5. Why is it necessary to pass the bacterial smear through the flame before stai

Question

5. Why is it necessary to pass the bacterial smear through the flame before staining? 6. Why is the timing of the decolorization by alcohol so important? After the alcohol treatment, what color is a Gram positive organism? What color is a Gram negative organism? 7. 8. Is the Gram stain useful in observation of spore forming bacteria? Explain. 9. Name another Gram positive organism Name another Gram negative organism. 10. Why would methylene blue not work as well as safranin for counterstaining in the Gram stain procedure? What part of the bacterial cell is responsible for whether or not an organism is Gram positive? 11. 46

Explanation / Answer

5. It is useful to evaporate excess water, fix, and adhere the bacterial cells in glass slide before staining. Hence, the bacteria are not washed away during washing steps.

6. Decolorization step involves the cell wall to dehydrate and shrink which closes the pores in the cell wall and prevents the stain from exiting the cell. Therefore, overtime of decolorization results in the loss of the primary stain, causing Gram positive bacteria to appear Gram negative.

7. Gram positive bacteria retain the primary stain that colored purple and Gram negative bacteria will be colorless since it leaks crystal violet-iodine complex after alcohol treatment.

8. In general Gram staining is used to divide eubacteria into two large groups; Gram positive and Gram negative. Most of the spore forming bacteria is Gram positive but still their spores are difficult to destroy with heat or other physical and chemical disinfecting agents. Therefore, endospore staining is an appropriate technique than Gram stain.

9. Gram positive organism – Saphylococcus aureus, Streptococcus pneumonia

    Gram negative organism – Escherichia coli, Haemophilus influenzae

10. Since, methylene blue stains the cells as same as the color produced by primary stain, it is difficult to differentiate gram positive bacteria from gram negative bacteria.

11. The cell walls for Gram positive microorganisms have a higher peptidoglycan and lower lipid content than gram negative bacteria. While staining and subsequent washing in gram positive bacteria retain the stains since it has thick cell wall. But gram negative cells lose primary stain after alcohol treatment due to removal of lipid layer.

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