How to do a Gram Stain… What a difference a wall makes! (Day 215)

Proteus Mirabilis on Blood Agar

Today’s Diary Entry is sponsored by Pet Hooligans

Well today was interesting as we are looking at Gram positive bacteria which are different to the Gram negative bacteria that we have been studying previously. The grouping into gram negative and gram positive bacteria is the most basic step in the identification of bacteria using a technique called Gram Staining (or Gram’s method). The gram stain technique was invented by Hans Christian Gram in the Berlin city hospital in 1984 whilst working in the morgue. Hans originally designed gram staining to make it easier to view bacteria in lung tissue, and noted that it did not stain all the types of bacteria.

Proteus Mirabilis on Blood Agar

This phenomenon is due to differences in the structure of the cell wall when exposed to different chemicals. There are four different chemicals used in the gram stain process; a basic dye, a mordant, decoloriser, and counterstain.

The basic dye is applied first before the mordant which is a substance that increases the bond between the dye and cell wall helping to fix the dye inside the cell. This makes it more difficult to wash off the dye and in the gram stain fixes the gram positive blue colour. A decoloriser is then used which is a substance that removes the dye from the stained cells, this ability varies between the cell wall type and so only gram negative cells lose their dye. The final counterstain is another dye that is applied and fills the cells that have just been cleaned by the decoloriser giving the gram negative red colour.

How to do a Gram Stain

Doing a gram stain is one of the most basic procedures in the microbiology laboratory, and so I wanted to make sure that I had covered it here. The video below demonstrates the steps that are outlined here:

  1. Apply bacteria to a slide, if using a culture plate add a drop of saline solution to the slide to allow the bacteria to be easily spread and then air dry this over a bunsen flame.
  2. Fix the bacteria to the slide by passing the slide back and forth through the bunsen flame.
  3. Apply crystal violet to the slide and let it react for 30 seconds.
  4. Rinse the slide thoroughly under running water
  5. Apply Grams Iodine (or Lugols solution) for 30 seconds
  6. Rinse the slide under running water and then apply 95% ethyl alcohol for 10 seconds
  7. Rinse the slide under running water.
  8. Apply the counterstain; in our case carbolfuchsin for 30 seconds
  9. Rinse the slide under running water and then allow to dry.
  10. Examine the slide under the microscope – I prefer the 100x Oil Immersion lens here!

The video demonstrating how to do a gram stain is here: