MAKING THE MOST OF GRAM STAIN

INTERPRETATIONS

Richard (Tom) Thomson, Jr., Ph.D., D(ABMM), FAAM

Evanston Hospital
NorthShore University HealthSystem
University of Chicago Pritzker School of Medicine
rthomson@northshore.org
 Community Acquired Pneumonia

68 yo male admitted to hospital with findings

suggestive of CAP
History of COPD (30 pack-year smoking history)
Blood and sputum specimens submitted to
the Microbiology Laboratory
 Sputum Gram Stain
Technologist Report
Gram stain report:

< 25 SECs per 10X Field

2+ Polymorphonuclear Leukocytes
4+ Gram-negative coccobacilli
2+ Gram-positive cocci
2+ Gram-positive rods

How will the doctor interpret this report?

How should the doctor interpret this report?
 Sputum Gram Stain
Review with Interpretation
Gram stain report:

< 25 SECs per 10X Field

3+ Polymorphonuclear Leukocytes
3+ Gram-negative coccobacilli suggesting
Haemophilus (some intracellular)
2+ Mixed Respiratory Flora

How will the doctor interpret this report?

How should the doctor interpret this report?
The Gram Stain: One Powerful Tool

Goals
Standardize technical details
Standardize reporting
Introduce interpretive reporting
Microbiology consultations are our future
 Improving the Gram stain

Standardize technical details

Selecting a portion of the specimen

Preparing the smear
Low power (10X) examination
High power (100X) examination
Quantitation of cells and microorganisms
Examination and reporting based on specimen type
Slides for review and interpretation
 Improving the Gram stain

Standardize Reporting

Reporting Level I:
Minimum competency for everyone
Reporting Level II:
Advanced competency for senior technologists and
supervisors
 Improving the Gram Stain

Introduce Interpretive Reporting:

laboratory directors, pathologists (medical

microbiologists)
Pathogenesis and indicators of pathology
Pattern recognition of specific diseases
 HOW TO STANDARDIZE THE
GRAM STAIN PROCEDURE

Selecting a portion of the specimen

Preparing the smear
Low power (10X) examination
High power (100X) examination
Quantitation of cells and microorganisms
Examination and reporting based on specimen
type
Slides for review and interpretation
Selecting a Portion of the Specimen
Selecting a Portion of the Specimen

Visualize specimen
Consult technologist, director or pathologist
as necessary
Select best portion
Purulence
Mucous/tenacious
Blood
Necrosis (black)
PREPARING THE SMEAR WITH
SEMI-SOLID SPECIMEN
Make a Monolayer of Cells
PREPARING THE SMEAR WITH A FLUID SPECIMEN
Concentrating fluids using a cytospin
LOW POWER (10X) EXAMINATION

10-20 fields; Quantitate Cells;

Select Area for High Power Examination
HIGH POWER (100X) EXAMINATION

20-40 fields; Confirm Cells, Quantitate Microorganisms

 GRAM STAIN QUANTITIES

1+ (very rare)
Less than 10 in all fields examined
2+ (few)
More than 10 in all fields but less than 1/field
3+ (moderate)
More than 1/field but less than 25/field
4+ (many)
More than 25 in one field

1-3+ best for clinician

1-4+ best for laboratory
 Procedures to Standardize

Standard gram stain reagents and method to

establish a normal against which variations
can be compared
Crystal Violet 30 sec
Gramss Iodine 30 sec
Acetone Alcohol Clear runoff plus 5-10 sec
Safranin 1 min
Standard ocular and objective with
micrometer to standardize magnification and
field size
GRAM STAIN REPORTING BASED ON
SPECIMEN SOURCE
Sterile fluid or tissue (presumed)
Report PMNs
Report all microorganisms
> 3 morphologically typical shapes before
reporting
Leave for review
Non-Sterile specimen source
Report PMNs
Name microorganisms only if potential pathogen
Quantitate and report normal flora as a group
SLIDES FOR REVIEW and INTERPRETATION

