Staining Techniques (Lab)

Question 1

Why do we need to stain bacterial cells?

Answer 1

They’re transparent and hard to see

Question 2

What are the 4 types of stains we used in this lab?

Answer 2

1. Simple stains
2. negative stains
3. Differential stains
4. structural stains

Question 3

What are simple stains?

Answer 3

General stains that stain whole cells and stain every cell

Question 4

How do simple stains work?

Answer 4

They are often positively charged and will interact with the negatively charged membrane

Question 5

What type of stain is methylene blue?

Answer 5

Simple stain

Question 6

What type of stain is crystal violet?

Answer 6

Simple stain

Question 7

What type of stain is safranin?

Answer 7

Simple stain

Question 8

What are negative stains?

Answer 8

Stains that stain the background instead of the cell, so the cell sticks out like a silhouette against a dark background

Question 9

What are the 2 types of negative stains?

Answer 9

Colloid stains and acidic stains

Question 10

How do colloid stains work?

Answer 10

Dark particles of ink that are neutrally charged but are physically too big to get into the cell

Question 11

How do acidic stains work?

Answer 11

Negatively charged stains that are repelled by the cell envelope

Question 12

What type of stain is India Ink?

Answer 12

Colloid negative stain

Question 13

How do differential stains work?

Answer 13

Use multiple stains that will bacteria with different properties differently

Question 14

What type of staining is gram staining?

Answer 14

Differential. Gram-positive bacteria look purple because they retain the CV-I complexes, and gram-negative bacteria look pink because they become counterstained by safranin

Question 15

What type of staining is acid fast staining?

Answer 15

Differential. Acid-fast bacteria look pink and everything else is counterstained by methylene blue

Question 16

What are the 3 dyes used in gram staining?

Answer 16

Crystal violet, iodine, safranin

Question 17

What does the crystal violet stain do in gram staining?

Answer 17

Primary stain. Stains everything

Question 18

What does the iodine do in gram staining?

Answer 18

Mordant. Forms complexes with the crystal violet

Question 19

What does the ethanol wash do in gram staining?

Answer 19

Dehydrates the cell membrane and causes gram-negative cells to lose the CV-I complexes while gram-positive cells have thicker cell walls and retain them

Question 20

What does the safranin do in gram staining?

Answer 20

Counterstains the gram negative cells that lost the CV-I complexes

Question 21

What causes over-decolourization in gram-staining?

Answer 21

Washed with ethanol for too long, and caused the gram-positive cells to lose the CV-I complexes

Question 22

What happens to a gram stain if there was too much heat fixation?

Answer 22

The cell walls and membranes get destroyed and the CV-I complexes don’t get retained by anything, so everything looks gram negative

Question 23

What happens to a gram stain if the smear was too thick ?

Answer 23

The stains can’t get inside the layers, so we get a mixture of purple and pink

Question 24

What happens to a gram stain if there was improper washing between the steps?

Answer 24

The extra CV-I complexes don’t get washed away and everything looks purple

Question 25

How does culture age affect gram stains?

Answer 25

Older cultures have more dead cells, which don’t have intact membranes and look pink from the counterstain

Question 26

What happens to a gram stain if there was an impure culture?

Answer 26

Mixture of both colours

Question 27

Why can’t acid fast bacteria be gram stained?

Answer 27

The mycolic acid cell wall keeps all the stains out

Question 28

How are acid fast bacteria stained?

Answer 28

The cells are heated with carbolfushin in phenol, which stains them pink. Wash with a mixture of acid and alcohol so only the acid fast cells retain the stain, then counterstain everything else with methylene blue

Question 29

What do structural stains stain?

Answer 29

Structural features like flagella, capsules, or endospores

Question 30

How do you get a stain into an endospore?

Answer 30

Malachite green + heat and steam