1. Chapter 40 Staining Specimens
Microbiology Unit 7
Chapter 40
Staining Specimens
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2. Stains A variety of stains available Most common
Gram stain
Ziehl-Neelsen (acid-fast) stain
Stain before culturing
Rapid identification
Determine appropriate medium
Determine appropriate antibacterials
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3. Staining Kits
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4. Stains (cont.) Simple stains
Crystal violet and methylene blue
Typically used for yeasts
Lactophenol cotton blue
Confirms identity of fungal organisms
Many stains available but most are performed only in large reference or research laboratories
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5. Gram Stain Used to categorize bacteria Requires four steps
Gram-positive or gram-negative
Based on cell wall structure
Requires four steps
Primary stain, a mordant, a decolorizer, and a counterstain
Mordant – fixes dyes to the structures (cell wall)
Primary stain usually crystal violet
Mordant – Gram’s iodine solution
Decolorizer – 95% ethanol or acetone
Counterstain – basic fuchsin or safranin
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6. Gram Stain Procedure Thin sample applied to slide
Swabs can be gently rolled on slide
Sterile wire to one young colony (24 hour)
Older colonies may not stain accurately
Mix sample from plates with a drop of water or saline
If broth – 2 to 3 loopfuls
If liquid – circle drop area with a wax pencil
No matter source – take care not to damage organisms
Air dry then heat fix by passing through a flame 2 to 3 times, specimen side up
Do not overheat – warm not hot
Prevents sample from washing off, preserves morphology, kills the bacteria, and renders them permeable to the stain
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7. Gram Stain Procedure (cont.)
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8. Gram Stain Interpretation
Gram-positive
Bacteria that retain the violet-iodine complex stain purple
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9. Gram Stain Interpretation (cont.)
Gram-negative
Lose the crystal-violet or purple color and stain red
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10. Potassium Hydroxide Test
Gram variable
Stain both gram-positive and gram-negative
May be due to excessive decolorization, an overly thick sample, excessive heat fixing, old cultures, or poor-quality stain
KOH test
Determines the true Gram status
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11. Potassium Hydroxide Test (cont.)
A loopful (or two) of 3% HOH solution placed on slide
A generous quantity of surface growth from culture transferred to the KOH
Stir specimen (30 seconds to 2 minutes)
Then gently lift the loop
Gram-negative develop a mucoid appearance and produce a sticky strand
Gram-positive – mixture remains homogeneous, no strand
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12. Ziehl-Neelsen Stain Primarily used to detect acid-fast organisms
Mycobacterium and Nocardia species
Many stains available, but few work well in veterinary practice laboratories
Several steps
Primary stain – dimethyl sulfoxide (DMSO) and carbol fuchsin
Decolorizer – acid-alcohol
Counterstain – methylene blue
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13. Ziehl-Neelsen Stain Procedure
Slide is air dried and heat fixed
Primary stain is flooded on slide
Slide heated over flame until stain steams
Cool slide for 5 minutes, then rinse with tap water
Acid alcohol is used to decolorize – 1 to 2 minutes until red color is gone
Rinse slide
Counterstain added then rinsed with water and air dried
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14. Ziehl-Neelsen Stain (cont.)
Agents like DMSO allow the stain to penetrate stain-resistant cells such as Mycobacterium
If stain isn’t removed by acid alcohol then the organism is “acid-fast” and appears red
Non–acid-fast cells stain blue
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15. Ziehl-Neelsen Stain (cont.)
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16. Giemsa Stain
Used to detect spirochetes and rickettsiae and the capsule of Bacillus anthracis and the morphology of Dermatophilus congolensis
Smear fixed with absolute methanol for 3 to 5 minutes and air dried
Dip in diluted stain for 20 to 30 minutes
For Borrelia anserina – the smear is gently heated while covered with the stain for 4 to 5 minutes then rinsed and air dried
Purplish-blue-stained bacteria
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17. Specialized Stains
Flagella, capsule, and endospore stains are available but have limited application in the average veterinary practice
Expensive stains
Malachite green endospore
stain of Bacillus
anthracis
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18. Summary
Gram stain is the most common staining procedure in the microbiology laboratory
Gram stains require primary stain, mordant, decolorizer, and counterstain
Gram-positive – purple
Gram-negative – red
KOH test helps identify Gram variable organisms
Ziehl-Neelsen stain is used to identify acid-fast organisms
Flagella, capsule, and endospore stains are primarily used in reference laboratories
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