Microbiology and Infectious Disease / ERRORS IN GRAM STAIN INTERPRETATION

Errors in Interpretation of Gram Stains From Positive

Blood Cultures
Kenneth H. Rand, MD,1 and Maria Tillan, MD2*
Key Words: Gram stain; Misread Gram stain; Gram positive; Gram negative; Acinetobacter; Bacillus
DOI: 10.1309/V4KE2FPM5T8V4552

Abstract
We reviewed major errors in Gram stain reports
from positive blood cultures to identify patterns and
potential clinical impact. During a 23-month period,
blood cultures were misread for 57 (0.7%) of 8,253
patients. Of 5,885 read as gram-positive cocci, 6 (0.1%)
had only gram-negative organisms by culture, 3 of
which were Acinetobacter species. Of 1,959 read as
gram-negative bacilli, 25 (1.3%) had only grampositive organisms by culture. Of these, 9 were Bacillus
and 2 were Clostridium species. Nonrecognition of
mixed Gram stains accounted for 28 errors that most
often were associated with a reading of gram-positive
cocci. In 4 cases, there were delays of 14 hours to 3
days in starting appropriate antibiotic treatment; 2
deaths occurred, although the erroneous Gram stain
report probably was not contributory. Pathologists and
laboratory personnel need to be aware of these types of
misinterpretations and the potential effects on patient
outcome.

The Gram stain may be the oldest and most entrenched

technique still in use in the microbiology laboratory.
Because of its essential simplicity and its widespread familiarity, physicians almost never question its accuracy. Yet, no
laboratory test in existence is 100% accurate, and the Gram
stain is no exception owing to human interpretive error and
the exigencies of the staining properties of certain bacteria.
For example, it is well recognized that Bacillus species and
certain other gram-positive species often stain gram-negative
or gram-variable as cultures age because of cell wall changes
with loss of viability.1
Microbiologists are familiar with many of the problematic areas of the Gram stain, such as underdecolorization and
overdecolorization and which species are likely to exhibit
interpretive issues. However, we were unable to find any systematic studies in the literature of the incidence or clinical
impact of misinterpretation of Gram stains. We reviewed the
initial Gram stain readings for all blood cultures reported
during a 23-month period, January 1, 2002, through
November 30, 2003, because such errors could lead directly
to the choice of an incorrect antibiotic or discontinuation of
an appropriate one.

Materials and Methods

Blood cultures were performed with the Bactec 9240
using the Bactec Plus Aerobic/F Medium, the Bactec lytic/10
anaerobic/F medium, and the Bactec Peds Plus/F medium
(BD Diagnostic Systems, Sparks, MD). All positive blood cultures were manually recorded in a log book in the Shands
Hospital at the University of Florida Clinical Microbiology
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Laboratory (Gainesville). This record included the date,

time, Gram stain result, and person to whom the stat result
was called. When the culture identification was complete,
the final result was entered into the log book alongside the
original Gram stain reading. Both of us independently
reviewed all positive blood cultures obtained between
January 1, 2002, and November 30, 2003. All cases in which
the original Gram stain reading that was called to a care
provider was inconsistent with the known Gram stain characteristics of the organism(s) based on final culture identification were recorded. All misread Gram stains were found
within 24 to 48 hours when the organisms recovered from
the subculture plates did not match those described in the
initial Gram stain reading. In all cases, the errors were
reported as soon as they were recognized, corrected reports
were issued, and the proper procedures and interpretation
were reviewed with the technologist involved.
Because of the retrospective nature of the study, there was
no way to systematically determine whether the error was due
to an interpretive error, ie, different individuals would interpret the slide differently, or a clerical error, eg, inputting the
wrong comment into the computer. Technical errors such as
overdecolorization were not recorded systematically.
We focused on the types of errors that had the greatest
potential for patient harm, ie, errors in which a gram-negative
result was reported but the final identification was only grampositive or vice versa. We did not record cases in which grampositive bacilli were reported but only gram-positive cocci
ultimately were found (and likewise for the gram-negatives)
because such reports were less likely to lead to changes in
antibiotic coverage with serious harmful effects on outcome.
All cases were recorded in which only gram-positive cocci
were reported but gram-negative species (alone or in mixed
culture with a gram-positive organism) ultimately were identified by culture; only gram-positive bacilli were reported but
gram-negative species (alone or in mixed culture with a
gram-positive organism) ultimately were identified by culture;
and only gram-negative bacilli were reported but gram-positive species (alone or in mixed culture with a gram-negative

organism) ultimately were identified by culture. There were so

few blood cultures with gram-negative cocci that no interpretive errors were found. Yeasts were excluded from the analysis.
Gram Stain
Gram stain of positive blood cultures was performed as
described2 with reagents from Becton Dickinson, Sparks,
MD, according to the instructions from the manufacturer.
Briefly, slides were air dried, fixed with methanol, and
allowed to air dry on a slide warmer. Crystal violet was
applied for 45 to 60 seconds, slides were rinsed in tap water,
and Gram iodine was overlayed for 1 to 3 minutes.
Decolorization was performed with acetone:95% ethanol,
25:75 by volume for less than 10 seconds. Slides were counterstained with safranin for 40 to 60 seconds.
Outcome Studies
After obtaining approval from our institutional review
board for waiver of informed consent, the charts of patients
with misinterpreted initial Gram stains were reviewed.

