Microbiology

Gram Stain & Acid Fast Stain

Gram Stain: Introduction

• Interpretation
• Staining Procedure
• Theory
• Microscopy

Interpretation

Clinical Signifance

• Gram stain results can have dramatic effect on patient care.
• Hospitalization may be required when the Gram stain results indicate bacteria are present in a normally sterile body fluid.
• The initial choice of antibiotic therapy is guided by the Gram stain results.

Gram Stain

• The Gram stain is a technique for staining and detecting bacteria and yeast.
• It is one of the most commonly performed procedures in the clinical microbiology laboratory.

Gram Stain: Procedure

Four reagents are used to perform a gram stain: crystal violet, Gram's Iodine, acetone - alcohol, and safranin.

Direct Smear Preparation

If the specimen is received on a swab, gently role the swab on a clean glass slide to avoid rupturing host cells. Allow to air dry.

Direct Smear Pap

If the specimen is a fluid, place a drop of fluid on a clean glass slide and allow to air dry. *In both cases, the specimen is fixed to the glass slide by passing it a few times over a flame.

Control Slide

Fishers Band Gram Slide has control Gram positive cocci and Gram Negative rods.

Staining Procedure

Step 1. Flood the slide with crystal violet for 1 minute. Rinse with water. Step 2. Flood the slide with Gram's iodine for 1 minute. Rinse with water.

Staining Procedure

• Step 3. Decolorize the slides by gently rinsing with an acetone - alcohol solution for 1 to 10 seconds dependent on content of acetone in solution. Rinse with water.
• step4. Flood the slide with saffranin, the counterstain, for 1 minute. Rinse with water and dry air.

Theory: Cell Wall Construction

• Gram positive and gram negative bacteria stain differently because of the structure of their cell walls.
• Gram - positive bacteria and yeasts stain purple.
• Gram negative bacteria and host cells stain pink.

Microscopy

Proper adjustment of the microscope is essential.

Microscopy

• Scan slide on low power (10X) to find the best area of the slide.
• Than go to oil immersion (100X)

Scan slide on low power 10X: Many Neutrophils seen	Oil (100X): Neutrophils with gram positive cocci

Focusing Technique

• Focusing techniques: Often it is necessary to focus through a specimen since organisms, and cells can be found on different planes.

  Oil (100X): Slide shows neutrophils and gram positive cocci.

   

Morphology

• Bacteria
• Host Cells: WBC's

  Macrophages, RBC's, Epithelial Cells

• Yeast
• Artifact

Gram Positive Rods

• Gram positive rods can be found in found in 4 clinically significant forms
  • long, wide
  • lonf, narrow
  • coccobacillus
  • Branched

Gram Positive Rods

 

 

Gram Positive Rods

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Gram Negative Rods

• Gram negative rods can be present in 5 Clinically significant forms:
  • Long and narrow
  • Coccobacillus
  • Curved
  • Fusiform
  • Spiral

Gram Negative Rods

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Gram Negative Rods

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Mixed Populations

Avian Stool, Gram stained

80% Gram positive rods

20% Gram negative rods

 

Find the Gram positive and Gram negative rods in this field.

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Gram negative spiral rods (spirochetes)

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Gram positive Cocci: a spherical bacterium

Gram positive Cocci may be present as:

• diplococci - pairs
• Chains of cocci
• Tetrad appear as a cluster of exactly four cocci
• Cluster - Groups of cocci with variable numbers

  

Bacteria: Gram positive Cocci

• Gram positve Cocci

Gram positive Cocci

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Gram positive Cocci

Gram positive cocci in cluster (top black arrow)       Gram positive cocci in tetrad formation (botton black arrow)

 

Gram Negative Cocci

Diplococci Do not form typically chains or clusters

Host Cells

• Epithelial Cells
• Stain Gram Negative
• White Blood Cells

Yeast

 

Stains Gram Positive Can be budding or in branching hyphae form

 

 

 

 

Artifact

Crystal violet precipitate on epithelial cell: May be confused with Gram positive cocci.

Crystal violet precipitate crystal on gram stain.

Underdecolorization

• Neutrophils that should stain pink are underdecolorized at acetone alcohol step and stained purple

underdecolorized neutrophils

Overdecolorization

Part of this slide (pink area) has been overdecolorized with the acetone alcohol giving the false impression of gram negative rods being present.

Reporting Results

Use systematic, descriptive terminology to report gram stain results. * For example, this Gram stain is properly described as "Gram - positive cocci (clusters) and white blood cells present.

Reporting Results: Avian Stool

Avian Gram Stain

Report percentages of Gram positive,

Gram negative bacteria and yeast.

 

• Normal Psittacine stool consist of 95% gram positive rods and cocci and up to 5% gram negative rods.
• Occasional yeast is normal.

Gram Stain: Normal

Avian (Psittacine) Stool

Normal Psittacine: 100% gram positive rods and cocci. No yeast seen.

Avian Stool: Increased numbers of Gram negative rods

Abnormal Avian gram stain: 50% Gram negative rods 50% Gram positive rods

Reporting Results

• * It is NOT possible to determine the species of bacteria from Gram stain results alone!
• Difinitive identification requires culture and biochemical testing.

Acid fast stain: Ziehl Neelson Method

• Procedure
• Morphology

Acid fast Organisms

• Contains waxlike lipoidal material affecting staining quality.
• Carbolfuchsin is primary stain.
• Acid fast organisms resist decolorization with acid alcohol.
• After decolorization, methyelene blue is added to organisms to counterstain any material that is not acid fast.

Ziehl Neelsen Acid fast Stain method

Acid Fast Organisms on wrights stain

Acid Fast Stain: Ziehl neelsen method

Acid fast organisms stain red. Non acid fast organisms and tissue cells stain blue.

Acid fast stain: Cryptosporidium

 Question?

email instructor: Sue Dempsey

webpage-Mary Kennedy-Dec 1,2000