Microscopic Examination of Infected Material
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| Gram Stain Procedure *principle* | show 🗑
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| show | 1. dry the material on the slide
2. place smear on a staining rack, overlay in sequence
3. place smear upright on rack and allow to dry
4. examine under low-power; then under oil objective
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| Gram Stain Procedure *application* | show 🗑
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| show | Gram-positive bacteria stain dark blue to blue-black. all other elements stain safranin red. Structures have different avidity to safranin. Gram -negative bacteriahas a strong avidity to safranin and will stain bright red.
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| show | always check the quality of the stain before moving to interpretation. Leave stain on for the required amount of time to avoid false positives/negatives
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| show | stain binds to mycolic acid in the cell walls of the mycobacteria and is retained after the decolorizing step with acid alcohol. The counter stain does not penetrate the mycobacteria to affect the color of the primary stain.
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| show | 1. cover smears with TB auramine-rhodamine T stain for 25 minutes
2. wash
3. move to rack on acid alcohol collection container
4. flood smears with 0.5% acid alcohol for 2 min
5. wash smears
6. move to original staining rack
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| Acid-Fast Stain procedure *short procedure Fluorescent stain(cont'd)* | show 🗑
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| Acid-Fast Stain procedure *application* | show 🗑
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| Acid-Fast Stain procedure *results Fluorescent stain* | show 🗑
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| show | Kinyoun and Ziehl-Neelsen stains:
Mycobacteria stain red, whereas the background material and non-acid-fast bacteria stain blue
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| show | Mix equal amounts of the smear positive sputa with, 4% glutaraldehyde
Vortex well
30 min@ RT
DI water to 40 mLs.
Centrifuge 20 minutes.
Decant completely
Add DI water
If growth repeat
Label slides
Spread on slide
dry and heat fix them.
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| Acid-Fast Stain procedure *stains used* | show 🗑
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| show | 1. cover smear with carbolfuchsin stain for 5 min
2. wash slide
3. move to staining rack
4. Decolorize w/acid alcohol
5. wash
6. flood with methylene blue counter stain for 1 min
7. wash, drain, air dry
8. examine with oil
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| Acid-Fast Stain procedure *short procedure Modified Kinyoun stain (partial acid-fast)) | show 🗑
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| show | 8. counterstain with methylene blue for 30 sec.
9. rinse and air dry
10. examine with X100 oil immersion objective
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| Acid-Fast Stain procedure *short procedure Ziehl-Neelsen stain* | show 🗑
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| show | crystal violet; iodine; decolorizer; safranin
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| show | Mycobacterium sp.; Nocardia sp.; Actinomyces sp.; Streptomyces sp.; Mycoplasma sp.; L-forms
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