Practical 3 - Techniques for staining bacteria

Question 1

Visualisation of living microorganisms is difficult, not only because they are minute, but also because they are…

Answer 1

Transparent and practically colourless when suspended in an aqueous solution

Question 2

Staining methods can be either

Answer 2

Simple or differential

Question 3

What is the difference between simple and differential staining?

Answer 3

Simple staining shows you the morphology of the organisms whereas differential staining allows different types of bacteria to be separated

Question 4

What is the first step in any staining procedure?

Answer 4

To prepare a smear on a slide

Question 5

When preparing a smear on a slide the aim is to produce a…

Answer 5

Unicellular layer of organisms

Question 6

Why is a unicellular layer of organisms (thin smear) important when preparing a smear on a slide?

Answer 6

This allows their shape and size to be clearly seen

Question 7

How is a bacterial smear fixed?

Answer 7

Passed through a bunsen flame three times

Question 8

Why is it important to fix a bacterial smear onto the slide?

Answer 8

The smear will wash away during the staining procedure otherwise

Question 9

What does heat fixation serve to do?

Answer 9

To kill the bacteria and cause them to adhere to the surface of the slide - the bacterial proteins coagulate and fix to the surface of the slide

Question 10

In simple staining, the bacterial smear is stained with a…

Answer 10

single reagent

Question 11

In simple staining what can be seen?

Answer 11

A distinctive contrast between the organism and its background

Question 12

Why are basic stains with a positively charged chromogen preferred in simple staining?

Answer 12

The bacterial nucleic acids and certain cell wall components carry a negative charge that strongly attracts and binds to the cationic chromogen

Question 13

Name the three most commonly used basic stains

Answer 13

1. Methylene blue
2. Crystal violet
3. Carbol fuchsin

Question 14

Negative staining requires the use of an ______ stain

Answer 14

acidic

Question 15

Give an example of a stain that can be used for negative staining

Answer 15

Nigrosin

Question 16

Acidic stains have a ________ charged chromagen

Answer 16

negatively

Question 17

Why do acidic stains not penetrate the cells of bacterial cells?

Answer 17

The bacterial cells have a negative charge on the bacterial cell surface

Question 18

In negative staining the unstained bacterial cells are easily seen on a

Answer 18

coloured background

Question 19

What is an advantage of no heat fixation step in negative staining?

Answer 19

The bacterial cells natural size and shape can be observed

Question 20

Why should the bacteria be handled with care in negative staining?

Answer 20

They are still alive

Question 21

Some bacteria are difficult to stain such as some spirilli, why is negative staining ideal here?

Answer 21

Because heat fixation is not carried out

Question 22

What is the most important staining procedure used in determinative bacteriology?

Answer 22

Gram stain

Question 23

The Gram stain reaction divides bacteria into

Answer 23

Gram positive and gram negative

Question 24

The staining reaction in gram stain depends upon the chemical ad physical structure of the bacterial

Answer 24

cell wall

Question 25

Gram positive bacteria have walls which will retain

Answer 25

crystal violet

Question 26

Gram negative bacteria cells fail to retain the

Answer 26

crystal violet

Question 27

What is the primary dye?

Answer 27

Crystal violet

Question 28

What is the mordant?

Answer 28

Iodine solution

Question 29

The iodine combines with the crystal violet in the cells to form a compound referred to as…

Answer 29

The crystal violet-iodine complex

Question 30

What is used as the decolouriser?

Answer 30

Ethanol

Question 31

Gram negative bacteria are counterstained with…

Answer 31

safranin

Question 32

Gram positive bacteria should appear purple because they will have retained the

Answer 32

CVI complex during the ethanol treatment

Question 33

Gran negative bacteria should appear red since they will have been

Answer 33

decolourised by the ethanol and would have taken up the safranin

Question 34

What method can be used to differentiate between the bacterial spore and vegetative cell forms?

Answer 34

Schaeffer-Fulton method

Question 35

Give an example of an anaerobic bacteria which is able to form endospores

Answer 35

Clostridium

Question 36

Give an example of aerobic bacteria which are able to form endospores

Answer 36

Bacillus

Question 37

What are endospores?

Answer 37

Highly resistant, metabolically inactive cell types, when the environment becomes unfavourable they form and remain to give rise to the metabolically active vegetative cell form when the right conditions return

Question 38

What is the endospore surrounded by?

Answer 38

Impervious layers called the spore coat

Question 39

Is heat fixation required in the Schaeffer-fulton method?

Answer 39

Yea babes

Question 40

What colour will the endospores stain and what colour will the rest of the cell stain?

Answer 40

Endospore - green

rest of cell - pink/red

Question 41

What is the primary stain used in the Schaeffer fulton method used for staining endospores?

Answer 41

malachite green,

Question 42

What is the counterstain used in the schaeffer fulton method for staining the rest of the cell?

Answer 42

safranin