Gram Staining Protocol

• The Gram staining method was named after a Danish inventor Hans Christian Gram, who invented it as an approach to differentiating bacteria species in 1875.
• The method is often used in modern histology especially in paraffin fixatives for tissue sectioning. In a recent case in Kuwait, the Gram staining technique was particularly effective in the diagnosis of Gonorrhea giving 99.4% effective results. The method is still used today especially with paraffin sections and has been modified to suit different substances.

• Preparation of the Crystal Violet solution. For preparing solution A (Crystal Violet stock solution), add 20 gm of 85% crystal violet dye in 100 ml ethanol (95%) and dissolve by mixing thoroughly. For preparing solution B (Oxalate Stock solution), add 1 gm ammonium oxalate in 100 ml distilled water and dissolve by mixing thoroughly. For preparing the working solution, add 1 ml of crystal violet stock solution in 10 ml distilled water and add 40 ml of oxalate stock solution. Let the solution sit for 24 hours at room temperature and store the solution in a dark bottle for use.
• Preparation of Gram's Iodine solution. Dissolve 1 gm of Iodine, and 2 gm of potassium iodide in 300 ml distilled water. Mix properly till the iodine dissolve and keep the solution in a dark bottle.
• Preparation of decolorizing solution. Mix 50 ml of acetone with 50 ml of 95% ethanol.
• Flood crystal violet solution over fixed smear.
• After 30-60 seconds, pour off the CV solution and rinse with gentle running water.
• Flood the Gram's Iodine solution over the smear.
• Leave the iodine solution for 30-60 seconds and pour off the excess iodine and rinse with gentle running water, shake off the excess water over the smear.
• Decolorize the smear by passing the decolorizing solution till the solution runs down in clear form. Alternatively, add a few drops of decolorizing solution and shake gently and rinse with distilled water after 5 seconds.
• Rinse with distilled water to wash decolorizer, and shake off the excess water over the smear.
• Pour counter stain over the smear.
• Leave for 30-60 seconds and wash with gentle running water. Air dry or blow-dry the smear.

• Require multiple reagents.
• Over-decolorization may result in the identification of false gram-negative results, whereas under-decolorization may result in the identification of false gram-positive results.
• Smears that are too thick or viscous may retain too much primary stain, making the identification of proper Gram stain reactions difficult. Gram-negative organisms may not decolorize properly.

Special Staining Services

Immunohistochemistry (IHC), Immunofluorescence (IF) Service

Transmission Electron Microscopy (TEM) Service

For research use only. Not for any other purpose.