5. Schaeffer and Fulton spore stain Staining procedure 5% aqueous solution of malachite green Make and heat fix. Stain smears in a 1:10 solution of the stock 0,5% aqueous solution of safranin carbol fuschin for 15 mins. Wash in running tap water to remove excess Staining procedure stain. Decolourize with acetic acid for 20 - 30 secs. Prepare smear and heat fix as for Gram’s stain. Wash in running tap water. Flood with malachite green. Counterstain with Loeffler’s methylene blue for Steam gently over a flame for 30 sec (don’t 30sec. allow to boil). The stain should be just Wash and air dry. Examine. steaming. Placing the glass smear over a jar with boiling water in the area of the steam will Interpretation have the same effect. Wash with water and stain with safranin for Red: Brucella, Nocardia, Chlamydia, Coxiella 30 sec. Wash under running tap water, allow drying Blue: Other organisms and background and examine. 7. Ziehl-Neelsen Acid-Fast Stain (rapid method) Interpretation Solutions: Endospores stain a bright refractive green and Carbolfuchsin stain bacteria protoplasm pink. 0.3 g 100 ml Basic fuchsin Stamp’s stain (modified acid-fast for Ethanol, 95% Brucella) 45 g/ 900 ml water This solution is mixed with Stock carbol fuschin (weak) Basic fuschin 1g 5% Phenol 32 ml Absolute methanol 10ml 970 ml Acid alcohol Phenol (5%) 90ml 2.0 g Hydorchloric acid Decolourizer 1000 ml (concentrated) Acetic acid 0,5ml Ethanol, 95% Distilled water 95,5ml Counterstain For Nocardia 0,05% aqueous sulphuric acid Malachite Counterstain green/methylene blue Loeffler’s methylene blue (see Ziehl Neelsen Distilled water method) Procedure
5. Schaeffer and Fulton spore stain 5% aqueous solution of malachite green 0,5% aqueous solution of safranin Staining procedure Prepare smear and heat fix as for Gram’s stain. Flood with malachite green. Steam gently over a flame for 30 sec (don’t allow to boil). The stain should be just steaming. Placing the glass smear over a jar with boiling water in the area of the steam will have the same effect. Wash with water and stain with safranin for 30 sec. Wash under running tap water, allow drying and examine. Interpretation Endospores stain a bright refractive green and bacteria protoplasm pink. Stamp’s stain (modified acid-fast for Brucella) Stock carbol fuschin (weak) Basic fuschin 1g Absolute methanol 10ml Phenol (5%) 90ml Decolourizer Acetic acid 0,5ml Distilled water 95,5ml For Nocardia 0,05% aqueous sulphuric acid Counterstain Loeffler’s methylene blue (see Ziehl Neelsen method) Staining procedure Make and heat fix. Stain smears in a 1:10 solution of the stock carbol fuschin for 15 mins. Wash in running tap water to remove excess stain. Decolourize with acetic acid for 20 - 30 secs. Wash in running tap water. Counterstain with Loeffler’s methylene blue for 30sec. Wash and air dry. Examine. Interpretation Red: Brucella, Nocardia, Chlamydia, Coxiella Blue: Other organisms and background 7. Ziehl-Neelsen Acid-Fast Stain (rapid method) Solutions: Carbolfuchsin stain Basic fuchsin Ethanol, 95% This solution is mixed with 5% Phenol Acid alcohol Hydorchloric acid (concentrated) Ethanol, 95% Counterstain Malachite green/methylene blue Distilled water 0.3 g 100 ml 45 g/ 900 ml water 32 ml 970 ml 2.0 g 1000 ml Procedure
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