JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2007, p. 37543758 Vol. 45, No.

11
0095-1137/07/$08.000 doi:10.1128/JCM.01632-07
Copyright 2007, American Society for Microbiology. All Rights Reserved.

NOTES
Mechanisms To Assess Gram Stain Interpretation Proficiency of
Technologists at Satellite Laboratories
Erik Munson,1,2* Timothy Block,1 Janice Basile,1 Jeanne E. Hryciuk,1 and Ronald F. Schell3,4,5
Wheaton Franciscan and Midwest Clinical Laboratories, Wauwatosa, Wisconsin 532261; College of Health Sciences, University of
WisconsinMilwaukee, Milwaukee, Wisconsin 532012; and Wisconsin State Laboratory of Hygiene3 and Departments of
Bacteriology4 and Medical Microbiology and Immunology,5 University of Wisconsin, Madison, Wisconsin 53706
Received 15 August 2007/Accepted 16 August 2007

Downloaded from http://jcm.asm.org/ on October 29, 2016 by guest

To address Gram stain interpretation proficiency in a satellite/centralized microbiology laboratory para-
digm, two programs were devised. In quality assurance program 1, nonmicrobiology technologists at satellite
laboratories were required to interpret standardized Gram-stained specimens of clinical material prepared by
an experienced microbiologist at a central laboratory. In quality assurance program 2, clinical Gram stains
prepared and read by the satellite laboratorians were reviewed by experienced microbiologists at the central
laboratory. Satisfactory performance (94%) was achieved in quality assurance program 1. In contrast, quality
assurance program 2 had a significantly lower overall performance (89%; P < 0.0001) due to poorer identi-
fication of host cells (93%) and bacteria (84%). A variety of intervention mechanisms, including continuous
monitoring, resulted in overall performance improvement (P < 0.006). While a technologist challenge has
educational merit, having a microbiologist review previously read slides is a better indicator of the technolo-
gists Gram stain interpretation proficiency.

Gram staining of primary clinical specimens has tremendous
clinical utility. Studies have shown that Gram staining of spu-
tum can be predictive for several etiologies of lower respiratory
tract disease (1, 10, 11, 15, 16, 17, 19, 23). Gram stain data also
influence the treatment of clinically significant bloodstream
infections (8, 13). In addition, the proper performance of the
primary Gram stain procedure can guide the processing of
expectorated sputum specimens (14) and aid in the interpre-
tation of cultured skin and soft tissue specimens (3, 26).
Bartlett (2) cited the virtual elimination of house staff
laboratories (as an indirect result of the Clinical Laboratory
Improvement Amendments of 1988) and the outsourcing of
primary specimens as two factors contributing to the decline in
clinical microbiology studies for patients with lower respiratory
tract disease. Others have raised concerns over the clinical
diagnostic value of the Gram stain in terms of protocol stan-
dardization (5, 7, 18). As a result, many have discouraged the
utilization of Gram-stained smears as predictors of certain
clinical infections, particularly those involving the lower respi-
ratory tract (10, 20, 21).
Technologist competency has become of even greater im-
portance with the centralization of microbiology testing and
the creation of satellite processing (rapid-response) laborato-
ries staffed by nontraditional microbiologists (6). Moreover,
accrediting agencies mandate the proficiency documentation
of all technologists who execute clinical Gram staining proto-
cols. In this report, we describe a mechanism for objectively
* Corresponding author. Mailing address: Midwest Clinical Laborato-
ries, 11020 West Plank Court, Suite 100, Wauwatosa, WI 53226. Phone:
(414) 256-1479. Fax: (414) 256-5566. E-mail: Erik.Munson@wfhc.org.
Published ahead of print on 29 August 2007.
assessing the Gram stain interpretation competency of nonmi-
crobiology laboratorians and present a quality assurance pro-
gram for monitoring and increasing the proficiency of Gram
staining.
Full-spectrum clinical laboratories were present at three
Milwaukee, Wisconsin, inpatient facilities until 1988, when two
hospitals (B and C) consolidated their clinical microbiology
laboratories into a freestanding building (a central laboratory).
Subsequently, the central laboratory assimilated the clinical
microbiology services of a third hospital (A) in 1999. Algo-
rithms were devised in which the primary clinical specimens for
routine bacteriology were processed onto appropriate media
(22) and smeared for Gram staining and interpretation (25) by
trained nonmicrobiology technologists at the satellite labora-
tories. Within 24 h, Gram-stained smears were forwarded to
the central laboratory for confirmation of results.
