Simple Stain Lab 1. What is the purpose of a simple stain? The purpose of a simple stain is to put color into the cytoplasm (bacteria cell) to enhance our ability to observe it. 2. What is the purpose of heat fixing the smear? Heat fixation adheres the cells to the slide and coagulates the bacterial proteins, effectively killing the bacteria. 3. Another method of fixing smears is to use methanol instead of heat. How does methanol chemically fix the bacteria? Methanol chemically fixes the bacteria by denaturing the enzymes which prevents lysis. 4. In heat fixing what would happen if too much heat were applied? If too much heat was applied during heat fixing, the heat could damage the organism. 5. Methylene blue can be prepared as a basic stain or an acidic stain. How would the pH of the stain affect the staining of bacteria? The pH of the stain will determine if the bacteria will have a particular charge. (e.g. acidic is negative) 6. Can dyes other than methylene blue be used for staining? Briefly explain. Yes, crystal violet, basic fuchsin and safranin are all dyes that can be used in direct staining because they are cationic with a positive charge. 7. Bacteria can be seen without staining. Why are fixing and staining important? Because staining enhances our ability to observe the bacteria. 8. What mistakes do you need to avoid when doing a simple stain? Do not apply excess force when blotÝng dry, Do not rub bacteria smear off slide when blotÝng dry, and do not rinse with alcohol. 9. Quality control staff in a sterilization unit of a hospital used a simply stain to determine whether bacteria were present in sterilized materials. A simply stain of sterile saline used for respiratory therapy revealed the presence of bacteria. Is the saline contaminated? Yes it is, the H2O should be free of bacteria. The bacteria could only come from saline.