General Staining Techniques

1.    APPARATUS

1. Hot air Oven
2. Laminar Air flow
3. Spirit Lamp / Bunsen Burner
4. Media bottles
5. Glass slides   

2.     REAGENTS   AND  MEDIAS

1. Grams Stain kit
2. Crystal Violet

Use commercially available solution.

If the above is not available prepare solution as follows.

Solution A

                                    Crystal Violet                          2.0 g

                                    Ethyl alcohol                           20.0 ml

Solution B

                                    Ammonium Oxalate                0.8 g

                                    Distilled water                         80.0 ml

Dissolve crystal Violet in ethyl alcohol and the ammonium Oxalate in distilled water. Mix solutions A and B.     

1. Grams Iodine

Use commercially available solution.

If the above is not available prepare solution as follows.

                                    Iodine                                     1.0 g

                                    Potassium Iodide                    2.0 g

                                    Distilled water                         300.0 ml

Dissolve Iodine and Potassium Iodide in distilled water.

1. Ethyl Alcohol ( 95%)

Ethyl alcohol ( 100%)             95.0 ml

Distilled water                        5.0 ml

1. Safranin

Use commercially available solution.

If the above is not available prepare solution as follows.

             Safranin ( 2.5% solution in95% ethyl alcohol )                 10.0 ml

             Distilled water                                                                100.0 ml

1. Fungal Stains                         
   1. Water –Iodine solution ( As in grams Stain)
   2. Use commercially available solution.

If the above is not available prepare solution as follows.

            Iodine                                     1.0 g

            Potassium Iodide                    2.0 g

            Distilled water                         300.0 ml

Dissolve Iodine and Potassium Iodide in distilled water.

1. Safranin
2. Isopropyl Alcohol
3. Lactophenol cotton blue
4. The morphological characters of bacteria shall be observed by two methods.
   1. Method A : Simple staining –  Stain the bacteria using a single staining solution.
      1. Prepare the bacterial smear over the slide .
      2. Prepare the staining solutions (Crystalviolet, Methylene blue and Safranin) .
      3. Hold the slide using a staining tray.
      4. Cover the smear with Crystal violet or Methylene blue or Safranin for 30 – 60 sec.
      5. Wash the smear with Distilled water for a few seconds, using wash bottle.
      6. Allow the stained slides to air dry.
      7. Observe the smear under Low power and high power objectives of a microscope.
      8. Apply a drop of Chedar wood oil over the Smear.
      9. Examine the smears under oil-immersion objective of a Microscope.
      10. Make drawings for each Microscopic fields.
      11. Describe the size, shape and arrangement of cells.
      12. Most of bacteria come under the following Morphological categories.

..  ..  ..              ……                            :::.                               - - - - -  | |||

Diplococci       Strptococci                  Staphylococci              Rods

1. Method - B  : Grams staining – Stain the bacteria using more number of staining solutions.
   1. Prepare the bacterial smear over the slide.
   2. Prepare the staining solutions (Crystal violet, Grams Iodine, 95% Ethyl alcohol and Safranin) .
   3. Hold slide using the slide rack.
   4. Cover smear with crystal violet for 30 seconds.
   5. Wash the smear with distilled water for a few seconds, using a wash bottle.
   6. Cover the smear with Iodine solution for 30 seconds.
   7. Add ethyl alcohol drop by drop over the Iodine solution.
   8. Wash off the Iodine solution with ethyl alcohol until no color flows down from the smear.
   9. Wash the smear with distilled water and drain.
   10. Cover the Smear with Safranin for 30 seconds.
   11. Wash the Smear with Distilled Water.
   12. Dry the Smear with a tissue paper.
   13. Allow the stained slides to air dry.
   14. Examine the slide under low power and high powder objectives of a Microscope.
   15. Apply a drop of Chedar wood oil over the smear.
   16. Examine the slides under the oil immersion objective of a Microscope.
   17. Draw the representative Microscopic fields
   18. Identify the gram reaction on the basis of color.
   19. Those bacteria appear dark  blue or Violet described as Gram positive.
   20. Those bacteria appearing pink described as Gram negative.
   21. Describe the morphological shape, size, arrangement of cells and Grams reaction.
2. The morphological characters of Fungi shall be observed by Lactophenol cotton blue staining
   1. Lactophenol cotton blue Staining :
      1. Place a drop of Lactophenol cotton blue on a slide.
      2. Hold the Inoculating needle with one hand.
      3. Light the Bunsen Burner/ Spirit lamp with lighter.
      4. Flame the loop at a 360⁰ angle into the upper core of the Bunsen Burner/ Spirit Lamp.
      5. Heat the loop till redness of entire wire
      6. Remove and allow it to cool for one minute.
      7. Take culture slant into other hand.
      8. Remove the plug in front of the flame with the little finger.
      9. Sterilize the mouth of the test tube.
      10. Insert the sterilized loop into a slant culture and touch over the surface growth, take a small tuft.
      11. Sterilize the mouth of the culture tube and replace the plug.
      12. Spread and mix the Mold cultures on the Lactophenol cotton blue.
      13. Apply wax to all sides of cover glass.
      14. Place the cover glass over the preparation present on glass slide.

Examine the preparation under Low power and high power objectives of a Microscope.

General testing procedure, techniques, bunsen burner, spirit lamp