal and shape such as oval or round. Spores may
occasionally be seen in certain Gram-positive rods. Spores do not stain
with Gram stain reagents but will appear as clear areas within the cells.

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5. Quantitate organisms as follows:

• Bailey & Scott’s, 2014 (6):

Many 4+ 10 to 20 per oil immersion field

Moderate 3+ 6 to 10 per oil immersion field
Few 2+ 3 to 5 per oil immersion field
Rare 1+ Fewer than 10 identified on complete smear
None

• Hashimoto, 2013 (8):

Many 4+ More than 30 bacteria per field

Moderate 3+ 6–30 bacteria per field
Few 2+ 1–5 bacteria per field
Rare 1+ Less than one bacterium per field
None 0 No bacteria

• Thomson, 2013 (5):

Many 4+ More than 25 in one field

Moderate 3+ More than 1/field but less than 25/field
Few 2+ More than 10 in all field but less than 1/field
Very Rare 1+ Less than 10 in all fields examined

5. Quantitate cells (WBCs, RBCs, and epithelial) as follows:

Many 4+ 25 or greater per low-power field

Moderate 3+ 10 to 25 per low-power field
Few 2+ 2 to 10 per low-power field
Rare 1+ Fewer than 2 per low-power field
None

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B. Indirect Smear
Gram stain also plays a key role in the identification of bacteria grown in
culture. Report the Gram stain organism’s cellular shape, morphology, and
Gram reaction.

2.2 QUALITY CONTROL OF GRAM STAIN (1, 2, 6)

There are several steps for quality control (QC) for Gram stain. First, the
suggested strain for Gram stain is using Staphylococcus aureus (Gram-
positive) and Escherichia coli (Gram-negative). Second, the quality of
Gram stain can be monitored using the results of direct smear examination
and the adequacy culture interpretation, which should be an ongoing work
activity. Correlation between the two results should be made for each
patient. Explanations for discrepant results should be sought within the
work material. This repeated inspection of results enables each observer
to practice self-education and improve observation skills. Review of these
QC activities allows corrections to be made in specimen collection,
specimen management, and culture management.

2.3 PITFALLS OF GRAM STAIN (1, 2, 6)

1. Over-decolorization may result in the identification of false gram-
negative results, whereas under-decolorization may result in the
identification of false gram-positive results.
2. Smears that are too thick or viscous may retain too much primary
stain, making identification of proper Gram stain reactions difficult.
Especially, the Gram-negative organisms may not decolorize properly.
3. Cultures older than 16 to 18 hours will contain living and dead cells.
Cells that are dead will be deteriorating and will not retain the stain
properly.
4. Stain may form precipitate with aging. Filtering through gauze will
remove excess crystals. e.g. interpreting thin crystal/gentian violet
precipitate needles for gram-positive bacillus-shaped bacteria.

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5. Gram stains from patients on antibiotics or antimicrobial therapy may

have altered Gram stain reactivity due to the successful treatment.
6. Occasionally, pneumococci identified in the lower respiratory tract on a
direct smear will not grow in culture. Some strains are obligate
anaerobes.
7. Toxin-producing organisms such as Clostridia, staphylococci, and
streptococci may destroy white blood cells within a purulent specimen.
8. Faintly staining Gram-negative organisms, such as Campylobacter
and Brucella, may be visualized using an alternative counterstain
(e.g., basic fuchsin).

3. SUMMARY
The Gram stain is the most frequently used rapid diagnostic test as the
cornerstone in the clinical bacteriology. Standards of reporting and
interpretation should be developed and implemented for Gram stain of all
types of clinical specimens. Therefore, this will enhance the accurate
result of Gram stain, as well as gives impact to the quality of patient care.

REFERENCE
1. Thairu Y, Nasir IA, Usman Y. Laboratory Perspective of Gram Staining
and its Signi cance in Investigations of Infectious Diseases. Sub-
Saharan Afr J Med. 2014;1(4):168-74.
2. Ayers LW. Microscopic Examination of Infected Materials. In: Mahon
CR, Lehman DC, Manuselis G, editors. Textbook of Diagnostic
Microbiology. 4 ed. Maryland Heights, Missouri: W.B. Saunders
Company, Elsevier; 2011.
3. Church D, Melnyk E, Unger B. Quantitative Gram Stain Interpretation
Criteria Used by Microbiology Laboratories in Alberta, Canada. J Clin
Microbiol. 2000;38(11):4266–68.

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4. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW,
Schreckenberger PC, et al., editors. Koneman's Color Atlas and
Textbook of Diagnostic Microbiology. 7 ed. Philadelphia: Wolters
Kluwer Health; 2017.
5. Thomson R. Making the Most of Gram Stain Interpretations 2013
[cited 2017 March 30]. Available from:
http://www.scacm.org/free/Gram Stain SCACM Workshop 2013.pdf.
6. Tille PM, editor. Bailey & Scott’s Diagnostic Microbiology. 13 ed. St.
Louis, Missouri: Mosby Elsevier; 2014.
7. Samuel LP, Balada-Llasat J-M, Harrington A, Cavagnolod R.
Multicenter Assessment of Gram Stain Error Rates. J Clin Microbiol.
2016;54(6):1442-47.
8. Hashimoto S, Shime N. Evaluation of semi-quantitative scoring of
Gram staining or semi-quantitative culture for the diagnosis of
ventilator-associated pneumonia: a retrospective comparison with
quantitative culture. J Intensive Care. 2013;1(1):2.

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