47 lab2－细菌的芽孢染色.ppt

第1页：实验二细菌的芽孢染色一教学要求了解芽孢方法的原

实验二细菌的芽孢染色一教学要求了解芽孢方法的原理，掌握操作技术。二实验原理Afewgeneraofbacteria,suchasBacillusandClostridiumhavetheabilitytoproduceresistantsurvivalformstermedendospores.Unlikethereproductivesporesoffungiandplants,theseendosporesareresistanttoheat,drying,radiation,andvariouschemicaldisinfectants.

第2页：Duetotheresistantnat

Due to the resistant nature of the endospore coats,endospores are difficult to stain. Strong dyes and vigorousstaining conditions such as heat are needed. Once stained,however, endospores are equally hard to decolorize. Sincefew bacterial genera produce endospores, the endosporestain is a good diagnostic test for species ofBacillusandClostridium.

第3页：三材料与器材

• 三材料与器材
• 菌种：蜡样芽孢杆菌约2 d营养琼脂斜面培养物，球形芽孢杆菌(B.sphaericus) 1-2 d营养琼脂斜面培养物。
• 染色剂：5％孔雀绿水溶液，10.5％番红水溶液。
• 小试管，滴管，烧杯，载玻片，显微镜等。

第4页：Heat-fixasmearofBaci

1. Heat-fix a smear ofBacillus megateriumas follows:a. Using the dropper bottle of distilled water found in yourstaining rack, placea small drop of wateron a clean slide bytouching the dropper to the slide.b. Aseptically remove asmall amountof the culture fromtheedge of the growthon the agar surface andgenerouslymix itwith the drop of water until the water turns cloudy.c.Burn the remaining bacteria off of the loop.d. Using the loop,spread the suspension over the entire slideto form a thin film.e. Allow this thin suspension tocompletely air dry.f. Pass the slide (film-side up) through the flame of the bunsenburner 3 or 4 times to heat-fix.四操作步骤

第5页：Placeapieceofblottin

2. Place a piece of blotting paper over the smear andsaturate withmalachite green.3. Let the malachite green sit on the slide forone minuteand proceed to the next step.4. Holding the slide with forceps, carefully heat the slidein the flame of a bunsen burner until the stainjust beginsto steam. Remove the slide from the heat until steamingstops; then gently reheat. Continue steaming the smear inthis manner forfive minutes.As the malachite greenevaporates, continually add more. Do not let the paperdry out.

第6页：Afterfiveminutesofst

5. After five minutes of steaming,washthe excess stainand blotting paper off the slidewith water. Don't forgetto wash of any dye that got onto thebottomof the slide.6. Blot the slide dry.7. Now flood the smear withsafraninand stain foroneminute.8. Wash off the excess safranin with water, blot dry, andobserve using oil immersion microscopy. With thisendospore staining procedure, endospores will staingreenwhile vegetative bacteria will stainred.

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第8页：五注意事项

• 五注意事项
• 注意掌握供试验菌种的培养时间。
• 染色加热过程要及时补充染液，切勿让涂片干涸。六实验报告绘出显微镜下观察到的结果。七思考题
• 用简单简单染色法是否能观察到细菌的芽孢？为什么？