INING:             Salts of colored compounds, the ionized form either is basic (positive) or acidic (negative):

Stains usually dissolved in an alcohol or water solution,

SIMPLE STAINS

Basic dyes:            are positive when ionized, stain negatively charged materials such as bacteria

                  examples:      crystal violet

methylene blue

safranin

Acid  stains:             are negative when ionized, stain positively charged materials (zB: glass)

examples:      eosin

nigrosin (spirochete)

results in negative staining because background is usually positive, and so is stained

Fluorescence microscopy:  stain with fluorochromes:

auramine O                   glows yellow in UV, absorbed by Mycobacterium tuberculosis

fluorescein isothiocyanate      apple green for Bacillus anthracis

DIFFERENTIAL STAINS:   Usually four steps:  primary stain, mordant, decolorize, secondary stain)

Gram Stain (p 70):            by Hans Christian Gram (1884): Hucker=s stain, Iodine, 95% EtOH, safranin O(  Fankhauserser's page)

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Acid fast: (p 70)            1) primary stain:            steam carbolfuchsin (fuchsin is a red dye) on specimen, several min.

(Ziehl-Neelsen)            2) decolorize             acid-alcohol, removed color if not acid fast.

3) counter stain             methylene blue

If red (p 69), may be either Mycobacterium tuberculosis or leprae. (or Nocardia, a closely related bacterium)

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Negative stain (p 71):              demonstrate capsules, usually not stainable,  add India ink (or other acid dyes?), capsule shows up as halo (stains background)

Endospore staining:             five genera of bacteria make spores.

(P 71)                          Very difficult to stain, although easily seen due to different refractive index.

Schaeffer-Fulton endospore stain: (p 71)

malachite green steamed for five minutes wash 30 seconds with water (spores stay green)

safranin counter stain

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 power10X: 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 o long, wide o lonf, narrow o coccobacillus o 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: o Long and narrow o Coccobacillus o Curved o Fusiform o 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

Diplococci Clusters

 

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