Chapter 3: Staining

Question 1

Staining

Answer 1

• Bacteria are negatively charged with a pH of 7.
• Bacteria are stained with basic stains
  What is a stain?
  Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes.
  Chromosphere: a very intense dyed color
  Acidic vs. Basic dye:
  Staining the background instead of the cell is called negative staining

Question 2

Different Types of Stains

Answer 2

Simple/Differential Stain
 a. Gram Stain
 b. Acid Fast Stain
Special Stains
a. Capsule Stain
b. Endospore

Crystal Violet is a mordant
A mordant is a substance (like iodine) that enlarges the specimen.

Question 3

Gram Stain

Answer 3

• Gram stain: classifies bacteria into gram positive or gram negative.
• H.C. Gram 1888
  1. Primary Stain: Crystal Violet ~1
  a. Results in purple for Gram + and Gram -
  2. Mordant: Iodine
  a. Results in purple for Gram + and Gram -
  3. Decolorizing agent: Alcohol-acetone or 95% ethanol.
  a. Purple for Gram +, Colorless for Gram -
  4. Counterstain: Safranin
  a. Purple for Gram +, red for Gram -
  Gram +
  1. Has thick PG (cell wall)
  2. Forms a CV + I2 complex
  3. Doesn’t escape the wall

Gram -

1. Has only 1 layer of PG (thin cell wall)
2. CV + I2 complex
3. Outer membrane falls apart
4. Forms holes, CV+I2

Question 4

Peptidoglycan

Answer 4

Abbreviated PG

- Forms a mesh-like layer outside the plasma membrane of most bacteria, forming the cell wall.

Question 5

Acid Fast

Answer 5

Ziehl-Neelsen Stain

• Can be used to detect tuberculosis and leprosy-causing organisms of the Mycobacterium species.
• A thick layer of PG
• They use this technique because they have an extra layer fo mycolic acid.
  1. Primary stain: Carbolfuchsin
  a. Heat for 10-15 minutes over boiling water
  2. Decolorizing agent: Acid-alcohol
  3. Counterstain: Methylene blue

Acid Fast Non-Acid Fast
1. red 1. red
2. red 2. colorless
3. red 3. blue
If red, it’s leprosy or TB

Question 6

Capsule Stain

Answer 6

Special stain

1. Primary stain: Acidic stain - India ink, nigrosin
2. Decolorize: Water
3. Counterstain: Safranin

Will have a blue background and red cells.

Question 7

Endospore

Answer 7

Special stain (Scheffer-Fulton Staining)

1. Primary: Malachite green for ~ 5 mins
2. Decolorize: wash with water
3. Counterstain: Safranin

Green cells – small green dots – red cells with green centers

Question 8

Flagella Staining

Answer 8

Special stain

1. Carbol fuchsin
2. Potassium Alum (mordant)

Question 9

Mordant

Answer 9

A substance, typically an inorganic oxide, that combines with a dye or stain and thereby fixes it in a material.