1. Requested by technologist
2. Requested by laboratory director
 GRAM STAIN POLICY FOR
SLIDE REVIEW AND INTERPRETATION
Director-Requested Review
Sterile fluid or tissue
Microorganism reported
Non-sterile source
Report is diagnostic
Gram stain and culture results do not agree
Technologist-Requested Review
Technologist unsure of finding
Physician-requested review
Interpretation
Results of review added to lab work card or patient
report
GRAM STAIN SLIDE REVIEW AND
INTERPRETATION
 Gram Stain Report with Clinician Requested
Review and Interpretation by Medical Microbiologist
 BACTERIAL MORPHOLOGIES
LEVEL I
MINIMUM COMPETENCY-EVERYONE
MORPHOLOGY REPORTED
Gram-positive cocci clusters
Gram-positive coccci pairs/chains
Gram-positive cocci
Gram-positive rod
Gram-negative diplococci
Gram-negative coccobacilli
Gram-negative rod
Yeast cells
Yeast cells with pseudohyphae
OTHER FINDINGS-LEAVE FOR REVIEW
ORGANISM IMPLIED
Staphylococcus
Streptococcus
Staphylococus/Streptococcus
Any Gram-positive rod
Neisseria/Moraxella
Haemophilus/Bacteroides
Any Gram-negative rod
Yeast, usually Candida
Candida, not C. glabrata
 CELL MORPHOLOGIES
LEVEL I
MINIMUM COMPETENCY-EVERYONE

PMNs
Squamous epithelial cells
Sputum screens
 BACTERIAL MORPHOLOGIES
LEVEL II
Senior Technologists/Supervisors

MORPHOLOGY REPORTED ORGANISM IMPLIED

Level I plus
Gram-positive diplococci (lancet) S. pneumoniae
Gram-positive rod diphtheroid Corynebacterium, etc.
Gram-positive rod boxcar Bacillus/Clostridium
Gram-positive rod endospores Bacillus/Clostridium
Gram-positive rod filamentous/branching
Nocardia/Actinomyces
Hyphae septate Aspergillus, etc.
 BACTERIAL MORPHOLOGIES
Level II
Senior Technologists/Supervisors
MORPHOLOGY REPORTED
Continued
Gram-negative rod thick
Gram-negative rod thin
Gram-negative rod pleomorphic
Gram-negative diplobacilli
Gram-negative rod fusiform
Gram-negative rod curved
Gram-negative cocci tiny
BV Screens
OTHER FINDINGS-LEAVE FOR REVIEW
ORGANISM IMPLIED
Enterobacteriaceae-type
Nonfermenter-type
Bacteroides-type
Acinetobacter-type
Fusobacterium-
/Capnocytophagia-type
Campylobacter/Other
Brucella/Francisella
 Interpretations
Laboratory Directors/Pathologists
Medical Microbiologists

Level I and Level II plus

Indicators of pathology
Disease pattern recognition
Interpretation added to patient
record
 Interpretations
Laboratory Directors/Pathologists
Medical Microbiologists

Cells
White blood cells (polymorphonuclear
leukocytes)
Alveolar macrophages
Squamous/columnar epithelial cells
 Interpretations
Laboratory Directors/Pathologists
Medical Microbiologists
Indicators of Pathology
Inflammatory cells
Kirschmanns spirals and corpora amylacea
Casts and crystals
Cell necrosis (elastin and collagen)
Intracellular bacteria/yeasts
Other oddities
Contaminated agar in transport tubes
Gram Stain
Gram Stain
Diagnosis
Pap Stain
 Pap Stain
Pelger-Huet Anomaly

Is a benign dominantly inherited defect of terminal neutrophil differentiation

secondary to mutations in the lamin B receptor (LBR) gene.
How To Make Gram Stain Reporting
Relevant in the Future

