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
reagents of acid fast staining
primary stain- strong carbol fuchsin
decolourizer- acid alcohol
counter stain-meth blue
prep of 3% acid alcohol for ZN
75mls of conc HCl
2425mls of 95% ethanol
place flask in 5-8cm cold water
add alcohol, add acid and cover top, leave for 10 minutes, final conc of HCl should be 3%
prep of Loeffler’s methylene blue
300mls of saturated methylene blue in alcohol
1000mls of KOH (0.01%) in water.
when this is allowed to ripen it’s called polychrome methylene blue
ZN procedure
-prepare smear
- flood in carbol fuchsin and heat unti. fumes rise, about every 15 seconds for 4 minutes
(repeat if necessary)
- let cool for 5 minutes
- flood in 3% acid alcohol for 1 minute
- wash gently with tap water
- flood 2 more times in acid alocohol, rinisng in between
- flood with meth blue for 1 minute
-wash gently, rinse with tap water and air dry
- view with oil immersion lense
modifications of ZN to make kinyoun stain
when preparing carbol fuchsin: higher conc of basic fuchsin and phenol used
3% HCl in acid alcohol is replaced by 1% sulphuric acid for the partial acid fast bacteria (nocardia)
kinyoun stain procedure
-flood with carbol fuchsin for 5-10 minutes
- rinse with tap water
- flood with acid alcohol for 3 minutes
- rinse with tap water
- flood with mth blue for 3 minutes
-rinse gently with tap water and air dry
when fixing the slide, heating kill all bacteria apart from which type?
Mycobacteria- methanol would be used to kill it
prep of 1% acid alcohol decoulorizer for flourochrome stain
20mls of conc HCl
1980 mls of 95% ethanol
flask in cold water in sink, add alcohol, then acid, cover for 10 min and final conc of 1% HCl
reagents of flourochrome stain
auramine O dye
acid alcohol 1% decolourizer
potassium permanganate counter stain
prep of auramine phenol stain
3g of auramine O
30g of phenol crystals and 1000mls of distilled water
filter before storing and store in dark bottle
prep of potassium permangante for flourochrome stain
2g of KMnO4
2000mls of distiled water
shake well and final conc of 0.1%
flourochrome stain procedure
cover slide in aruamine O dye (auramine phenol) for 10 mins at room temp
- wash gently w/ tap water
- cover slide with excess 1%acid alcohol and leave for 5 minuets
- rinse with tap water
-cover slide with 0.1% KMnO4 and leave for 30 sec
- wash gently and allow to air dry
-examine with flourescent microscopy
tubercle bacilli seen as yellow luminisce rods
why cant the gram stain be used on acid fast bacteria
the mycolic acid- lipid- in the cell wall repels the aqeous crystal voilet stain. therefore cannot be maintained
why is the slide heated in ZN stain
for stain to penetrate the cell wall.
