Materials Required:
Endospore Staining
- Clean glass slide in box
- Inoculation loop
- Test organism
- Absorbent paper
- Boiling water in tripod stand
- Water in wash bottle
- Bibulous paper
- Tweezers
- Microscope
- Primary Stain – Malachite green
- Counter stain - Safranin
Staining Steps:
- Aseptically transfer the bacterium with an inoculating loop to a clean glass slide and prepare a thin smear of the bacterium.
- Air dry and heat-fix the bacterial smear.
- Cover the bacterial smear with a piece of absorbent paper cut to fit the smear and slide.
- Place the slide over a container of boiling water.
- Saturate the absorbent paper with malachite green stain solution and steam for 5 minutes. Keep the paper moist by adding more stain as required.
- Remove the absorbent paper using forceps, allow the slide to cool, and rinse the slide with water for 30 seconds.
- Wash the slide with water.
- Counter stain with safranin for 30 seconds.
- Wash the slide with water and blot dry the slide.
- Examine the slide under the oil immersion lens for the presence of endospores.
Expected Result:
On microscopic observation endospores appear in green color and the vegetatives cells as pink.
- Test organism
- Glass slides in the box
- Inoculation loop
- Basic fuchsin
- Negrosin
- Microscope
- Water bath
Staining Steps:
- Mix an aqueous suspension of bacteria with an equal volume of basic fuchsin in a test tube.
- Keep the test tube in a boiling water bath for 10 minutes.
- Add a loopful of the boiled basic fuchsin-organism suspension to one side of the glass slide.
- 7% nigrosin stain is added over the boiled basic fuchsin-organism suspension.
- Make a thin smear of the culture using a second glass slide making a 45 degree angle with the first slide.
- Air dries the smear for some time.
- Examine the slide under the oil immersion lens for the presence of endospores.
Expected Result:
On microscopic observation, endospores will appeared as red, and the vegetative cells as colourless, in a dark background.
Acid Fast Staining
- Clean glass slide in the box
- Inoculation loop
- Test organism
- Absorbent paper
- Beaker with water in tripod stand
- Tweezers
- Bibulous paper
- Microscope
- Primary stain - Carbol fuschin
- Decolorizer - 25% sulphuric acid
- Counter stain - Methylene blue
Staining Steps:
- Aseptically transfer the bacterium with an inoculating loop to a clean glass slide and prepare a thin smear of the bacterium.
- Heat- fixes the bacterial smear.
- Cover the bacterial smear with a piece of absorbent paper cut to fit the smear and slide.
- Place the slide over a container of boiling water.
- Saturate the paper with carbol fuschin steam for 5 minutes. Keep the paper moist by adding more stain as required.
- Remove the absorbent paper using tweezers, the excess stain is washed with water and allow the slide to cool for some time.
- Decolorize the slide with 25 % sulfuric acid.
- Rinse the slide with water.
- Counter stain the cells with Methylene blue stain for 45 seconds.
- Wash the slide with water and blot dry the slide.
- Examine the slide under the oil immersion lens to observe the acid fast or non acid fast-cells
Expected Result:
On microscopic observation, the acid fast bacterium will appear as pink coloured cells.
Capsule Staining
- Positive Capsule Staining
- Glass slides in the box
- Inoculation loop
- Test organism
- Crystal violet
- 20% copper sulfate
- Bibulous paper
- Microscope
Staining Steps:
- 2 drops of Crystal Violet (primary stain) is added to one side of the glass slide.
- A loopful of bacterial culture is mixed with Crystal Violet stain.
- Make a thin smear of the culture using a second glass slide making a 45 degree angle with the first slide.
- Decolorize and counter stain the bacterial smear with 20% copper sulfate.
- Air dries the smear for some time.
- Examine the slide under the oil immersion lens for the presence of capsules of bacteria.
- Negative Capsule Staining
- Glass slides in the box
- Inoculation loop
- Bunsen burner
- Test organism
- Negrosine
- Microscope
Staining Steps:
- Add one drop of nigrosin onto the end of a clean slide.
- A loopful of bacterial culture is added onto the drop of nigrosin and mix well.
- Make a thin smear of the culture using a second glass slide making a 45 degree angle with the first slide.
- Air dries the smear for some time.
- Examine the slide under the oil immersion lens for the presence of capsules of bacteria.
Expected Result:
On microscopic observation, the capsulated bacterium can be observed in a dark background.
Metachromatic Granule Staining
- Glass slides in the box
- Inoculation loop
- Test organism
- Albert stain A
- Albert stain B
- Water in the wash bottle
- Bibulous paper
- Microscope.
Staining Steps:
- Aseptically transfer the bacterium with an inoculating loop to a clean glass slide and prepare a thin smear of the bacterium.
- Air dries the smear for some time.
- Cover the air dried smear with Albert Stain A.
- Remove the excess stain by washing with water.
- Cover the bacterial smear with Albert Stain B. Let it stand for 2 minutes.
- Wash the slide with water and blot dry the slide.
- Examine the slide under the oil immersion lens for observing the metachromatic granules of bacteria.
Expected Result:
The metachromatic granules appear as bluish black and the bodies of the bacillus appear as green.
Flagella Staining
- Clean glass slides in the box
- Inoculation loop
- Test organism
- Leifson’s stain
- Water
- Microscope
Staining Steps:
- Make a rectangular border on the surface of the glass slide with a grease pencil.
- Aseptically transfer the bacterium with an inoculating loop to a clean glass slide.
- Allow the smear to run down in an inclined slide.
- Flood the slide with Liefson's Flagella Stain and allow staining for 10 – 15 minutes. A fine rust colored precipitate forms throughout the slide.
- Air dries the slide.
- Examine the slide under the oil immersion lens to observe the type of flagella.
- Nutrient agar plate with test organism
- Scalpel
- Tweezers
- Glass slides in the box
- Basic fuschin
- Microscope
Staining Steps:
- Cut a small portion of the agar with the test organism using a sterile scalpel.
- Raise the agar block with the aid of a forceps.
- Place the agar block in a sterile glass slide, with the bacterial culture facing downwards. Keep it undisturbed for some time.
- Take the agar block and discard it in hazardous box.
- Allow the bacterial culture in the glass slide to air dry for some time.
- Flood the bacterial culture with basic fuchsin until a golden precipitate is observed.
- Gently wash with water.
- Examine the slide under the oil immersion lens to observe the type of flagella.
Expected Result:
On microscopic observation the flagella present around the bacterial cells are observed.
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