Practical 3 - Techniques for staining bacteria Flashcards Preview

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Flashcards in Practical 3 - Techniques for staining bacteria Deck (42):
1

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

Transparent and practically colourless when suspended in an aqueous solution

2

Staining methods can be either

Simple or differential

3

What is the difference between simple and differential staining?

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

4

What is the first step in any staining procedure?

To prepare a smear on a slide

5

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

Unicellular layer of organisms

6

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

This allows their shape and size to be clearly seen

7

How is a bacterial smear fixed?

Passed through a bunsen flame three times

8

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

The smear will wash away during the staining procedure otherwise

9

What does heat fixation serve to do?

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

10

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

single reagent

11

In simple staining what can be seen?

A distinctive contrast between the organism and its background

12

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

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

13

Name the three most commonly used basic stains

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

14

Negative staining requires the use of an ______ stain

acidic

15

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

Nigrosin

16

Acidic stains have a ________ charged chromagen

negatively

17

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

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

18

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

coloured background

19

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

The bacterial cells natural size and shape can be observed

20

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

They are still alive

21

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

Because heat fixation is not carried out

22

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

Gram stain

23

The Gram stain reaction divides bacteria into

Gram positive and gram negative

24

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

cell wall

25

Gram positive bacteria have walls which will retain

crystal violet

26

Gram negative bacteria cells fail to retain the

crystal violet

27

What is the primary dye?

Crystal violet

28

What is the mordant?

Iodine solution

29

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

The crystal violet-iodine complex

30

What is used as the decolouriser?

Ethanol

31

Gram negative bacteria are counterstained with...

safranin

32

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

CVI complex during the ethanol treatment

33

Gran negative bacteria should appear red since they will have been

decolourised by the ethanol and would have taken up the safranin

34

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

Schaeffer-Fulton method

35

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

Clostridium

36

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

Bacillus

37

What are endospores?

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

38

What is the endospore surrounded by?

Impervious layers called the spore coat

39

Is heat fixation required in the Schaeffer-fulton method?

Yea babes

40

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

Endospore - green
rest of cell - pink/red

41

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

malachite green,

42

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

safranin