The document describes the Gram stain procedure and interpretation. It discusses how Gram staining classifies bacteria based on differences in cell wall structure and composition that determine whether they appear gram-positive or gram-negative. The Gram stain procedure and interpretation are explained in detail over multiple pages, including slide preparation, staining steps, microscopic evaluation, and reporting of results. Examples of various bacteria visualized by Gram stain from different specimen types are also shown.

1. Module 4: Gram Stain Principle,
Procedure, & Interpretation

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Learning Objectives
At the end of this session participants should be able to :
 Describe the differences between gram-positive & gram-negative
bacteria that determine how they will stain with the Gram stain
 Properly prepare smears for Gram staining, both from clinical
specimens & cultures
 Properly perform the Gram stain procedure
 Interpret Gram stains based on sample type
 Recognize the morphological groups of organisms based on Gram
stain appearance
 Recognize the appearance of human cells associated with infectious
processes
 Prepare a set of slides to be used for Gram stain quality control (QC)

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Content Overview
Uses & advantages of Gram stains
Principle of the Gram stain
Specimens for Gram stain
Gram stain procedure
Examination & interpretation
Reporting of Gram stain results
QC of Gram stains

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 A critical test for a rapid, presumptive
diagnosis of infectious agents
 Used to classify bacteria on the basis of
their Gram reaction, morphology (shape),
& arrangement
 Serves to assess the quality of clinical
specimens
Gram Stain
 Originally developed by Christian Gram in 1884
 With minor modifications still used 126 years later

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Advantages of the Gram Stain
Rapid procedure
Cost effective
Accessible
Effective screening technique
Reliable
Semi-quantitative
Provides culture clues
Presumptive diagnosis of infection

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Gram positive Gram negative
Peptidoglycan Thick Thin
Teichoic acid Yes No
Outer membrane No Yes
Periplasm No Yes
Why do Bacteria Stain Differently?
It’s in the Cell Wall!

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Principle of the Gram Stain
 Bacteria stain either Gram-positive or Gram-
negative on the basis of differences in their cell wall
composition
 Gram positives have a thick peptidoglycan layer &
large amounts of teichoic acids
 Gram negatives have a thin peptidoglycan layer &
an outer membrane composed of a lipid bilayer
 The outer membrane of Gram-negative organisms
is damaged by the decolorizer (organic solvents)

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Specimens for Gram Stain
Clinical specimens (direct smears): wounds,
eye lesions, sterile fluids, body tissues,
discharges
Not done on: blood, throat swabs, nasal
swabs, stool (why?)
Young colonies on solid medium
Broth & blood cultures (turbid) - Arrangement
is best seen in broth

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Gram Stain Reagents
Crystal violet – initial stain
Iodine – mordant/binding agent
Alcohol – decolorizer
Safranin or diluted basic or carbol fuchsin –
counterstains

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Slide Preparation
Prepare a thin smear & make sure it is dry before
processing. To speed drying you can place the slide
on a warming tray (~ 50 ºC).
 In Module 5, Specimen Processing, We will discuss in more
detail how to prepare smears from clinical specimens.
Fix the slide with methanol (allow to dry) or by gently
passing through a flame 3 or 4 times. Touch the
slide after it has been passed through the flame, it
should be warm, NOT hot. Allow slide to dry.
You are now ready to stain the slide.

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Basic Gram Stain Method
 Flood smear with Crystal Violet - 10 seconds
 Rinse clear with tap water & drain the excess
 Flood with Iodine solution - 10 seconds
 Rinse as above
 Hold slide at a 45 degree angle & run the
acetone/alcohol mixture over the slide watching the
spilloff, when the spill off is no longer blue, rinse
immediately. DO NOT OVER DECOLORIZE!
 Flood slide with counterstain - 10 seconds
 Rinse & allow to air dry (may be carefully blotted)
Recommended for general bacteriology use

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Microscopic Evaluation of Gram-stained Smears
Evaluate smear to determine:
 If specimen/smear is acceptable
 e.g. reject sputum with many epithelial cells
 Check for stain deposits
 Determine if smear is too thick or too thin
 Was the smear properly stained?
 Background, epithelial cells (EC), polymorphonuclear leucocytes
(PMN) – red
 Gram positive organisms – deep violet
 Gram negative organisms – pink or red
 Gram variable – both Gram-positive & Gram-negative with the same
morphology
Note: When examining slides from clinical specimens, different
areas of the slide may stain quite differently depending on the
thickness of the area & the cells, proteins or mucous present.

