Chapter 3: Clinical use of the Light Microscope (Stains)

Question 1

Why are stains used when viewing microorganisms in a microscope?

Answer 1

• because microorganisms are colourless when seen through a microscope

Question 2

What are stains composed of? (2)

Answer 2

• positively and negatively charged ions
• one of which is coloured - chromophore

Question 3

What is a simple stain?

Answer 3

• only one dye used to highlight the entire microorganism

Question 4

What are the steps to a simple stain?

Answer 4

1. Smear the sample (organism) of the slide
2. Fix with heat
3. Add stain
4. Wash, dry and view

Question 5

How do stains work? (2)

Answer 5

• bacteria have a net negative charge on their outer surface
• This charge attracts stains with positively charged chromophores, and repels stains with negatively charged chromophores

Question 6

What is a chromophore?

Answer 6

it is the dye/stain

Question 7

What will occur to positive stains? (2)

Answer 7

• Stains will bind to the bacterium
• bacterium appears coloured
• background appears clear
  ex. crystal violet

Question 8

What will occur to negative stains? (3)

Answer 8

• will not bind to the bacterium
• bacterium appears clear
• background in coloured
  ex. nigrosin

Question 9

What are differential stains?

Answer 9

• react differently with different bacteria, thus can be used to distinguish between them
  ex. Gram stains

Question 10

What are gram stains?

Answer 10

• differentiates bacteria based on the structure of the cell wall

Question 11

What are gram positive stains?

Answer 11

• Bacteria with a thick cell wall retain the primary stain colour violet and appear purple
  ex. Streptococcus Pyogenes

Question 12

What are gram negative stains?

Answer 12

• Bacteria wth a thin cell wall lose crystal violet during destaining
• take on the colour of the counterstain safranin and appear pink
  ex. E. coli

Question 13

What are spore stains?

Answer 13

• stains an internal structure of some bacteria

Question 14

What will the primary stain of spore stains colour the endospores?

Answer 14

• will colour the endospores green

Question 15

What will the counterstain (safranin) colour the rest of the cell in a spore stain?

Answer 15

• red (pink)
  ex. bacillus anthracis (gram +)

Question 16

What is a flagella stain? (2)

Answer 16

• Stains an external structure
• Mordant thickens the flagella so they can be observed under a light microscope
  I think mordants are dye

Question 17

What is an acid-fast stain used for? (2)

Answer 17

• detects the presence of a waxy compound in the cell wall
• used to identify the genus Mycobacterium
  ex. Mycobacterium tuberculosis

Question 18

In an acid-fast stain, what will the mycobacterium retain?

Answer 18

• retains the dye carbol fushcin

Question 19

What will counterstain an acid-fast stain?

Answer 19

• methlene blue stains non-acid fast bacteria and tissues blue

Question 20

Is mycobacterium gram + or gram -? (3)

Answer 20

• neither!!!
• they have a completely different cell wall
• mycolic acid in the cell wall and therefore is not stained

Question 21

What is a capsule stain?

Answer 21

• detects a thick layer of polysaccharide outside the cell (capsule)

Question 22

What will staining do in a capsule stain? (3)

Answer 22

• A negative stain colours the background
• a positive stain colours the cell
• the capsule does not take up most dyes and remains colourless
  ex. streptococcus pneumoniae

Question 23

Can a capsule be present in both gram positive and gram negative stains?

Answer 23

yes

Question 24

How would we identify a capsule under a microscope?

Answer 24

a halo means there is a presence of a capsule