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
Heat fix impression smears Cover slides with carbolfuchsin stain and heat steam for 5 minutes Rinse with tap water Decolorise by rinsing with acid alcohol for 2 minutes Rinse with tap water Counterstain with malachite green for 0.5 – 1 minute Rinse with tap water and leave to air dry Interpretation Red: Mycobacteria Blue (or green); Other organisms and background material Modified acid-fast stain for Nocardia species. Staining procedure Make a smear of the organism from growth media and heat fix. the slide with Kinyoun carbol fuchsin for 5 minutes. Pour off excess stain. Decolourize with 1% aqueous sulphuric acid. Wash with tap water. Counterstain with methylene blue for 1 minute. Rinse with water and dry. Examine with the 100X oil immersion optics. Acid-fastness can be enhanced by growth on Middlebrook 7H11 agar. Interpretation Acid-fast (red): Most Nocardia spp. Negative (blue): Other actinomycetes. Diene’s stain for mycoplasma cultures Methylene blue 2.5g Azure II 1.25g Maltose 10g Sodium carbonate 0.25g Distilled water 100ml Staining procedure Place a small amount of the stain next to a suspected colony (with a loop or cotton swab). The stain will diffuse and the colony will become blue if it is mycoplasma. Live bacteria decolourize the stain within a few minutes. Staining procedures for fungi India ink wet mount Clinical material or organisms from cultures are mixed on a slide in a drop made up of loopfuls of distilled water and India ink or permanent black pen ink. Experience will indicate the amount of India ink to use. A coverslip is added, and the preparation examined. The large capsules of Cryptococcus neoformans show up. An alternative method is to suspend the material in distilled water under a coverslip and then add the India ink to the edge of the coverslip. The ink will seep under the coverslip and provide areas of variable density. Lactophenol cotton blue Lactophenol blue is used both as a mounting fluid and a stain. Lactic acid acts as a clearing agent and aids in the preservation of fungal structures, phenol