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Unit 1 Chapter 7

Microscopic Examination of Infected Material

Gram Stain Procedure *principle* colors all cells and background material a deep blue and for Gram iodine to provide the larger iodine element to replace the smaller chloride in the stain molecule.
Gram Stain Procedure *short procedure* 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
Gram Stain Procedure *application* used routinely and as requested in the clinical microbiology laboratory for the primary microscopic examination of specimens submitted for smear and culture.
Gram Stain Procedure *results* 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.
Gram Stain Procedure *QC* 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
Acid-Fast Stain procedure *principle* 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.
Acid-Fast Stain procedure *short procedureFluorescent stain* 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
Acid-Fast Stain procedure *short procedure Fluorescent stain(cont'd)* 7. Flood smear for 4 minutes in potassium permanganate counter stain 8. wash smears 9. air dry 10. examine with the X16 and X40 objectives of the fluorescent microscope equipped with a filter system comparable to a BG-12 exciter filterand an OG-1 barri
Acid-Fast Stain procedure *application* the direct smear examination is a valuable diagnostic procedure for the detection of mycobacteria in clinical specimens
Acid-Fast Stain procedure *results Fluorescent stain* Fluorescent stain: Mycobacteria stain bright orange. Count the number of acid-fastbacilli seen on the smear and report as follows: 1-20 -> number seen 21-80 -> few 81-300 -> moderate >300 -> numerous
Acid-Fast Stain procedure *results Kinyoun and Ziehl-Neelsen stains* Kinyoun and Ziehl-Neelsen stains: Mycobacteria stain red, whereas the background material and non-acid-fast bacteria stain blue
Acid-Fast Stain procedure *QC* 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.
Acid-Fast Stain procedure *stains used* Fluorescent stain Kinyoun stain Modified Kinyoun stain (partial acid-fast) Zeihl-Neelsen stain
Acid-Fast Stain procedure *short procedure Kinyoun stain* 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
Acid-Fast Stain procedure *short procedure Modified Kinyoun stain (partial acid-fast)) 1. flood with carbolfuchin stain for 5 min 2. rinse 3. flood with 70% ethanol, rinse, repeat as needed 4 move to collection rack 5. drop 1% sulfuric acid on smear until colorless 6. rinse 7. move to original rack
Acid-Fast Stain procedure *short procedure Modified Kinyoun stain (partial acid-fast) cont'd* 8. counterstain with methylene blue for 30 sec. 9. rinse and air dry 10. examine with X100 oil immersion objective
Acid-Fast Stain procedure *short procedure Ziehl-Neelsen stain* 1. cover with appropriate size filter paper 2. layer paper with carbolfusin stain. heat until steam occurs. stain for 5 min 3. proceed with Kinyoun method @ step 2
Gram Stain Procedure *stains used* crystal violet; iodine; decolorizer; safranin
Acid-Fast stain used on Mycobacterium sp.; Nocardia sp.; Actinomyces sp.; Streptomyces sp.; Mycoplasma sp.; L-forms
Created by: luceroapril