Question

1. Why do acid-fast organisms give gram-variable results?

2. Explain how the negative staining technique works.

3. Which waterborne parasite oocysts can be identified using staining techniques?

4. When should you used negative staining instead of a simple stain?

5. Give an example of a bacterial structure that can't be staied with basic dyes but can be seen with negative staining techniques.

6. What are other arrangements of flagella, and why can't flagella be observed with a simple stain?

Answer 1

Ans 1: These microorganisms have a waxy cell wall due to mycolic acid (a lipid) that makes them resistant to decolorization. Acid-fast organisms give Gram variable results due to this high lipid content (mycolic acid) in their cell wall.

2: Negative stain is a structural stain, used to see the shape and size of microorganisms that cannot withstand heat-fixing. The main purpose of Negative staining is to study the morphological shape, size and arrangement of the bacteria cells that is difficult to stain.

Negative staining requires an acidic dye such as India Ink or Nigrosin. India Ink or Nigrosin is an acidic stain. This means that the stain readily gives up a hydrogen ion (proton) and the chromophore of the dye becomes negatively charged. Since the surface of most bacterial cells is negatively charged, the cell surface repels the stain. The glass of the slide will stain, but the bacterial cells will not. The bacteria will show up as clear spots against a dark background.

3: Cryptosporidium is a waterborne parasite oocysts that can be identified using staining technique. The best common technique of detecting Cryptosporidiosis is acid-fast staining,  as Cryptosporidium is acid-fast, it retains a red/pink color.

4: The negative stain is particularly useful for determining cell size and arrangement. It can also be used to stain cells that are too delicate to be heat-fixed. Example: Spirilla. So for staining the structural component or to stain heat sensitive bacterial species, we use negative stain.

5:  Certain bacteria and yeasts have a protective outer structure called a capsule. Capsules do not absorb most basic dyes; therefore, a negative staining technique (staining around the cells) is typically used for capsule staining.

6: There are basically four different types of flagellar arrangements:

a. A single flagellum can extend from one end of the cell - if so, the bacterium is said to be monotrichous.

b. A single flagellum (or multiple flagella; see below) can extend from both ends of the cell - amphitrichous.

c. Several flagella (tuft) can extend from one end or both ends of the cell - lophotrichous

d. Multiple flagella may be randomly distributed over the entire bacterial cell - peritrichous.

Flagella are very thin, so they don't retain much stain and this makes the simple staining non useful for observing flagella.