2.1.1c The Use of Staining in Microscopy

Question 1

Why do we need to stain samples in microscopy?

Answer 1

The cytosol of cells and other cell structures are often transparent. Stains increase ocntrast as different components within a cell take up stains to different degrees. The increase in contrast allows components to become visible so they can be identified.

Question 2

How do you prepare a sample for staining?

Answer 2

Place the sample on a slide and allow it to air dry, then heat-fix it by passing it through a flame

Question 3

What is the charge on crystal violet and methylene blue dyes and how do they work?

Answer 3

These dyes are positively charged and so are attracted to the negatively charged materials in cytoplasm, leading the the staining of cell components.

Question 4

What is the charge on nigrosin and Congo red dyes and how do they work?

Answer 4

These dyes are negatively charged and so are repelled by the negatively charged cytosol (aqueous cell interior). These dyes stay outside of the cell, leaving the cells unstained, which make them stand out against the stained background.

Question 5

Differential staining

Answer 5

where more than one dye is used to differentiate between different types of microorganisms on a slide

Question 6

What is the Gram stain technique used for?

Answer 6

to seperate bacteria into Gram- positive and Gram-negative bacteria

Question 7

Gram stain technique method

Answer 7

• Crystal violet is applied to a bacteria specimen on a slide
• Then iodine is added, which fixes the dye
• The slide is then washed with alcohol
• Gram-positive bacteria retain the crystal violet stain and will appear blue or purple under a microscope
• Gram-negative bacteria have thinner walls and so lose the stain
• Gram-negative bacteria are then stained with safranin dye, which is a counterstain
• Gram-negative bacteria will now appear red

Question 8

What is the Acid-fast stain technique used for?

Answer 8

to differentiate species of Mycobacterium from other bacteria

Question 9

Acid-fast staining technique method

Answer 9

• A lipid solvent is used to carry carbolfuchsin dye into the cells being studied
• The cells are then washed with a dilute acid-alcohol solution
• Mycobacterium are not affected by the acid-alcohol and retain the carbolfuchsin stain, which is bright red
• Other bacteria lose the stain and are instead exposed to methylene blue