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Differential and Cytological Staining Techniques
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Materials Required:

 

Endospore Staining

 

  • Schaeffer-Fulton Method
  1. Clean glass slide in box
  2. Inoculation loop
  3. Test organism
  4. Absorbent paper
  5. Boiling water in tripod stand
  6. Water in wash bottle
  7. Bibulous paper
  8. Tweezers
  9. Microscope
  10. Primary Stain – Malachite green
  11. Counter stain - Safranin
     

Staining Steps:

 

  1. Aseptically transfer the bacterium with an inoculating loop to a clean glass slide and prepare a thin smear of the bacterium.
  2. Air dry and heat-fix the bacterial smear.
  3. Cover the bacterial smear with a piece of absorbent paper cut to fit the smear and slide.
  4. Place the slide over a container of boiling water.
  5. Saturate the absorbent paper with malachite green stain solution and steam for 5 minutes.  Keep the paper moist by adding more stain as required.
  6. Remove the absorbent paper using forceps, allow the slide to cool, and rinse the slide with water for 30 seconds.
  7. Wash the slide with water.
  8. Counter stain with safranin for 30 seconds.
  9. Wash the slide with water and blot dry the slide.
  10. 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.

 

 

  • Dorner Method

 

  1. Test organism
  2. Glass slides in the box
  3. Inoculation loop
  4. Basic fuchsin
  5. Negrosin
  6. Microscope
  7. Water bath

 

Staining Steps:

 

  1. Mix an aqueous suspension of bacteria with an equal volume of basic fuchsin in a test tube.
  2. Keep the test tube in a boiling water bath for 10 minutes.
  3. Add a loopful of the boiled basic fuchsin-organism suspension to one side of the glass slide.
  4. 7% nigrosin stain is added over the boiled basic fuchsin-organism suspension.
  5. Make a thin smear of the culture using a second glass slide making a 45 degree angle with the first slide.
  6. Air dries the smear for some time.
  7. 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
 

  • Ziehl Neelsen Method

 

  1. Clean glass slide in the box
  2. Inoculation loop
  3. Test organism
  4. Absorbent paper
  5. Beaker with water in tripod stand
  6. Tweezers
  7. Bibulous paper
  8. Microscope
  9. Primary stain - Carbol fuschin
  10. Decolorizer    - 25% sulphuric acid
  11. Counter stain - Methylene blue
     

 

Staining Steps:

 

  1. Aseptically transfer the bacterium with an inoculating loop to a clean glass slide and prepare a thin smear of the bacterium.
  2. Heat- fixes the bacterial smear.
  3. Cover the bacterial smear with a piece of absorbent paper cut to fit the smear and slide.
  4. Place the slide over a container of boiling water.
  5. Saturate the paper with carbol fuschin steam for 5 minutes.  Keep the paper moist by adding more stain as required.
  6. Remove the absorbent paper using tweezers, the excess stain is washed with water and allow the slide to cool for some time.
  7. Decolorize the slide with 25 % sulfuric acid.
  8. Rinse the slide with water.
  9. Counter stain the cells with Methylene blue stain for 45 seconds.
  10. Wash the slide with water and blot dry the slide.
  11. 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

 

  1. Glass slides in the box
  2. Inoculation loop
  3. Test organism
  4. Crystal violet
  5. 20% copper sulfate
  6. Bibulous paper
  7. Microscope

 

Staining Steps:

 

  1. 2 drops of Crystal Violet (primary stain) is added to one side of the glass slide.
  2. A loopful of bacterial culture is mixed with Crystal Violet stain.
  3. Make a thin smear of the culture using a second glass slide making a 45 degree angle with the first slide.
  4. Decolorize and counter stain the bacterial smear with 20% copper sulfate.
  5. Air dries the smear for some time.
  6. Examine the slide under the oil immersion lens for the presence of capsules of bacteria.
     
  • Negative Capsule Staining
     
  1. Glass slides in the box
  2. Inoculation loop
  3. Bunsen burner
  4. Test organism
  5. Negrosine
  6. Microscope

 

Staining Steps:

 

  1. Add one drop of nigrosin onto the end of a clean slide.
  2. A loopful of bacterial culture is added onto the drop of nigrosin and mix well.
  3. Make a thin smear of the culture using a second glass slide making a 45 degree angle with the first slide.
  4. Air dries the smear for some time.
  5. 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

 

  • Albert’s Staining
  1. Glass slides in the box
  2. Inoculation loop
  3. Test organism
  4. Albert stain A
  5. Albert stain B
  6. Water in the wash bottle
  7. Bibulous paper
  8. Microscope.
     

Staining Steps:

 

  1. Aseptically transfer the bacterium with an inoculating loop to a clean glass slide and prepare a thin smear of the bacterium.
  2. Air dries the smear for some time.
  3. Cover the air dried smear with Albert Stain A.
  4. Remove the excess stain by washing with water.
  5. Cover the bacterial smear with Albert Stain B. Let it stand for 2 minutes.
  6. Wash the slide with water and blot dry the slide.
  7. 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

 

  • Liefsons Flagella Stain
  1. Clean glass slides in the box
  2. Inoculation loop
  3. Test organism
  4. Leifson’s stain
  5. Water
  6. Microscope

     

Staining Steps:

 

  1.  Make a rectangular border on the surface of the glass slide with a grease pencil.
  2. Aseptically transfer the bacterium with an inoculating loop to a clean glass slide.
  3. Allow the smear to run down in an inclined slide.
  4. Flood the slide with Liefson's Flagella Stain and allow staining for 10 – 15 minutes. A fine rust colored precipitate forms throughout the slide.
  5. Air dries the slide.
  6. Examine the slide under the oil immersion lens to observe the type of flagella.
     
  • Gray Method
  1. Nutrient agar plate with test organism
  2. Scalpel
  3. Tweezers
  4. Glass slides in the box
  5. Basic fuschin
  6. Microscope
     

 

Staining Steps:

 

  1. Cut a small portion of the agar with the test organism using a sterile scalpel.
  2. Raise the agar block with the aid of a forceps.
  3. Place the agar block in a sterile glass slide, with the bacterial culture facing downwards. Keep it undisturbed for some time.
  4. Take the agar block and discard it in hazardous box.
  5. Allow the bacterial culture in the glass slide to air dry for some time.
  6. Flood the bacterial culture with basic fuchsin until a golden precipitate is observed.
  7. Gently wash with water.
  8. 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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