Gram stain procedure

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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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