Results
As shown in Table 1, the Gram stains of 57 (0.7%) of
8,253 positive blood cultures during the 23-month study period were misread. After omitting Bacillus species, which were
almost certainly contaminants, there were 46 (0.6%) of 8,253
misread Gram stains. Of the 46, 28 were mixed cultures in
which the second (or third) organisms were not recognized in
the initial Gram stain reading.
Data for the remaining 18 cases are given in Table 2.
Chart review revealed that there were no adverse consequences for 12 of 16 patients. For the remaining 4, there
were 14- to 72-hour delays in switching to appropriate
antibiotics. Two patients died of septic shock, but the delay
in reporting correct Gram stain results in case 4 was 14 hours
and in case 13 was 48 hours. Empiric gram-negative coverage was ineffective in the former case because slow growth

Table 1
Summary of Gram Stains of All Positive Blood Cultures*
Blood Culture Gram Stain

No. of Positive
Blood Cultures

No.
Misread

5,885
367
1,959
42
8,253

22 (0.4)
10 (2.7)
25 (1.3)
0 (0)
57 (0.7)

Gram-positive cocci
Gram-positive rods
Gram-negative rods
Gram-negative cocci
Total

No. of Major
Errors

No. of Mixed
Cultures

6 (0.1)
7 (1.9)
5 (0.36)
0 (0)
18 (0.2)

16 (0.3)
3 (0.8)
9 (0.5)
0 (0)
28 (0.3)

No. of Bacillus
Species

11 (0.6)

11 (0.1)

Data are given as number (percentage).

major error was defined as a single organism whose Gram stain result was opposite that found in culture results.
See Discussion, last sentence of first paragraph.
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of the organism delayed identification (Capnocytophaga),

whereas in the latter case, appropriate treatment failed even
after susceptibility information was available.
Two systematic errors were observed. First, in 11 cases,
Bacillus species were read as gram-negative bacilli. The case
shown in Image 1 illustrates well how gram-negative

Bacillus species can sometimes appear. Second, in 5 of 13

cases in which Gram stains were read as only gram-positive
cocci or gram-positive bacilli, an Acinetobacter species was
identified as the sole organism (4 cases) or with another gramnegative bacillus (1 case). As shown in Image 2,
Acinetobacter can appear unmistakably as gram-positive. The

Table 2
Patient Characteristics, Underlying Diagnoses, Interpretive Error, and Outcome of Misread Gram Stains
Case No./
Sex/Age(y)

Diagnosis

Original Gram
Stain Result

Drug at Time of
Antibiotic Change
Final
Erroneous Gram Stain
Based on Erroneous
Culture Result
Interpretation
Gram Stain Interpretation

1/F/75

Diverticulitis with
sepsis

Gram+ rods

Bacteroides sp;
Escherichia coli

2/F/78

Urosepsis, CAP

Gram+ rods

3/F/48

Endocarditis

Gram+ rods

4/M/45

Neutropenic fever,
mucositis

Gram+ rods

Klebsiella
pneumoniae
Acinetobacter
lwoffi; Enterobacter sp
Capnocytophaga

5/M/54

Respiratory failure

Gram+ rods

6/M/73

Diabetes, chronic
bronchitis
Line sepsis

Gram+ rods

7/F/38

Adverse
Effect

Outcome

Ticarcillin disodium/
clavulanate
potassium
Ceftriaxone,
azithromycin
Not available

Ciprofloxacin;
metronidazole
added
None

None

Survived

None

Survived

Not available

Probably none

Survived

Ciprofloxacin,
vancomycin

None

Haemophilus
influenzae
E coli

Trimethoprim
sulfamethoxazole
Not available

14-h delay until

Died; septic shock
another bottle
13 d after blood
positive for gram culture drawn,
rods and cefepime despite imipenem
and gentamicin
for 5 d begun 4 d
started
after culture first
known to be positive
None
Survived

None; thought to
be a contaminant
Not available
Not available

Gram+ rods

Acinetobacter
baumannii

Vancomycin

Gram rods

Enterococcus
faecalis

Discontinued line; None

discontinued
vancomycin
Ceftriaxone,
None; EnteroNone
azithromycin
coccus thought
to be a contaminant
Cefepime, piperacillin Gentamicin added 3-d delay in
sodium/tazostarting
bactam sodium
metronidazole
Vancomycin,
None
None
ampicillin
Vancomycin
Gatifloxacin added None

