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Gram staining Procedure


                      GRAM STAIN

Gram stain or Gram staining, also called Gram's method, is a method of staining used to distinguish and classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique
                       
 Gram Stain Procedure

1.      Place slide with heat fixed smear on staining tray.
2.      Gently flood smear with crystal violet and let stand for 1 minute.
3.      Tilt the slide slightly and gently rince with tap water or distilled water using a wash bottle.
4.      Gently flood the smearwith Gram’s iodine and let stand for 1 minute.
5.      Tilt the slide slightly and gently rince with tap water or distilled water using a weash bottle. The smear will appear as a purple circle on the slide.
6.      Decolorize using 95% ethyl alcohol runs almost clear. Be careful not to over-decolorize.
7.      Immeduate rince with water.
8.      Gently flood with safranin to counter-stain and let stand for 45 seconds.
9.      Tilt the slide slightly and gently rince with tap water or distilled water using a weash bottle.
10.  Blot dry the slide with bibulous paper.
11.  View the smear using a light-microscope under oil-immersion.

                                Summary of Gram stain
Application of
Reagent
Cell color
Gram-positive
Gram-negative
Primary dye
crystal violet
purple
purple
Trapping agent
iodine
purple
purple
Decolorizer
alcohol/acetone
purple
colorless
Counter stain
safranin/carbol fuchsin
purple
pink




    Note     
Occasionly Gram positive organism lose their ability to retain crystal violet and stain Gram negatively. The reasons may be as follows:

  1. Over decolorization of the smear.
  2. Use of iodine solution which is too old (yellow instead of brown).
  3. Preparation of smear from old culture.
  4. cell wall damage due to excessive heat fixation of the smear or due to antibiotic therapy.                                                                                
                                                                                                                         RANDHIR KUMAR 
                                                                                                                                                 RDKPARAMEDICALSTUDENTSTUDY

                      




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