1. Experiment 5 acid-fast stain ， Culture and stain of fungus

2. Content 1.acid-fast stain of M.tuberculosis 2. Culture of fungus 3.Simple stain of fungus

3. acid-fast stain of M.tuberculosis Cell wall of M.tuberculosis is impermeability to stains and dyes. But M.tuberculosis can be stained by acid-fast stain with long time heating. Bacteria except M.tuberculosis can be decolorized by 3% acid alcohol. So the color of M.tuberculosis is red and that of other Mycolic acid negtive bacteria is blue after Counterstain with methylene blue

4. 1.Make a smear of the sputum, dry and fix it. 2.Ziehl-Neelsen acid-fast stain: (1) Flood the slide with carbolfuchsin. Heat the slide to steaming for 5mins. Do not boil or allow the smear to dry. As stain evaporated from the slide, replenish with additional carbolfuchsin. Allow the slide to cool and rinse it thoroughly with water. (2) Decolorize the slide with acid alcohol until the red color no longer comes off in the decolorizer. It takes about 30secs. Rinse the slide with water. (3) Counterstain with methylene blue, allow the stain to react for 1min, rinse as above. (4) Bolt the slide carefully. Examine under microscope.

5. Culture of Fungi Sabouraud’s dextrose agar is commonly used medium for cultivation of fungi. Colony types: (1)Yeast-form colony: e.g. Yeast; Cryptococcus neoformans. (2)Yeast-like colony: e.g. Candida albicans. (3)Hyphomycete-form colony: e.g. Dermatophytes.

6. 白假丝酵母 ( 血平板 ) 黄曲霉

7. Microslide cultivation technique (1)Place a block of Sabouraud’s dextrose agar (about 1cm2) on a sterile slide. (2)Using a straight inoculating needle, inoculate the specimen to be assayed on each side of the block. (3)Gently heat a coverslip by passing it quickly through the flame of an alcohol burner and immediately place it directly on the surface of the inoculated agar block. (4)Place the slide in a petri dish on a slide supporter glass rods above a moistened filter paper disk, incubate at 28 ℃ for 3 ～ 5 days. (5)The slide can be microscoped at intervals during cultivation to observe the dynamics of hyphae and spore growth.

8. Simple stain of fungus （ Candida albicans ） precedure 1.Smear preparation （ same to gram stain ） 2.stain ： add crystal violet (or methylene blue), stain for 1 min ， rinse ， blot ， Microscopic examination 。

9. Experimental report: 1.acid-fast staining method: 2.Simple stain of fungus experimental principle, procedures, and analysis of experimental results, drawing