Survived
Survived

8/F/60

CAP, heart failure,

COPD, diabetes

9/F/3

Stage IV neuroGram rods

blastoma, neutropenic fever, mucositis
Fever; subarachnoid Gram rods
bleeding
Urosepsis,
Gram rods
lymphoma
Fever, infected
Gram rods
decubitus, ESRD
ESRD, renal trans- Gram+ cocci
plant, diabetes,
fever, cellulitis vs.
Sweet syndrome

Clostridium
clostridioforme

Abdominal abscess/ Gram+ cocci

sepsis bladder
cancer, post bilateral
nephrectomy
Myelodysplastic
Gram+ cocci
syndrome,
fever sepsis
Fever, sepsis,
Gram+ cocci
pneumatosis coli,
35%-40% burn
Fever, MVC,
Gram+ cocci
multiple trauma
Ruptured appendix Gram+ cocci
and pelvic abscess

Bacillus fragilis

Imipenem,
amikacin,
vancomycin

None

None

A baumannii

Gatifloxacin

Vancomycin

Survived

K pneumoniae

Imipenem

None

24-h delay in
starting
cefepime
None

A baumannii

Cefazolin,
gentamicin
Amoxicillin/clavulanate potassium

Vancomycin

None

Survived

None

None

Survived

10/F/52
11/M/58
12/F/78
13/F/51

14/M/68

15/M/64

16/M/44

17/F/21
18/F/66

Corynebacterium
Clostridium sp

Staphylococcus
Ertapenem
coagulase-negative
A baumannii
Vancomycin

Bacteroides sp

Vancomycin

None

Survived

Survived

Survived
Survived
Survived

Ciprofloxacin;
36- to 48-h delay
trimethoprim
in starting
sulfamethoxazole imipenem
added

Died; sepsis, multiorgan failure, 30%

body surface
bullous lesions,
bullous fluid
positive for ciprofloxacin-resistant
Acinetobacter
Survived

Survived

CAP, community-acquired pneumonia; COPD, chronic obstructive pulmonary disease; ESRD, end-stage renal disease; MVC, motor vehicle crash.

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Image 1 Bacillus species staining gram-negative from a

positive blood culture. Although this smear could be
interpreted as gram-variable, it is easy to understand how it
could be read as gram-negative (1,000).

Image 2 Acinetobacter staining gram-positive from a

positive blood culture (1,000).

morphologic features of the gram-positive Acinetobacter

organism were somewhat variable, most often recorded as
gram-positive cocci and less often as gram-positive cocci in
pairs or pairs and chains.
In 13 cases, mixed gram-positive and gram-negative cultures initially were read as gram-positive cocci only. The
gram-positive
organisms
were
coagulase-negative
Staphylococcus species (6 cases), Enterococcus species (5
cases), or both (2 cases). In these cases, the original Gram
stains were misread only in the sense that an unsuspected
organism of opposite staining characteristics also was present.
These second organisms may not have been present in high
enough quantities to be visualized on Gram stain, but the
results of restaining or reviewing the original stain after culture results became available were not recorded systematically. The following gram-negative organisms were found by
subculture: Acinetobacter species, 3; Pseudomonas aeruginosa, 3; Enterobacter aerogenes, 2; Serratia marcescens, 2;
and Klebsiella pneumoniae, 1. Chart review revealed 4
patients with a clinically recognized abdominal focus of infection or illness, and all had infections with Enterococcus
species and an enteric gram-negative organism. In the other
cases, the unrecognized gram-negative organism was related
to another known site of infection (line infection, 2; sternal
wound infection, 1) or had no obvious source for the gramnegative organism (4 cases). Two charts were unavailable.
In 14 cases, there were only gram-negative bacilli in the
initial Gram stain reading but final cultures grew gram-negative
and gram-positive organisms. In 8 of these cases, coagulase-

negative staphylococci were found later by culture and were

not clinically significant. Two patients had Streptococcus
viridans that was not considered pathogenic in 1 case but
was recovered from peritoneal fluid along with Serratia
species, which were the gram-negative organisms in the
blood culture. In this case, infection with the same organisms recurred 3 months later. One patient had a
Staphylococcus aureus infection in addition to the gram-negative P aeruginosa, and both organisms were considered
pathogenic in a catheter-related bacteremia. One patient had
a Streptococcus pneumoniae infection, but the chart could
not be obtained to determine the clinical relevance, and the
remaining 2 patients had infections with Lactobacillus and
Bacillus species that were not pathogens. The last patient
had a culture read as gram-positive bacilli but was found to
have infection with Clostridium and Bacteroides species by
culture. This patient had a small bowel perforation repaired
2 days after the blood culture was obtained.

