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:

Looking at Bacteria and Microbiological Staining (Day 22)


So Tuesday is solely Microbiology and Immunology, I kinda enjoy it as many of the why’s that previously confused me are being made clear. The morning starts with a lecture, before then having a practical session a couple of hours later. The lecture today was on the differences between the cell walls of Gram Negative and Gram Positive bacteria, I previously knew there was a difference, so am pleased to now actually understand it.

Talking about praticals here something I really do enjoy are the small group sizes, so for Microbiology there are just 9 of us in the lab meaning there is plenty of space to work. More importantly though it means that you get more time with the lecturer which means that stupid mistakes are corrected. Today for example when looking at motility (movement) of bacteria under the microscope I had pressed down a cover slip onto the microscope slide too hard squeezing all the saline suspension from the slide. After spending 5 minutes trying (and failing) to find the bacteria I was corrected and told to do it again without drying out the slide. When repeating it I rapidly found the bacteria, it may seem basic however its definately not a mistake that I will be making again.

microbiology-staining-practical-setupThe most basic way to look at bacteria is without staining, however like this you cannot view details. Probably the most common stain used is a gram stain, this is done in stages and uses different dyes to stain different bacteria different colors. The first dye is crystal violet which can be absorbed by both gram negative and gram positive bacteria and stains everything blue. The slide is then washed and Lugol solution is applied which makes the cell nuclie more visible before that is then also washed off. 95% ethyl alcohol is then applied which removes any excess dye, and in the case of gram negative bacteria can also cross the cell membrane to wash the crystal violet out from with the cell. The last dye applied is Carbolfuchsin which is red in color which then enters the empty gram negative cells to stain them red.

Gram Negative Bacteria
Gram Negative Bacteria

This evening I went to the anatomy self study which was pretty cool, basically the university opens up the dissection rooms between 5pm – 8pm on Tuesdays, Wednesdays and Thursdays for students to use under the supervision of student supervisors. We got to experience searching the bone cuboard for the bones from the thoracic limb (which was good revision in itself identifying the different bones). Being self study you are basically free to revise whatever you need to revise, and several second year students were revising the digestive tract and stomach and so had a goat dissection going on in one of the rooms which was pretty interesting to look at. I am still amazed at how vivid the colours of internal organs are!!!

Anyways I can relate all the structures on the Scapula now, and have started on the Humerus and Radius and Ulna (which is interesting as they are connected differently in different species). I will be back again tommorow night for sure as I am still not feeling as confident as I would like!