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