3. Does the stain penetrate the bacteria? Why or why not? 4. Briefly describe ho

Question

3. Does the stain penetrate the bacteria? Why or why not? 4. Briefly describe how to prepare a smear using heat fixation. 5. What are the two main purposes of heat fixation? 6. Why is it important to the slide air dry before heat fixing? 7. Describe another method of fixing a smear other than heat fixing. 8. During heat fixing, what would happen to the bacteria in the smear if a. too little heat were applied? b. too much heat were applied? 9. A small group of bacterial species has a preponderance of positive charges on the outside of their cells. How would these cells stain with a dye such as crystal violet? Why? 10. Why is it preferred to use an inoculating loop when making smears from liquid media?

Explanation / Answer

1) The molecules of stain like crystal violet are very small in size that allows it to easily penetrate by diffusion through the cell wall of bacteria.

2) Prepare a thin smear of bacteria on a glass slide by taking a small inoculum from the fresh. fix the slide by passing it (cell side up) through a flame to warm the glass. Do not let the glass become hot to the touch.

3) two purpose of hit fixation is 1) it denatures bacterial enzyme which may digest the cells part, therefore, preventing them from autolysis . and 2) it firmly adheres the bacteria to the slide and therefore, allow the sample to take up stain more readily.

4) It is very important to the slide air dried before heat fixing .becaue heat fixing a wet or damp slide will cause cell lysis and therefore, will break the cell wall of bacteria.

5) bacterial cell can be fixed on a slide by using absolute methanol. prepare the smear followed by air dry. flood little amount (200ul-300ul) on the smear and keep it for 2min. Decant the excess methanol in proper waste disposal container followed by air drying and primary staining.

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