Three primary clinical specimens from a variety of sources
were smeared and Gram stained quarterly by an experienced
microbiologist at the central laboratory (quality assurance pro-
gram 1). The number of cells (viewed at 100 magnification
for eukaryotic cells and 1,000 magnification for prokaryotic
cells) was identified as rare (1 per field), few (1 to 5 per field),
moderate (5 to 20 per field), or abundant (20 per field).
Included within these primary clinical specimens were sputa to
be evaluated for culture processing acceptability, based on the
criteria of Murray and Washington (14). Seventy satellite lab-
oratory technologists were made aware of the specimen source
and were asked to provide Gram stain interpretation and semi-
quantitation of the eukaryotic and prokaryotic cells from the
challenge slides. The results were forwarded to the central
laboratory microbiologist. Furthermore, the central laboratory
microbiologists (on a rotating basis) daily selected a number of

3754
VOL. 45, 2007
FIG. 1. Gram stain interpretation proficiencies of three satellite laboratories, determined by quality assurance program 1 and delineated by
interpretation of bacteria (black bars) and host cells (white bars) and the sum of the two categories (shaded bars).
the satellite-laboratory-prepared smears that was commensu-
rate with the relative microbiology volume generated by that
hospital (quality assurance program 2). A descriptive Gram
stain report was documented by the microbiologist and for-
warded to the microbiology supervisor.
Reviews of the Gram-stained preparations in quality assur-
ance programs 1 and 2 were documented using the following
system: 1 point was given for each correct Gram stain reaction,
1 point for each bacterial morphology correctly identified, 1
point for each host cell (squamous epithelial or inflammatory)
morphology correctly identified, and 1 point for the quantity of
each host or bacterial cell type correctly identified. The accept-
able range for quantitation was 1 gradation of the expected
value. Credit for quantity, Gram reaction, or cell type involved
was not granted if a sputum specimen was rejected for culture;
the technologist received a slide value of 0. In addition, points
were subtracted from a score when additional cell types or
bacteria were erroneously reported. Finally, point values for
host cells (quantity plus presence/absence) and bacteria (quan-
tity plus Gram stain result and morphology) were summed per
preparation and expressed as a percentage of the expected
value. The standard error of the mean was calculated for all
measures of Gram stain interpretation proficiency testing.
Comparisons of interlaboratory performances, cell types, and
the two measures of Gram stain interpretation proficiency
testing were facilitated by the t test for independent samples.
Assessment of the temporal change (improvement) in labora-
tory performance was facilitated by one-way analysis of vari-
ance (24). The alpha level was set at 0.05 before investigations
commenced, and all P values were two-tailed.
The overall Gram stain interpretation proficiency rate for 70
satellite laboratory technologists, as determined by quality as-
surance program 1, was 94% (Fig. 1). However, host cells were
NOTES 3755
Downloaded from http://jcm.asm.org/ on October 29, 2016 by guest
interpreted with a higher success rate than bacteria (98% ver-
sus 89%; P 0.0001). When data from individual hospitals
were examined (Fig. 1), personnel at hospital C showed the
lowest proficiency for host cell recognition and identification of
bacteria. Upon the review of 2,517 randomly chosen Gram-
stained preparations by quality assurance program 2, no sig-
nificant differences in proficiency for the detection of host cells
or bacteria were found among the laboratorians of the three
satellite laboratories. Similar to the results for quality assur-
ance program 1, greater proficiency in interpretation of host
cells than in bacterial observations was demonstrated by sat-
ellite laboratorians (P 0.001).
When the results of the two quality assurance programs were
compared, the proficiency in interpretation of Gram stain re-
sults of quality assurance program 2 was 5.4% lower than that
of quality assurance program 1 (P 0.0001; Fig. 2). Analogous
differences were detected between the programs for identifi-
cation of bacteria and host cells (P 0.03). Upon delineation
of individual hospital data by cell type, the rates of proficiency
noted for quality assurance program 1 were generally higher
than those for quality assurance program 2 (P 0.04).
The implementation of quality assurance programs 1 and 2
caused a significant increase in Gram stain interpretation pro-
ficiency in the fourth quarter of 2006 (P 0.006; Fig. 3).
