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Negative Staining Technique
Technical Training Centre
Lund, Sweden
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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 cm2 area
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 microscopist or if no phase microscope is available. With a good phase contrast
microscope trained microbiologists prefer a wet preparation (some organismmaterial dispersed in water), a cover glass and then using the phase
microscope with the 100 objective.
Most often size and shape can be viewed in the gram stain preparation. But if
this is difficult you can use negative staining or wet preparation.
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Catalase Test
Technical Training Centre
Lund, Sweden
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Catalase Test
Reagent:
10 vol % H2 O2
Add 1 drop of H2 O2 to the surface of one colony.
Positive result:
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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
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Oxidase Test
Kits are availablesafe and convenient
Technical Training Centre
Lund, Sweden
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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:
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Not recommended for use in wet or solution form
since this reagent is carcinogenic.
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Gram Differentiation
Technique
Technical Training Centre
Lund, Sweden
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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
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Gram Staining Technique (1)
Technical Training Centre
Lund, Sweden
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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
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Gram Staining Technique(2)
Technical Training Centre
Lund, Sweden
FiS Q A
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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.
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Examples of colonial Morphology
(1)
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Lund, Sweden
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Examples of colonial Morphology
(2)
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Lund, Sweden
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Moulds and Yeasts
Technical Training Centre
Lund, Sweden
Technical Training Centre 2/9908
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Fungi
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Laboratory Requirements
Equipment
Technical Training Centre
Lund, Sweden
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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-
Trolleys
(1 x small for laboratory. A walk in refrigerator
rator
for storing ready made culture media etc is also
useful/convenient
(for transporting sample packs)
(Water distillation equipment)
Bunsen burner
Top loading balanced scale (weighing media - only 2 d-place necessary)
Water bath and/or oven
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