Gram Stain

Principle:

Bacteria will differentially retain crystal violet depending on their cell wall structure after decolorization.

Method:

• Fix the smear by gentle heating , allow to cool before stain
• Crystal violet for 30-45 sec
• Rinse with tap water
• Iodine for 30-45 sec
• Rinse with tap water
• Decolorize 10 sec ( Acetone 400cc +ethanol 95% 200cc)
• Counterstain with safranin 30 sec.
• Rinse with tap water and dry.

Acid fast (ziehl-neelsen) stain

Principle:

Cell wall micolic acid retain the basic dye carbolfuchsin despite acid alcohol rinsing.

Method:

• Use only new clean slides
• Fix smear with heating 60°C 10 min
• Flood smear with carbolfuchsin (0.3 gr basic fuchsin+ethanol 95% 10cc)
• Heat the smear almost to boiling. don’t allow the smear to dry
• Wash with DW
• Decolorize for 1 min (ethanol 95% 97cc+Hcl conc.3cc)
• Wash thoroughly
• Flood with counterstain (methylen blue 0.3 gr+100 cc DW)
• Wash with DW and air dry

Auramine Rhodamine stain (fluorochrom)

Method:

• Fix slide by gentle heating
• Auramine rhodamine for 15 min
• Rinse with DW
• Decolorize with acid alcohol for 2-3 min
• Rinse with DW
• Counterstain with potassium permanganate for 3-4 min
• Rinse with DW and dry
• Under UV light the organism is bright yellow orange

    Flagella stain (gray method)

    Method:

·        Use grease free clean slide

·        Select younger colony and mix with drop of DW

·        Allow to air dry. Don’t heat

·        Add the following solution for 10 min: potassium aluminum 5cc+tannic acid 20% 2cc+mercuric chloride 2cc+alcoholic solution of basic fuchsin 0.4cc

·        Wash gentle with DW

·        Add ziehl neelson carbolfuchsin 5-10 min

·        Wash with tap water and air dry

   Capsule stain (Anthony method)

Method:

·        Make a thin even smear of a culture colony in lithmus milk to give uniform background

·        Air dry. Don’t fix with heat

·        Stain with 1% crystal violet for 2 min

·        Wash with 20% copper sulfate

·        Air dry

·        Capsule is unstained in a purple background, the cells are deeply stained

Spore satin (dorner method)

Method:

·        Make a heavy suspension of organism in DW in a test tube

·        Add equal volume of carbolfuchsin

·        Place tube in boiling water 5-10 min

·        Mix a loopful of boiled suspension with 10% nigrosin on a clean slide

·        Spread out and dry film quickly by gentle heat

·        The spore stain red and bacteria cell are colorless in a dark background

Methylen blue stain (loeffler’s method)

·        Prepare smear and fix with heat

·        Flood smear with methylen blue for 1 min

·        Wash and blot dry

·        The granules readily obsorb the dye and appear deep blue

Indian ink method

*for the capsule of Cryptococcus neoformans

• Minute amount of colony in a loopful saline or direct CSF fluid
• Mix well and add 1 loopful of Indian ink
• Cover a thin cover glass
• Examine with reduced light , capsule when present are clear halo against a dark background

KOH method

• Place the skin or nail scrape or hair on slide
• Add KOH 10% (10gr KOH+10cc glycerin+ 80cc DW) 1 drop
• A drop of Indian ink will enhance the contrast
• Cover slip at room temperature for 10 min. gentle heating allow better visualization
• Examine for hyphae….

Giemsa stain

*stock preparation:

giemsa powder    600 mg

Absolute methanol  50ml

glycerin                  50ml

Mix the material in a flask. Stopper the flask by cotton plug and place in 60°C water bath for 2 hours. Shake gently at 30 min intervals. Allow to cool. Store in brown bottle for 2-3 weeks. Filter before use.

Method:

• Prepare the smear
• Flood the slide by giemsa for 20 min. don’t allow drying.
• Wash with tap water thoroughly and air dry
• Examine for malaria, leishmania or…