Improvements occurred in host cell and bacterial identification
among the participants of quality assurance program 1 (P
0.01) and in host cell identification among the participants of
quality assurance program 2 (P 0.03). Among the hospitals,
the greatest improvement occurred with hospital C (P 0.005;
Fig. 4A and B). The nonmicrobiology laboratorians at hospital
C performed consistently less skillfully than laboratorians at
hospitals A and B prior to the implementation of the quality
assurance programs.
3756 NOTES
FIG. 2. Comparison of quality assurance programs 1 and 2 for
measure of overall Gram stain interpretation proficiency.
FIG. 3. Gram stain interpretation proficiency of nonmicrobiology laboratorians as determined by quality assurance programs 1 (squares) and
2 (diamonds) for four quarters of 2006.
J. CLIN. MICROBIOL.
The diagnostic value of the Gram stain has been questioned
from a variety of perspectives. In a meta-analysis, Reed et al.
(21) reported Gram stain sensitivity of 15% to 100% for the
diagnosis of pneumococcal pneumonia, with specificity ranging
between 11% and 100%. In a regional proficiency testing pro-
gram, Church et al. (5) reported a significant lack of standard-
ization in reporting quantities of host cells and bacteria and
raised concerns about how this could impact patients over a
continuum of care. Cooper et al. (7) documented low inter-
technologist agreement rates for cell quantitation from Gram-
stained lower respiratory tract specimens. In a broad sense,
following a regional microbiology laboratory restructuring, a
number of subsequent proficiency challenges revealed that er-
ror rates increased in a number of laboratories, such as rural
facilities (noted to possess neither a technologist devoted to
Downloaded from http://jcm.asm.org/ on October 29, 2016 by guest
microbiology duties nor on-site pathologist or medical micro-
biologist oversight) that took on additional microbiology re-
sponsibilities (6). Combined with the clinical and bench-level
utility of the Gram stain procedure, the preceding factors pro-
vide an impetus for evaluating and maintaining the proficiency
of microbiologists and technologists who perform this process.
The central laboratory microbiologists who determined the
expected values for slides throughout these quality assurance
programs were subjected to in-house Gram stain interpreta-
tion proficiency testing. On a quarterly basis, all microbiolo-
gists reported findings for a randomly selected slide set. Data
were pooled, and the expected value for each slide was derived
from the majority of responses for each parameter. Unlike
quality assurance program 1, which granted credit for results
within 1 gradation of an expected value for quantitation,
in-house proficiency testing used higher stringency (i.e., only
one acceptable value for quantitation). The microbiologist who
prepared the proficiency material and graded the responses for
quality assurance program 1 was one of two microbiologists
VOL. 45, 2007
FIG. 4. Gram stain interpretation proficiency of hospital C nonmi-
crobiology laboratorians, measured at the onset and later stages of
quality assurance programs 1 (A) and 2 (B).
who performed at 100% of the expected value for in-house
proficiency testing during the time frame of this investigation.
The central laboratory microbiologists demonstrated a mean
in-house proficiency rate of 95%. No significant differences
existed between the average microbiologist proficiency score
and the mean demonstrated by the microbiology laboratory as
a whole (P 0.06). These data confirmed the validity of the
expected values.
Data from both quality assurance programs demonstrated
the competency of satellite laboratorians in the interpretation
of Gram-stained material. However, greater proficiency was
NOTES 3757
found in host cell analysis (93%) than in bacterial analysis
(84%). The rate of successful interpretation of host cells was
markedly higher than that observed in an intralaboratory study
(7). Cooper et al. (7) limited their study to lower respiratory
tract smears and exercised more stringency in their grading of
quantitative measures. However, the satellite technologists in
our study may have had more breadth of knowledge in hema-
tology and sterile body fluid analysis.
Quality assurance program 1 yielded higher rates of profi-
ciency than did quality assurance program 2. This was true for
each component of the monitoring and was realized at all three
satellite laboratories. While intralaboratory technologist col-
laboration may or may not have played a role in the increased
frequency of correct responses in quality assurance program 1,
one aspect that could have played a role was the nature of the
Downloaded from http://jcm.asm.org/ on October 29, 2016 by guest
Gram-stained smear itself. To ensure consistency in the survey,
specimens were preselected and smeared onto slides, with con-
tents stained, coverslipped, and previewed by the microbiolo-
gist at the central laboratory before presentation to the three
satellite laboratories. In contrast, Gram-stained smears en-
countered in quality assurance program 2 were prepared dur-
ing routine operations of the satellite laboratory. As such,
there may have been greater opportunity for microbiologists to
review overdecolorized gram-positive cocci and degenerated
leukocytescell types that may have been incorrectly reported
by satellite technologists.
