staining Flashcards
different types of staining techniques
simple: Methylene blue (WBC; neutrophils vs lymphocytes), Carbol Fuchsin strong (stool preperations), Neutral Red (view WBC, easily view RBC)
differential: GRam stain and Ziehl Neelson
Special: Negative, endospore and flaggelar
bright field microscopy and phase contrast microscopy
in brightfield microscopy light passes througha condensor then through the specimen, and then a series of lenses to magnify the image.
phase contrast illuminates the object with parallel beams of light that move out of phse relative to each other. the object therefore seen as a 3 dimensional structureand is useful for internal structures
stains for gram positive and negative and acid fast stuff
gram stain
ziehl neelson(acid fast bacilli)
kinyoun stain (mycobacteria and Nocardia)
Auramine-phenol stain (mycobacteria)
vincent stain for oral bacteria (test for Borrelia vincentii)
components of gram stain
primary stain- pararosaniline violet dye (ammonium oxalate crystal voilet)
mordant- lugol’s or gram’s iodine (aq soln of I)
decolourising solvent- iodine-acetone
counter stain- carbol fuchsin, safranin, neutral red
staining theory behind gram stain
primary stain and mordant form insoluble compound in protoplasm and cell wall of bacteria. decolouriser removes this compound. but it is a very slow process in gram +ve. nitially, all bacteria take up crystal violet dye; however, with the use of solvent, the lipid layer from gram-negative organisms is dissolved. With the dissolution of the lipid layer, gram negatives lose the primary stain. In contrast, solvent dehydrates the gram-positive cell walls with the closure of pores preventing diffusion of violet-iodine complex, and thus, bacteria remain stained. The length of decolorization is a critical step in gram staining as prolonged exposure to a decolorizing agent can remove all the stains from both types of bacteria.
preperation of Ammonium Oxalate xrystal voilet
soln A 1 part of ammonium oxalate and 100 parts of distilled water
Soln B 1 part of crystal voilet and 10 parts of Ethanol 99%
prep of Lugol’s /Gram’s Iodin
10g of iodine, 20g of KI crushed with mortar and pestle.. mix with 1000mls of distilled water
prep of decolouriser; iodine acetone
Liqour Iodi Fortis
10g of I
6g of KI
90mls of methylated spirit and 10 mls of distilled water
mix 35mls of LIF with 965mls of acetone
prep of of carbol fuchsin
5g of basic fuchsin
25g of phenol crystals
50mls of ethanol 99%
500mls of distilled wtaer
prep of neutral red
1g of neutral red
2mls of 1% acetic acid
1000mls of distilled wtaer
best phase to stain bacteria
when at end of stationary phase or beg of death phase- stain will not be retained -old bacteria
best is log phase of beg of stationary phase
gram stain prep and Preston and Morell’s procedure
reagents are prepared and timer
prepare smear:
if from direct specimen- place on slide- air dry
if from colony- label slide with diamond pen- emulsify with sterile distilled water and leave to air dry
fix passing 3 times on flame or 3 minutes in methanol
staining:
30sec flood in ammonium oxalate crystal voilet
30sec in gram’s lugol’s iodine
30 seconds in iodine-acetone
wash freely with tap water
flood for 30sec-1min in counter stain
wash with water, blot and dry (do not plot pus )
mycolic acid what is it, what synthesizes it and its affinity to stain
it is a fatty acid, synthesized by enoyl acp reductase, binds to arabinogalactose and has high affinity for primary stain
what does the acid fast stain detect
cells capable of retaining the primary stainwhen treated with acid alcohol
modifications or types of acid fast staining
ZN stain
Kinyoun stain (cold ZN)
stain for nocardia and actinomyces(mildly acid fast)
parasitology for cryptosporidia and isospora
