Technique GRAM STAIN 09/02/01
Principle The reaction to the Gram stain is a stable characteristic of a bacterium.

Cells that can resist decolorization with ethanol or acetone are Gram positive. Decolorized cells are Gram negative and must be visualized by a counterstain such as saffranine or Bismarck brown
Cautions Although the most common stain in microbiology, the Gram Stain is performed incorrectly about 40% of the time

A positive test is more significant than a negative one as a Gram negative reaction may be false due to
a) the age of the culture,
b) excessive decolourization,
c) incorrect heat fixation

Use positive and negative controls to detect the last two problems.

Examine young cultures before the end of the logrithmic growth phase,
Older cultures of Gram positive microbes can lose the ability to retain the stain.

The bacteria should be completely free of all deposits od dye particles

Some organisms have granules that resist decolourization
Method 1 Prepare a smear
2: Heat fix
3: Flood with crystal violet solution
4: Leave for 1 min
5: Rinse briefly with tap water (not more than 2 sec)
6: Drain off the excess water
7: Apply the iodine solution of 1 minute
8: Rinse with tap water
9: Rinse with Decolourizerl until the solvent on the slide is colourless
10: Counterstain with safranine for 10 to 60 seconds.
11: Rinse with water,
12: Blot dry
13: Examine under the microscope.
Results Gram positive organisms will appear blue or purple
Gram-negative organisms will appear red.
Positive control
Transfer cultures daily on Trypticase soy agar slants. Make new transfers before using the cultures as controls
Staphylococcus aureus ATCC 25923
Negative control Escherichia coli (ATCC 11229) Transfer cultures daily on Trypticase soy agar slants. Make new transfers before using the cultures as controls
Reagents
Hucker's Crystal Violet Solution
Solution A    
Crystal violet 20 g
ethyl alcohol (95%) 200 mL
Solution B    
Ammonium oxalate grams 8 g
Distilled water 800 mL
Mix solution A and B, store for 24 hours and filter
 
Gram's Iodine solution
resublimed iodine 1 g
potasium iodide 2 g
distilled water 300 mL
     
Decolourizers    
Gram alcohol solution 95% ethanol (slowest decolourizer)
acetone-alcohol (95% ethanol 100 ml; acetone 100 ml)( intermediate rate)
acetone (fastest declourizer)
any of the above decolourizers will give good results
     
Safranin Counterstain    
Saffranin O ( 2.5% in 95% ethanol) 10 mL
distilled water 90 mL
Reference Manual of Clinical Microbiology 1985 E. H. Lennette, A. Balows, W.J. Hausler Jr, H. J. Shadomy American Society for Microbiology Washington DC

Manual of Microbiological Methods by the Society of Americam Bacteriologists 1957 McGraw Hill New York

Hucker, G. J., and H.J. Conn 1923 Methods of Gram Staining. N.Y. State Agr Expt Sta Tech Bull 129
Hucker, G. J., and H.J. Conn 1927 Further studies on the methods of Gram Staining. N.Y. State Agr Expt Sta Tech Bull 128