Question

LAB REPORT 2- Selected questions from Exercise 3-7 (Acid-fast stain) Note: This is turned in after you have viewed your acid fast stain slides. Bacteria for this exercise are: Mycobacterium smegmatis (or M. phlei) and S. aureus. Q1. Acid fast bacteria are have some significant differences as compared to non-acid fast bacteria. What are the major chemical (structural) differences among these different bacteria that might explain the acid-fast stain? Hint: This structural difference also affects the way the bacteria grow on media (both broth and media containing agar) and the length of time they take to grow. Q2. Why do you suppose the acid fast stain in not as widely used as the Gram stain? When is it more useful than the Gram stain? Can you name the bacterial genera that are considered to be acid fast? Q3. (Use the lab manual to answer this) How does heating the bacterial smear during a Ziehl-Neelson stain promote entry of carbel frshsin into the acid fast cell wall? Why is the Kinyeun procedure referred to as the "cold" acid fast stais procedure? Explain. Q4. Are acid fast negative bacteria stained by carbol fuchsin? If so, explain why the acid fast stain is considered to be a differential stain? LAB REPORT 3-Selected Festions from Exercise 3-9 and hybrid of Ex. 3-5/3-8 (Spore and capsule stain exercises) Note: This lab report is turned in after you have viewed the slides for both the spore stain and the capsule stain (sometimes the capsule stain is dene as a demonstration but you are responsible for looking at a capsule stain slide and knowing how it is done and important to remember, we are using the nigrosin stain procedure as described

Answer 1

Answer1: The major significant difference between the acid-fast bacteria and non-acid fast stain bacteria are:

1. Acid-fast bacteria possess the mycolic acids in their cell wall whereas, non-acid fast bacteria do not possess mycolic acid.

2. Acid-fast bacteria also contains the fatty acids and complex form of lipids in large amounts.

3. They are resistant for certain conditions like disinfectants and dry conditions for which they are different than other types of bacteria.

4. They are needed special methods of staining like acid-fast staining because of its different properties.

Answer 2: Acid-fast stain is not as widely used other types of staining techniques because of the presence of mycolic acids and fatty acids in large amount as well as complex lipids that are present in them. All these features together form the cell wall so resistant for most of the staining compounds. In that case, it requires special kind of staining technique i.e. Acid-fast staining is appropriate for them to be used.

This particular method differentiates between the acid-fast and non-acid fast bacteria effectively.

The bacterial genera considered to be acid-fast organism is the member belonging to genera Mycobacterium (M. leprae, M. tuberculosis).

Answer3: By using the Ziehl-Neelsen method, the primary stain colour used is the compound called carbolfuchsin and it is a lipid-soluble compound. This compound contains the phenol that helps the stain to cross or penetrate the cell wall. Also, this is further treated with heat. After this step, the strong decolourizer is used like some acid alcohol. This decolourizer removes the strain from all non-acid fast bacteria. After washing off, the slide of bacteria is further treated with counterstain i.e. methylene blue usually. This stains the acid-fast bacteria red in colour whereas, the non-acid fast remains blue in colour.

While using the Kinyoun method, the heat treatment step for the bacteria is not needed and hence, the name i.e. cold method of staining. In this usually, the concentration for the carbolfuchsin is increased. In this staining method, the primary stain used is carbolfuchsin, the decolourizer used is acid alcohol and the counterstain remains same i.e. methylene blue.

This particular method of staining involves modification i.e. it uses sulfuric acid instead of hydrochloric acid. This helps the method to even stain for other organisms which are not able to take stains of carbolfuchsin after treatment with HCL.

Answer4: Acid-fast stain bacteria can be stained with this differential staining method because of its differential testing i.e. removal of colour as carbolfuchsin. This can be very easily removed from the cell walls of the negative cells i.e. acid-fast negative bacteria. Although, this is different in case for acid-fast positive organisms as they possess the mycolic acids.