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We ʼ ll counterstain the vegetative portion of the

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Unformatted text preview: We ʼ ll counterstain the Vegetative Portion of the Cell with Safranin. The End Result (Atlas Figure 5-22, Page 41) will be Red Bacteria with Green Endospores, which lends a Festive Touch to this particular Exercise. How to do a Spore Stain without getting slimed by Malachite Green 1. Make your Smear using Bacillus cereus from (A) The Center of a Colony, or (B) The Middle of the A Segment of the Plate. We usually go to the Edge of a Colony to get Bacteria as close as possible to Logarithmic Phase. But for Spores we want Bacteria in Stationary Phase, because that ʼ s when Sporulation occurs. 2. Make your Bacillus cereus Smear thicker than usual. Spore Staining involves steaming the Bacteria underneath a Paper Towel Cover. The Steaming Process itself tends to lift Bacteria from the Slide, plus removing the Paper Towel can all-too-easily “wax” Bacteria off the Slide. 3. Use 3 Layers of Paper Towel to cover your Bacillus cereus Smear The Paper Towel serves two Purposes: (A) It acts as a Spong and allows you to add a large Amount of Malachite Green Stain without the Malachite Green Stain sloshing all over the Slide, and (B) It acts like an Air Bag if the Slide shatters in the Bunsen Burner. Three Layers are recommended. 4. Keep the Malachite Green Stain away from the Clothespin. The Clothespin is merely a Heat-Proof Handle for steaming the Bacteria on your Slide. Whether the Clothespin is clean or dirty doesn ʼ t matter; just keep the Malachite Green from making Contact with the Clothespin. 5. Keep the DI Water Stream away from the Clothespin. One of the other five Students who use your Clothespin may have gotten Malachite Green (or Safranin) on this Clothespin. 6. Thoroughly rinse your Slide with DI Water after the 2 Minute Steaming with Malachite Green Steaming (~80°C) makes the Spore Coat permeable to Malachite Green. Cooling to Room Temperature (~25°C) “locks” the Malachite Green inside the Spores. You cannot rinse with DI Water for too long; the Malachite Green can ʼ t get out. But if you don ʼ t rinse for long enough, Malachite Green will remain in the Vegetative Cells (and you ʼ ll lose Points)....
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