Gram Stain and Colonial Morphology

Question 1

What happens to a bacteria when it has an abundance of nutrients?

Answer 1

The bacteria will divide exponentially

Question 2

What is a colony?

Answer 2

A group of thousands to billions of bacteria cell that have become visible.

Question 3

What is another word for a colony?

Answer 3

Clone

Question 4

What is a visible group of thousands/billions of bacteria cells called?

Answer 4

A colony or a clone

Question 5

What is colonial morphology?

Answer 5

The characteristics of a colony

Question 6

What are the characteristics of a colony known as?

Answer 6

Colonial morphology

Question 7

Under what headings is the colonial morphology of a bacteria described.
(9)

Answer 7

Form

Margin

Elevation

Size

Surface

Consistency

Colour

Optical consistency

Odour

Question 8

What does the gram stain differentiate bacteria into?

Answer 8

Gram positive and gram negative

Question 9

Besides being gram positive or gram negative, what does a gram stain show us?

Answer 9

It shows us the cellular morphology of the organism

Question 10

What does the gram reaction depend on?

Answer 10

It depends on the cell wall differences of peptidoglycan

Question 11

What colour is peptidoglycan stained?

Answer 11

It is stained purple

Question 12

What stains the peptidoglycan purple during a gram stain?

Answer 12

The crystal violet (CV) stains the peptidoglycan purple

Question 13

Describe the amount of peptidoglycan in gram positive bacteria and what affect this has.

Answer 13

Gram-positive cell walls have a thick later of peptidoglycan, approximately 90% of the cell wall is peptidoglycan

Question 14

How do gram positive bacteria resist decolourisation?

Answer 14

The cell wall of gram positive bacteria has numerous teichoic acid cross-linking which resists decolourisation

Question 15

Do gram positive or gram negative bacteria have a cell wall made of 90% peptidoglycan?

Answer 15

Gram positive bacteria do

Question 16

What percent of a gram negative bacteria’s cell wall is peptidoglycan?

Answer 16

About 10% of their cell wall is peptidoglycan

Question 17

Why is iodine used after crystal violet>

2

Answer 17

The addition of iodine creates a large CV-I complex which is not easily dislodged from the thick peptidoglycan layers

It ensures the colour from the crystal violet will remain after decolourisation of gram positive bacteria

Question 18

What is the decolourising agent used during a gram stain?

Answer 18

Ethanol

Question 19

Why is ethanol added after the iodine?

Answer 19

Ethanol interacts with the lipids of the membranes of all bacteria.

The ethanol has one of two affects on the bacteria depending on whether it is gram positive or negative

Question 20

What happens when ethanol is added to gram negative bacteria?

Answer 20

In gram negative bacteria the cell wall is dissolved by the ethanol, leaving the thin layer of peptidoglycan exposed and leaky

Question 21

What happens when ethanol is added to gram positive bacteria?

Answer 21

In gram positive bacteria the highly cross-linked peptidoglycan dehydrates and traps the large CV-I complexes (purple colour remains)

Question 22

What is added after the ethanol?

Answer 22

Carbol fuchsin is added

Question 23

Why is carbol fuchsin added?

Answer 23

Carbol fuchsin gives a pink colour to the gram negative bacteria who’s peptidoglycan is now exposed

Question 24

Give the order of the reagents used in a gram stain.

Answer 24

Crystal violet

Iodine

Ethanol

Carbol Fuchsin

Question 25

How do you emulsify a colony for gram staining?

3

Answer 25

Using a sterile loop prepare a smear of the bacteria by placing 2-3 drops of water on a slide

Then using a sterile loop pick up a well defined colony

Emulsify the colony in the water to create a smear the size of a 1 euro coin

Question 26

What should you do after you have emulsified a colony for gram staining?
(2)

Answer 26

Allow the smear to air dry, there should be a visible change

Then using a forceps pass the slide through a Bunsen flame to fix the bacteria