Stain Protocols-BIOL 2420 Acid-Fast Stain 1.Perform a bacterial smear,as discussed in Figure 3-52 on page 150 of your lab manual. 2.Place a small piece of bibulous paper over the smear and saturate the paper with carbolfuchsin. 3.Heat the slide gently over the Bunsen burner for 5 minutes.Be sure to keep the bibulous paper saturated with carbolfuchsin during heating.If the slide is steaming,you're okay;if it stops steaming,add more carbolfuchsin! 4.Remove the bibulous paper from the slide,and rinse the slide gently with water.Dispose of the used bibulous paper in the trash.DO NOT leave the bibulous paper in the sink or drain! 5.Decolorize the slide with acid-alcohol until the rinsate runs clear. 6.Rinse the slide gently with water. 7.Counterstain with methylene blue for 2 minutes. 8.Rinse the slide gently with water. 9.Carefully blot the slide dry with bibulous paper. 10.Observe the slide under the microscope,using proper microscope technique. Acid-fast cells will stain fuchsia.Non-acid-fast cells will stain blue. Endospore Stain 1.Perform a bacterial smear of Bacillus or the organism you want to stain,as discussed in Figure 3-52 on page 150 of your lab manual. 2.Place a small piece of bibulous paper over the smear.Saturate the paper with malachite green. 3.Heat the slide gently over the Bunsen burner for 5 minutes.Be sure to keep the bibulous paper saturated with malachite green during heating.If the slide is steaming,you're okay;if it stops steaming,add more malachite green! 4.Remove the bibulous paper from the slide,and rinse the slide gently with water.Dispose of the used bibulous paper in the trash.DO NOT leave the bibulous paper in the sink or drain! Parent cell or 5.Counterstain with safranin for 2 minutes. bacterium(red) 6.Rinse the slide gently with water. 7.Carefully blot the slide dry with bibulous paper. Endospore (green) 8.Observe the slide under the microscope,using proper microscope technique. Endospores will stain green.Parent cells will stain red. Smith-2010 Page 4 of 4 Stain Protocols – BIOL 2420 Smith – 2010 Page 4 of 4 Acid-Fast Stain 1. Perform a bacterial smear, as discussed in Figure 3-52 on page 150 of your lab manual. 2. Place a small piece of bibulous paper over the smear and saturate the paper with carbolfuchsin. 3. Heat the slide gently over the Bunsen burner for 5 minutes. Be sure to keep the bibulous paper saturated with carbolfuchsin during heating. If the slide is steaming, you’re okay; if it stops steaming, add more carbolfuchsin! 4. Remove the bibulous paper from the slide, and rinse the slide gently with water. Dispose of the used bibulous paper in the trash. DO NOT leave the bibulous paper in the sink or drain! 5. Decolorize the slide with acid-alcohol until the rinsate runs clear. 6. Rinse the slide gently with water. 7. Counterstain with methylene blue for 2 minutes. 8. Rinse the slide gently with water. 9. Carefully blot the slide dry with bibulous paper. 10. Observe the slide under the microscope, using proper microscope technique. Acid-fast cells will stain fuchsia. Non-acid-fast cells will stain blue. Endospore Stain 1. Perform a bacterial smear of Bacillus or the organism you want to stain, as discussed in Figure 3-52 on page 150 of your lab manual. 2. Place a small piece of bibulous paper over the smear. Saturate the paper with malachite green. 3. Heat the slide gently over the Bunsen burner for 5 minutes. Be sure to keep the bibulous paper saturated with malachite green during heating. If the slide is steaming, you’re okay; if it stops steaming, add more malachite green! 4. Remove the bibulous paper from the slide, and rinse the slide gently with water. Dispose of the used bibulous paper in the trash. DO NOT leave the bibulous paper in the sink or drain! 5. Counterstain with safranin for 2 minutes. 6. Rinse the slide gently with water. 7. Carefully blot the slide dry with bibulous paper. 8. Observe the slide under the microscope, using proper microscope technique. Endospores will stain green. Parent cells will stain red. Parent cell or bacterium (red) Endospore (green)