A key component of any continuous quality assurance mon-
itoring program is ad libitum intervention (9). Throughout this
study, four major means of correction were implemented.
First, Gram-stained smears from either quality assurance pro-
gram 1 or 2 that yielded an egregiously incorrect response from
a satellite laboratorian were returned to a designated point
person from that laboratory. This individual personally re-
viewed the slide with the technologist and discussed any dis-
crepancies. Second, one-on-one interactions at a dual-headed
microscope took place between a central laboratory microbi-
ologist and the satellite laboratorian. Third, personnel from
the satellite laboratories and the central laboratory formed a
system-wide microbiology subcommittee, which provided a
conduit for the exchange of data, policies, and concerns. Fi-
nally, digital images of interesting findings from quality assur-
ance program 2 were disseminated via interlaboratory elec-
tronic mail in the format of a case presentation. Comprising
this document were the chart review, organism epidemiology,
and discussion of the relevance of the Gram-stained smear in
the specific case. These mailings also provided a forum for
discussion of aberrant findings in Gram-stained preparations,
such as organisms partially treated with antimicrobial agents
(4, 12).
Analysis of variance data for the four quarters of quality
assurance program 1 (Fig. 3) revealed an increase in Gram
stain interpretation proficiency. Analogous improvement was
seen via quality assurance program 2. However, neither hos-
pital A nor hospital B demonstrated any improvement in qual-
ity assurance program 1 or 2 throughout the course of this
monitoring period. It is likely that these two laboratories had
the most experience with satellite microbiology processing. In
further support of this statement, hospitals A and B possessed
the two highest proficiency rates (Fig. 1). In contrast, hospital
C showed significant improvement in total Gram stain inter-
3758 NOTES
pretation proficiency (Fig. 4) and the interpretation of bacteria
in both quality assurance programs.
In conclusion, we describe a quantitative measure of Gram
stain interpretation proficiency that can be applied to chal-
lenges of satellite laboratorians and an off-site review of tech-
nologist performance. These monitors can assess proficiency to
the level of the laboratory, technologist, cell type, and quanti-
tative measure. Technologist challenge is best utilized as an
educational tool. However, an off-site review program is likely
a better indicator of overall day-to-day Gram stain interpreta-
tion proficiency. When combined, these measures enable a
multisite microbiology service to generate valid data both for
patient care and for the practices of the clinical microbiology
laboratory.
REFERENCES
1. Aitken, J. M., and P. E. Thornley. 1983. Isolation of Branhamella catarrhalis
from sputum and tracheal aspirate. J. Clin. Microbiol. 18:12621263.
2. Bartlett, J. G. 2004. Decline in microbial studies for patients with pulmonary
infections. Clin. Infect. Dis. 39:170172.
3. Bowler, P. G., B. I. Duerden, and D. G. Armstrong. 2001. Wound microbi-
ology and associated approaches to wound management. Clin. Microbiol.
Rev. 14:244269.
4. Chan-Tack, K. M., and J. K. Johnson. 2005. An unusual Gram stain finding.
Clin. Infect. Dis. 40:1649, 16771678.
5. Church, D., E. Melnyk, and B. Unger. 2000. Quantitative Gram stain inter-
pretation criteria used by microbiology laboratories in Alberta, Canada.
J. Clin. Microbiol. 38:42664268.
6. Church, D. L., C. Don-Joe, and B. Unger. 2000. Effects of restructuring on
the performance of microbiology laboratories in Alberta. Arch. Pathol. Lab.
Med. 124:357361.
7. Cooper, G. M., J. J. Jones, J. C. Arbique, G. J. Flowerdew, and K. R.
Forward. 2000. Intra and inter technologist variability in the quality assess-
ment of respiratory tract specimens. Diagn. Microbiol. Infect. Dis. 37:231
235.
