Download Veterinary Practice Laboratory Unit 1

Microbiology
Unit 7
Chapter 40
Staining Specimens
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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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Staining Kits
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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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Gram Stain
• Used to categorize bacteria
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Gram-positive or gram-negative
• Based on cell wall structure
• Requires four steps
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Primary stain, a mordant, a decolorizer, and a
counterstain
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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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Gram Stain Procedure
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Thin sample applied to slide
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Swabs can be gently rolled on slide
Sterile wire to one young colony (24 hour)
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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
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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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Gram Stain Procedure (cont.)
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Gram Stain Interpretation
• Gram-positive
• Bacteria that retain the violet-iodine complex
stain purple
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Gram Stain Interpretation (cont.)
• Gram-negative
• Lose the crystal-violet or purple color and
stain red
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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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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)
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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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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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Ziehl-Neelsen Stain Procedure
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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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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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Ziehl-Neelsen Stain (cont.)
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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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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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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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