Acid-fast/ Endospore/ Capsule staining

Question 1

Why are acid-fast bacteria named that?

Answer 1

They will retain the primary stain even after treated with acid alcohol

Question 2

What do the cell walls of acid fast bacteria contain?

Answer 2

mycolic acid

Question 3

What is mycolic acid?

Answer 3

a waxy lipid that makes the cell impermeable to most stains

Question 4

What does mycolic acid do?

Answer 4

protects the bacteria from desiccation and prevents digestion from phagocytes

Question 5

What two genus’ are involved with acid-fast bacteria?

Answer 5

Mycobacterium and Nocardia

Question 6

What are two examples of acid-fast bacteria?

Answer 6

Mycobacterium tuberculosis and Mycobacterium leprae

Question 7

What are the steps of an acid-fast stain?

Answer 7

1. normal procedures with heat fix etc. 2. bacteria are stained with carbolfuchsin 2. acid alcohol is then added 3. methylene blue is then used as a counterstain

Question 8

What happens to non acid-fast bacteria when acid alcohol is added?

Answer 8

they are quickly destained

Question 9

What happens to acid-fast bacteria when acid alcohol is added?

Answer 9

They hold onto the carbolfuchsin because the carbolfuchsin has phenol in it and phenol is more soluble in in fat vs acid alcohol. The lipid in mycolic acid will retain the dye even after the acid alcohol rinse.

Question 10

What happens to the non acid fast bacteria when methylene blue is added?

Answer 10

they will be stained blue

Question 11

What happens to the acid-fast bacteria when methylene blue is added?

Answer 11

Nothing. They will remain red since the mycolic acid is impermeable to methylene blue.

Question 12

What are vegetative bodies?

Answer 12

bacteria that are actively metabolizing

Question 13

What genus’ produce endospores?

Answer 13

Clostridium and Bacillus

Question 14

What are four examples of diseases caused by endospores?

Answer 14

C. tetani, C. botulinum, B. anthracis, C. difficile

Question 15

What are four characteristics of endospores?

Answer 15

1. smaller and more compact than vegetative bodies
2. thick outer layer of keratin
3. are resistant to heat, drying, detergents, harsh environments, UV
4. are not actively metabolizing

Question 16

Why do endospores form?

Answer 16

Harsh environments make them do this as a survival mechanism

Question 17

What happens when the environment is more appealing to the endospore?

Answer 17

It will germinate and re-form the vegetative body.

Question 18

Making an endospore is NOT ______, it is a ______.

Answer 18

reproductive mechanism, survival mechanism

Question 19

What are the steps of an endospore stain?

Answer 19

A standard fixed smear is prepared and then malachite green is added and gently heated over a flame for 5 minutes. Malachite green is then rinsed off and counterstain safranin is added for 2 minutes and then rinsed and dried.

Question 20

Why must the stain be gently heated during an endospore stain?

Answer 20

since the endospores have a thick layer of keratin, heating is required to allow for stain penetration

Question 21

What does safranin stain during an endospore stain?

Answer 21

It stains the vegetative bodies

Question 22

What are the four characteristics of capsules?

Answer 22

2. gelatinous coat surrounding certain bacteria
3. composed primarily of uncharged polysaccharides, some polypeptides
4. size is influenced by the media the organism is cultured in
5. increases bacterial virulence

Question 23

What are the four ways capsules increase bacterial virulence?

Answer 23

1. help evade phagocytosis
2. help bacteria attach to cause infection
3. function as an osmotic barrier, evade desiccation
4. function as a nutrient reserve

Question 24

What are the steps for a capsule stain?

Answer 24

Prepare a standard negative stain with Congo Red. Then flood the slide with 0.1N HCl as a fixitive. Then add safranin.

Question 25

What does congo red stain during a capsule stain?

Answer 25

the background only

Question 26

What does safranin dye during a capsule stain?

Answer 26

it stains the cell body only. This is because the capsule has no charge so a basic dye will not stain it.

Question 27

How does the capsule appear during a capsule stain?

Answer 27

as a halo around the stained bacterium against the dark background.