8. Cunney, R. J., E. B. McNamara, N. Alansari, B. Loo, and E. G. Smyth. 1997.
The impact of blood culture reporting and clinical liaison on the empiric
treatment of bacteraemia. J. Clin. Pathol. 50:10101012.
9. Dunne, W. M., Jr., and M. T. LaRocco. 2007. Laboratory management, p.
419. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. L. Landry, and M. A.
Pfaller (ed.), Manual of clinical microbiology, 9th ed. ASM Press, Washing-
ton, DC.
J. CLIN. MICROBIOL.
10. Flournoy, D. J. 1998. Interpreting the sputum Gram stain report. Lab. Med.
29:763768.
11. Kalin, M., A. A. Lindberg, and G. Tunevall. 1983. Etiological diagnosis of
bacterial pneumonia by Gram stain and quantitative culture of expectorates.
Scand. J. Infect. Dis. 15:153160.
12. Lorian, V., and B. Atkinson. 1975. Abnormal forms of bacteria produced by
antibiotics. Am. J. Clin. Pathol. 64:678688.
13. Munson, E. L., D. J. Diekema, S. E. Beekmann, K. C. Chapin, and G. V.
Doern. 2003. Detection and treatment of bloodstream infection: laboratory
reporting and antimicrobial management. J. Clin. Microbiol. 41:495497.
14. Murray, P. R., and J. A. Washington II. 1975. Microscopic and bacteriologic
analysis of expectorated sputum. Mayo Clin. Proc. 50:339344.
15. Musher, D. M., K. R. Kubitschek, J. Crennan, and R. E. Baughn. 1983.
Pneumonia and acute febrile tracheobronchitis due to Haemophilus influen-
zae. Ann. Intern. Med. 99:444450.
16. Musher, D. M., N. Lamm, R. O. Darouiche, E. J. Young, R. J. Hamill, and
G. C. Landon. 1994. The current spectrum of Staphylococcus aureus infection
in a tertiary care hospital. Medicine (Baltimore) 73:186208.
17. Musher, D. M., R. Montoya, and A. Wanahita. 2004. Diagnostic value of
microscopic examination of Gram-stained sputum and sputum cultures in
Downloaded from http://jcm.asm.org/ on October 29, 2016 by guest
patients with bacteremic pneumococcal pneumonia. Clin. Infect. Dis. 39:
165169.
18. Nagendra, S., P. Bourbeau, S. Brecher, M. Dunne, M. LaRocco, and G.
Doern. 2001. Sampling variability in the microbiological evaluation of expec-
torated sputa and endotracheal aspirates. J. Clin. Microbiol. 39:23442347.
19. Nicotra, B., M. Rivera, J. I. Luman, and R. J. Wallace, Jr. 1986. Branhamella
catarrhalis as a lower respiratory tract pathogen in patients with chronic lung
disease. Arch. Intern. Med. 146:890893.
20. Rahman, M. 1979. Quality of specimens and sputum culture results: a ret-
rospective study. Postgrad. Med. J. 55:553555.
21. Reed, W. W., G. S. Byrd, R. H. Gates, Jr., R. S. Howard, and M. J. Weaver.
1996. Sputum Grams stain in community-acquired pneumococcal pneumo-
nia: a meta-analysis. West. J. Med. 165:197204.
22. Thomson, R. B., Jr. 2007. Specimen collection, transport, and processing:
bacteriology, p. 291333. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. L.
Landry, and M. A. Pfaller (ed.), Manual of clinical microbiology, 9th ed.
ASM Press, Washington, DC.
23. Thorsteinsson, S. B., D. M. Musher, and T. Fagan. 1975. The diagnostic
value of sputum culture in acute pneumonia. JAMA 233:894895.
24. Triola, M. F. 1992. Elementary statistics, 5th ed., p. 544577. Addison Wes-
ley, Boston, MA.
25. York, M. K. 2004. Gram stain, p. 3.2.1.13.2.1.23. In H. D. Isenberg (ed.),
Clinical microbiology procedures handbook, 2nd ed. ASM Press, Washing-
ton, DC.
26. York, M. K., S. E. Sharp, and P. G. Bowler. 2004. Wound and soft tissue
cultures, p. 3.13.1.13.13.1.16. In H. D. Isenberg (ed.), Clinical microbiology
procedures handbook, 2nd ed. ASM Press, Washington, DC.