4. Following heating, discard the paper form the slide surface and genuy was WD

Question

4. Following heating, discard the paper form the slide surface and genuy was WD running water 5, Counter-stain the smear with 0.5% aqueous Safranin for 1 minute. 6. Tip the dye into the collection bowl. Rinse with water and carefully blot dry 7. Examine your slide with the 100x oil immersion objective Remember to start at 4x and move you way up in magnification to the 100x objective. a. Notes: Vegetative cellis stain pink/red in this procedure, whereas endospores stain green/bi Some endospores may be seen within the former vegetative portion of the cell ***Also preparc Gram stains of the endosporc forming and the non-spore forming bacteria the cultures provided for this exercise. Questions 1) Can everything on your slides be identified by Gram stain? Explain your answer 2) Why was a basic solution then an acidic solution used for the Acid-fast stain? 3) What environmental conditions might trigger endospore formation? 4) Why do the stained endospores appear difterent from the vegetative celils? Bacillus

Explanation / Answer

Q.1

Only Gram positive and gram negative bacteria can be identified and distinguished by gram staining method. This technique is not applicable for Fungi and protozoa. There are other micro-organism such as Mycobacterium tuberculosis which can be stained by acid fast staining only but not by gram staining. So the answer is No, not every organism in the slide can be stained by gram staining.

Q.2

The outer layer of the mycobacterium is covered by a large amount of lipids known as mycolic acid.

Initially the cells are stained with carbol fuchsin. Cells appear red after staining.

Then decolourizing agent which is an acid-alcohol is added.

Cells which doesn’t have mycolic acid (non-acid-fast organism) will get decolourize while adding decolourizing agent. Whereas acid-fast organism such as mycobacterium which has mycolic acid in its outer layer will retain the color.

Q.3

Nutrient depleted or harsh environment such as high/low pH, availability of oxygen triggers the formation of endospore.

Q.4

Malachite green, a water soluble compound is used as the primary staining in endospore staining. After the staining the sample, decolourizing agent - water is added. Spores retain the dye while vegetative wont. Then safranin a counter stain is added to stain the decolourized vegetative cells. Hence vegetative cells appear pink and endospore will appear dark green.

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