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Crystal violet precipitate on
epithelial cell:
May be confused with Gram
positive cocci
Crystal violet precipitate on
Gram stain
*stain may need to be filtered
Examples - Crystal Violet Precipitation

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Gram Stain: Under-decolorized Smear (1)
Note the dark nuclei of the polys

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Gram Stain: Under-decolorized Smear (2)

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Gram Stain: Over-decolorized Smear

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Examination & Interpretation (1)
For smears prepared from clinical specimens
 105 organisms/ml of un-centrifuged fluid (104
centrifuged) organisms are required to be visible on slide
 At lower concentrations, organism will not be revealed
even if culture is positive
 Organisms seen on Gram stain, but fail to grow in culture
 fastidious (require specific media for growth)
 anaerobic bacteria
 patient has received antibiotics

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Examination & Interpretation (2)
 Cells are enumerated using the low power objective
(LPO)
 Squamous epithelial cells (SEC’s), PMN’s, red blood cells
(RBC’s)
 Examine multiple areas of the slide under both low power
& oil immersion magnification.
 Bacteria & yeasts are enumerated using the oil
immersion objective (OIO)
 Ignore one or two microorganisms on the entire slide (oil
objective field) unless results can be reproduced on a
second smear &, only if it is from an invasively collected
specimen

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Examination & Interpretation (3)
The presence of microorganisms from a
normally sterile site is likely to indicate
infections with the organism that is seen
The presence of large numbers of a single type
of organisms in a non-invasively collected
specimen, especially if associated with white
blood cells (WBC’s), is likely to indicate infection

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Interpretation & Reporting (1)
 Perform counts only in areas representative of
inflammation or necrosis, if present
 Specify gram-reaction, morphology (be as descriptive as
possible), arrangement & other information that can lead
to the suggestion of a particular pathogen
 Examples:
 Gram-positive diplococci consistent with Streptococcus
pneumoniae
 Small Gram-negative coccobacilli, consistent with
Haemophilus
 Gram-negative diplococci, consistent with Neisseria
species

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Interpretation & Reporting (2)
 Enumerate cells per low power field (LPF) Epis, PMNs,
RBCs)
1+ (rare or occasional): <1/LPF
2+ (few): 1– 9/LPF
3+ (moderate): 10-25/LPF
4+ (many): >25/LPF
 Enumerate bacteria & yeast per OIF
1+ (rare or occasional): <1/OIF
2+ (few): 1– 5/OIF
3+ (moderate): 6-25/OIF
4+ (many): >25/OIF

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10X: Many Neutrophils seen
Scan Slide on Low Power

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Oil (100X): Neutrophils with Gram-positive
cocci in clusters
Then….

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Gram Stain: Wound
Streptococcus pyogenes Infection

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Gram Stain: Cerebrospinal Fluid (CSF)
Bacterial meningitis (Streptococcus pneumoniae)

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Gram Stain: Sputum
Pseudomonas aeruginosa infection

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Page  27
Gram Stain: Sputum
Haemophilus influenzae

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Gram Stain: Urethral Discharge
Urethritis Neisseria gonorrhoaea

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Gram Stain: Sputum
Nocardia infection

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Gram Stain: Sputum
Aspergillus

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Gram Stain: Vaginal discharge:
Candida albicans

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Gram Stain: Sputum
Streptococcus pneumoniae & Haemophilus influenzae

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Do All Bacteria Stain with the Gram Stain?
Organisms that do not stain
Chlamydia & others - too small & intracellular
Spirochetes - many are too thin
Mycobacteria - waxy cell wall
Poorly staining
Some anaerobes
Legionella
Brucella & others
Gram-stain modifications used for poorly staining
organisms

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Carbol Fuchsin Method
Crystal violet 30 seconds
Gram’s iodine 30 seconds
95% ethanol ~30 seconds
Carbol fuchsin or
0.8% basic fuchsin ≥1 minute
Bacteroides spp., Fusobacterium spp., Legionella
spp., Campylobacter spp., Brucella spp., & other
faintly staining Gram-negative organisms

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Kopeloff’s Method
1. Alkaline crystal violet: flood with solution A;
add 5 drops of solution B 2–3 minutes
2. Kopeloff’s iodine ≥2 minutes
3. 3:7 acetone-alcohol rinse immediately after
applying
4. Kopeloff’s safranin 10-30 seconds
Anaerobes, diagnosis of bacterial vaginosis

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Summary