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2/9908 Technical Training Centre Negative Staining Technique TM-00031:11 Negative Staining Technique Objective : To colour the background in order to provide a contrast against which to

2/9908 Technical Training Centre

Negative Staining Technique

TM-00031:11

Negative Staining Technique

Objective : To colour the background in order to provide a contrast against

which to observe microbial cells which remain colourless (they do not take up the stain)

Reagent : 10% nigrosin in water Alternative : 0.45% methylene blue in 30 ml saturated alcohol

mixed with 10% w/v potassium hydroxide in water

Method : Place 1 drop of negative stain solution on a clean

glass slide Emulsify a small sample from an agar plate culture

in the drop of stain solution and spread over a 1-2 cm2area Allow to air dry

Observe microscopically under oil immersion Note! Negative staining technique is a good alternative to investigate size and form of cells This technique is to prefer for the untrained micro-scopist or if no phase microscope is available With a good phase contrast

2/9908 Technical Training Centre

Catalase Test

TM-00031:12

Catalase Test

Reagent: 10 vol % H2O2

Add 1 drop of H2O2to the surface of one colony

Positive result: Vigorous bubbling from surface of colony

indicates the presence of the enzyme catalase which breaks down the hydrogen peroxide into water + oxygen gas, which is given off as bubbles

2/9908 Technical Training Centre

Oxidase Test

Kits are available-safe and convenient

TM-00031:13

Oxidase Test

Oxidase Reagents: 1% solution of tetramethyl-p-phenylenediamine

dihydrochloride in distilled water

Soak one filter paper with reagent and make a smear test of the bacteria culture or drop one drop

of reagent solution onto colony on agar plate

Positive result: If the bacteria are oxidase positive the colouring

substance is oxidized into a purple blue colour

The reagent is available in several forms

* dry powder (mix with distilled water to form solution)

* reagent kits available as sticks or plates/pads

Note: Not recommended for use in wet or solution form

since this reagent is carcinogenic

2/9908 Technical Training Centre

Gram Differentiation

Technique

TM-00031:14

Gram Differentiation Technique

Objective :

To validate the gram staining technique by means of a biochemical test which differentiates the physical differences, in cell wall structure between gram positive and gram negative cells

Method :

1 Place one droplet of 3% potassium hydroxide(KOH) on a microscope slide

2 Take a needle of organism material from a colony on an agar plate

3 Emulsify the organism material very well in the KOH solution

4 After about 10 seconds, raise the needle from the emulsified material and look for the presence of long sticky threads between the needle and the slide

5 Stop stirring if no threads are observed after 15-20 seconds

Interpretation of result:

•Gram positive bacteria do not form threads

•Gram negative bacteria will show sticky thread formation

Note : This validation test is most useful in cases where the gram stain result

is unclear, eg Coryneform group

2/9908 Technical Training Centre

Gram Staining Technique (1)

TM-00031:15

Gram Staining Technique (1)

Objective :

To differentiate between cell types with different cell wall structures by means

of a differential staining technique

Method :

Making a smear

1 Place a drop of water on a clean glass slide

2 Place a needle of microbial material from a colony on an agar plate in the water drop and mix/emulsify and spread over an area of approximately 1-2 square cm

3 Allow the smear to air-dry

4 Heat fix the smear to the slide by means of passing through the hot part of a flame 2-3 times

5 Allow to cool

6 Place the slide on a staining rack over a sink

2/9908 Technical Training Centre

Gram Staining Technique(2)

TM-00031:16

Gram Staining Technique (2)

Staining method

1 Apply solution 1(Crystal violet) liberally to the smear Allow to stand for

30 seconds Rinse with water

2 Apply solution 2 (Gram’s iodine) Allow to stand for 1 minute Rinse with water

3 Apply decolourising solution 3(Acetone/alcohol) Allow to stand for only a few seconds before rinsing with water

4 Apply solution 4 (Safranin - red counter stain) Allow to stand for 30 seconds Rinse with water

5 Gently pat dry with tissue/paper towel

Examine microscopically under oil immersion

Interpretation of result:

Gram positive cells are coloured deep purple/blue

Gram negative cells are coloured red

2/9908 Technical Training Centre

Examples of colonial Morphology

(1)

TM-00031:17

2/9908 Technical Training Centre

Examples of colonial Morphology

(2)

TM-00031:18

2/9908 Technical Training Centre

Moulds and Yeasts

TM-00031:19

Fungi

2/9908 Technical Training Centre

Laboratory Requirements

Equipment

TM-00031:21

Laboratory Requirements

Essential equipment:

Autoclave (1 essential, 2 convenient to have)

1 for making / sterilising media

1 for sterilising contaminated plates / media prior to disposal) Microscope (Binocular - must be of good quality e.g Zeiss, Leitz, Olympus,

Nikon) Incubator (need at least 2)

1 x 30°C operating temperature

1 x 25°C

1 x 55C

pH meter

Hot plate stirrer (for making media)

Refrige- (1 x small for laboratory A walk in refrigerator

rator for storing ready made culture media etc is also

useful/convenient