Salts of colored compounds, the ionized form either is basic (positive) or acidic
INING:
(negative):
Stains usually dissolved in an alcohol or water solution, SIMPLE STAINS
are positive when ionized , stain negatively charged materials such as
Basic dyes:
bacteria examples:
crystal violet methylene blue safranin
Acid stains:
are negative when ionized , stain positively charged materials ( zB : glass) examples:
eosin nigrosin (spirochete (spirochete))
results in 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 DIFFERENTIAL STAINS: Gram Stain (p 70):
apple green for Bacillus anthracis
Usually four steps: primary stain, mordant, decolorize, decolorize, secondary secondary stain) by Hans Christian Gram (1884): Hucker =s stain, Iodine, 95% EtOH,
safranin O( Fankhauserser's page) page)
Acid fast: (p 70)
1) primary stain:
steam carbolfuchsin (fuchsin is a red dye) on specimen, several min.
(Ziehl-Neelsen)
2) decolorize 3) counter stain
acid-alcohol, removed color if not acid fast. methylene blue
If red (p 69), may be either Mycobacterium tuberculosis or leprae . (or Nocardia , a closely related bacterium) Negative stain (p 71):
demonstrate capsules, usually not stainable, add India ink (or other acid dyes?), capsule shows up as halo (stains background) five genera of bacteria make spores.
Endospore staining:
(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
Control Slide
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.
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
Focusing Technique
Oil (100X): Neutrophils with gram positive cocci
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 o lonf, narrow o coccobacillus o Branched o
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 o Coccobacillus o Curved o Fusiform o Spiral o
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
Gram positive Cocci
Clusters
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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
Avian Stool: Increased numbers of Gram negative rods
Normal Psittacine: 100% gram positive rods and cocci. No yeast seen.
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 stain: Cryptosporidium
Acid fast organisms stain red. Non acid fast organisms and tissue cells stain blue.
