Examination methods (Staining) Question 28

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  1. Wet mount
    • direct examination 
    • sample is suspended in water or saline
    • unstained preparation
    • brightfield, darkfield, phase contraset
  2. 10% KOH
    • direct examination 
    • KOH is used to dissolve proteinaceous materal and faciliate detection of fungal elements  that are not affected by strong alkali solution 
    • dyes like iodine can be added to increase the contrast between fungal elements and background
  3. India ink
    • direct examination 
    • modification of KOH procedure in whcih ink is added as a contrast material
    • for detection of capsules 
    • used for detection of cryptococcus in CSF
    • polysaccharide capsule excludes the ink creating a halo around the cell
  4. Lugol iodine
    • direct examination 
    • iodine is used to wet preparation of parasitolgy specimens to engance contrast of internal structures 
    • faciliates the differentiation bt protzoa and WBC
  5. Gram stain
    • Differential stain
    • most commonly used in labs
    • forms basis of sperating makor groups of bacs (G- and G+)
    • 1) Fixation to a glass slide via heat or alc treatment
    • 2) crystal violet 
    • 3) percipitation w/ iodine - forms a complex w/ primary dyw 
    • 4) decolarization w/ acetone 
    • 5) retained in G+(purple) but lost in G-
    • 6) counterstiain w/ safranin to retain the G- (red)

    • why is it G+ purple?
    • stain gets trapped in the thick peptdioglycan layer 
    • Why is G- red?
    • they only have a thin peptidoglycan layer 
    • outer membrane (which is only found in G-) can be counterstained by safranin )red) ... i think 

    • Exceptions:
    • a) myobacs (+ nocardia?)- have a waxy outer shell --> do acid fast stain
    • b) mycoplasma - has no peptidoglycan layer
  6. Iron hematoxylin stain
    • Differential stain
    • used for detection and identication of: fecal protozoa
    • helminths eggs and larvae are identified by wet mount preps
  7. Methenamine silver
    • Differential stain
    • more in histo labs
    • detection of fungal elements  (+bacs)
  8. Toluidine blue O stain
    • used for pneumocystis orgs in resp. specimen
    • cyst is seen redish blue not trophozoites
    • background staining is removed by sulfation reagent
  9. Trichome stain
    • alternative to iron hematoxylin to stain protozoa
    • protozoa have bluish - green cytoplasm w/ red ncl and incusion bodies
    • specimen background is green
  10. Wright - Giemsa stain
    • is used to detect:
    • blood parasites,
    • viral  and chlamydial inclusion bodies 
    • Borrelia
    • Toxoplasma 
    • Pneumocystis
    • Rickettsia 
    • Principle: mix of methylene blue, azure B and eosin Y
    • eosine ions are neg charged and stain basic parts of the orange , wherares other dyes stain acidic cell structures blue
  11. Acid fast stains (2)
    • 1. Ziehl- Neelsen stain
    • used to stain myobacs and other  acid fast orgs
    • 1) staining by basic carbol fuchsin 
    • 2) resist decolarization w/ acid alkali solution 
    • 3) background is counterstained w/ methylene blue 
    • orgs appear red
    • requires heating for the organism to uptake carbol fuchsin 
    • Initially, Carbol Fuchsin stains every cell. When they are destained with acid-alcohol, only non-acid-fast bacteria get destained since they do not have a thick, waxy lipid layer like acid-fast bacteria. When counter stain is applied, non-acid-fast bacteria pick it up and become blue when viewed under the microscope. Acid-fast bacteria retain Carbol Fuchsin so they appear red.
    • kinyoun stain - cold method

    • 2. Modified acid fast stain 
    • - weak colorized agent is used w/ acid fast stains 
    • used for 
    • nocardia
    • Ispora
    • Sarcocystis 
    • cylcospora
    • "partially acid fast"
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Examination methods (Staining) Question 28
staining methods - principle and application p. 159 table
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