Standardize technical details

Standardize reporting
Establish and maintain levels of competency
Level I
Level II
Standardize Gram stain review
Interpretive Reports by Medical Microbiologist
 Interpretations
Laboratory Directors/Pathologists
Medical Microbiologists

Disease pattern recognition

Respiratory tract
Pneumonia/bronchitis
Aspiration pneumonia
Chronic lung disease/COPD
Urinary tract
UTI
Fistula
Meningitis
 Interpretations
Laboratory Directors/Medical
Microbiologists
Skin-Soft Tissue-Closed Space Abscess
Staphylococcal
Mixed aerobic/anaerobic
Streptococcus milleri/anginosis
Nocardia
Toxemia
Streptococcal necortizing fasciitis
Clostridium gas gangrene
Miscellaneous
BV (bacterial vaginosis)
Lemierres disease (jugular vein thrombosis)
Gonococcal urethritis (male)
Crystalline joint disease
Radiology Model
Cytology Model
Hematology Model
 How To Make Gram Stain
Reporting Relevant in the Future

MICROBIOLOGY MODEL???
Comment: Gram stain reviewed by
Dr. Thomson. Branching septate
hyphae also present among PMNs.
Morphology suggests Aspergillus or
morphologically similar mold. See
attached image.
 How To Make Gram Stain
Reporting Relevant in the Future

Gram Stain:
< 25 Squamous Epithelial Cells
3+ Gram-positive diplococci
2+ Mixed respiratory flora

Culture Report:
1+ Staph aureus
1+ Streptococcus pneumoniae
Gram Stain: Culture Report:
< 25 Squamous Epithelial Cells 1+ Staph aureus
3+ Gram-positive diplococci 1+ Streptococcus pneumoniae
2+ Mixed respiratory flora

Interpretation by Dr Thomson:
Gram stain suggests pneumococcal LRT disease. Many diplococci are Gram-
negative possibly reflecting early antimicrobial damage. Staphylococci in the
Gram-stain are a minor component equivalent in quantity to other usual bacterial
flora. Clinical correlation necessary. Images attached.
 Pelger-Hut Anomaly

10 homozygous individuals have been reported

so far
Variable clinical presentation
Psychomotor retardation, macrocephalus, ventricular septal
defect (VSD), polydactyly (extra digits)
Heterozygous state is benign
Only apparent phenotype alteration being in the
PMNs
 Morphology

Suppression of segmentation of PMNs

Heterozygotes:
55% of the PMNs show nuclear segmentation arrest
at the bilobed level
Small population of PMNs possess nonlobulated
peanut-shaped nucleus (Stodtmeister cells)
Homozygotes:
95% of PMNs contain single, round eccentric nucleus
with little or no segmentation
 Urinary Tract Infection

76 yo female admitted to hospital with

findings suggesting a severe UTI
History of multiple UTIs in the past year
Blood and urine specimens sent to the
Microbiology Laboratory
 Urine Gram Stain

Urine Gram stain

1+ squamous epithelial cells
4+ Polymorphonuclear Leukocytes
4+ Gram-negative rods
4+ Gram-positive rods
4+ Gram-positive cocci
How should the doctor interpret this?
 Urine Culture

Urine Culture Results

4+ E. coli
4+ Proteus mirabilis
4+ E. faecalis
4+ other mixed urogenital flora
How should the doctor interpret this?
 Urinary Tract Infection

Many laboratories would not perform a urine

Gram stain
Those that do report the Gram stain would
provide a reading but no interpretation
Many laboratories would report the urine
culture as greater than 3 pathogens, gross
contamination present, submit a new
specimen
 Urine Culture

Urine Gram stain reading

4+ Polymorphonuclear leukocytes
4+ Gram-negative rods
4+ Gram-positive rods
4+ Gram-positive cocci
Urine Gram stain interpretation
Comment by Dr. Thomson (pager 2932). In addition to the
PMNs and bacteria present, there is roughage and crystalline
material. This pattern of microorganisms and foreign material
suggests the presence of stool and a vesicointestinal fistula. Dr.
Cook notified.