Discussion
It is believed that bacterial cells stain gram-positive
because the thickness of their cell wall peptidoglycan layer prevents the elution of the crystal violetiodine mordant insoluble
complex in an organic solvent.1,3 In gram-negative cells, the
complex diffuses out because of the thin peptidoglycan layer
together with damage to the cell wall by the decolorizing solvent, leaving the cells visualized by the safranin counterstain.
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Bacillus and Clostridium species typically are 95% to

100% gram-positive early in their growth phase in broth cultures but become 40% to 50% gram-negative in the late
growth phase and 90% to 95% gram-negative in the stationary phase.1 Although one might expect that blood cultures
growing Bacillus species would be in mid growth phase and,
thus, gram-positive, the loss of Gram stain retention associated with aging of the culture most likely accounts for the frequency of misreading for these species. Even so, only 11
(7.6%) of 144 blood culture bottles that grew Bacillus species
during the study period were read as gram-negative instead of
gram-positive.
In contrast with the loss of positive Gram stain through
overdecolorization or the effects of growth phase on the cell
wall of gram-positive organisms, Acinetobacter has been
reported to stain gram-positive, despite proper Gram stain
technique,4 and was involved in 5 of our misread cases. In
addition, 3 of the mixed cultures read as only gram-positive
cocci had Acinetobacter in addition to a gram-positive coccus
in culture. During this 23-month period, there were 68 patient
blood cultures positive for Acinetobacter species, so 8 (12%)
of 68 Acinetobacter species in blood culture were read initially as gram-positive. No explanation is available in the literature for this phenomenon, although it is recognized among
microbiologists.5
Misread Gram stains from positive blood cultures should
be recognized by the laboratory in 18 to 36 hours following
the erroneous Gram stain report because at that time, there
should be sufficient growth on plates that the inconsistent
colony morphologic features or presence of a mixed culture
should be recognized. However, anaerobes and slow-growing
fastidious organisms, such as Capnocytophaga reported herein, may require an additional 24 hours before such errors
could be recognized.
Clinical microbiology laboratories must maintain a high
level of awareness for the possibility that blood culture Gram
stains can be misread. Not only can technical and interpretive errors occur but also, as demonstrated in this report, the
potential for certain organisms to stain opposite their characteristic manner must be kept in mind, eg, Acinetobacter,

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Clostridium, and Bacillus species. In 4 of our cases with the

morphologic features of enterococci (ie, gram-positive cocci
in pairs and chains) in the setting of a potential abdominal
source of infection, there also was an enteric gram-negative
rod found on culture.
The overall 0.7% error rate of misinterpreted Gram stains
from positive blood culture bottles is low, but laboratory professionals and pathologists must be aware of the potential
types of error that can occur.
From the Departments of 1Pathology and Immunology and
Laboratory Medicine, and 2Medicine, Division of Infectious
Disease, University of Florida, Gainesville.
Supported in part by the Departments of Pathology and
Immunology and Laboratory Medicine (Dr Rand) and the
Department of Medicine, Division of Infectious Disease (Drs Rand
and Tillan), University of Florida.
Presented in part at the 42nd meeting of the Infectious
Diseases Society of America, Boston, MA, September 2004.
Address reprint requests to Dr Rand: Dept of Pathology,
Immunology and Laboratory Medicine, University of Florida
College of Medicine, PO Box 100275, Gainesville, FL 32610.
* Dr Tillan is currently with the Division of Infectious
Diseases, University of Louisville, Louisville, KY.
Acknowledgment: We gratefully acknowledge the support of
the staff of the Shands Hospital at the University of Florida
Clinical Microbiology Laboratory.

References
1. Beveridge TJ. Mechanism of Gram variability in select
bacteria. J Bacteriol. 1990;172:1609-1620.
2. York MK. Gram stain. In: Isenberg HD, ed. Clinical
Microbiology Procedures Handbook. Washington, DC:
American Society of Microbiology; 2004.
3. Popescu A, Doyle RJ. The Gram stain after more than a
century. Biotech Histochem. 1996;71:145-151.
4. Harrington BJ, Plenzler M. Misleading Gram stain findings
on a smear from a cerebrospinal fluid specimen. Lab Med.
2004;35:475-478.
5. Schreckenberger PC, Daneshvar ME, Weyant RS, et al.
Acinetobacter, Achromobacter, Chryseobacterium, Moraxella,
and other nonfermentative gram-negative rods. In: Murray
PR, Baron EJ, Jorgensen JH, et al, eds. Manual of Clinical
Microbiology. 8th ed. Washington, DC: ASM Press; 2003.

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