Gram staining

1. Smear preparation
I made smear from the sputum sample of the patient. I have taken inoculation loop and i made this smear in the center of slide with sputum sample and heat fixed and and kept dry.
There are ATCC culture media plates which have all types of bacterial
colonies and each bacteria in each culture media plate.
I have taken 2 culture media plates which contains Gram positive bacteria and Gram negative bacteria and i have labelled "+" representing Gram positive bacteria and "-" representing Gram negative bacteria.
From the two culture media plates i made two small smears on either side of the sputum smear on the slide. These two small smears acts as controls. The "+" one will give violet color which tells its Grams positive bacteria and "-" gives pink color which tells Grams negative bacteria and the sputum at the end of procedure when we look under microscope we will come compare with positive and negative controls on either on the slide and interpret whether it is Gram positive bacteria or Gram negative bacteria.

You can see below the smear

2. Adding of Grams crystal violet
This is the Grams crystal violet. It is the primary stain and it stains violet/dark blue color of all bacteria.

We have to flood the stain with Grams crytsal violet and kept for 1 min

Then it is washed under tap water.

3. Flood with Gram's Iodine
Iodine acts as mordant. Flood the slide with iodine and keep it for 1 min.

Iodine is flooded on the slided like this

Wash the slide under tapwater

4. Add Gram's decolouriser
Decolourising agent used in Grams staining is ethanol. If ethanol is not there means we can use acetone but should be so careful while doing this step.
Decolourising agent is added and seen till violet/blue color which we added in 1st step removed completely or the violet color should not be seen and its then washed under tap water.
Principle of this step -In this step if the smear has Gram positive bacteria it will resist decolourisation and if it is Grams negative bacteria the crystal violet is removed(decolourisation happens).

5. Add counterstain safranin
Safranin is pink color reagent it gives pink color if there is Grams negative bacteria is there when we are looking under microscopy. This step is just for 20 seconds the slide is flooded with safranin and kept for 20 seconds and washed under tap water.

Principle of this step - If the smear has Grams negative bacteria and which was decolourised previous step will take up the safranin and gives pink color when we look under microscope while the Grams positive bacteria which didn't get decolourised and already has Crystal violet staining will give violet colour.

After this we will allow it for dry.

So these are all the steps that i have show with images about Gram staining.

Finally we will take the slide after it gets dried and will look under microscope under oil immersion.
As i have taken sputum sample i have to check bartlett's score whether the sputum sample is adequate or inadequate which means whether it is saliva or sputum.
I will count number of neutrophils and epithelial cells under 10X magnification and will calculate and see the total value according to bartlett score and consider whether the sample is saliva or sputum.
If the sample is saliva means we can ask for repeat the sample.
If it is sputum sample means we will look under oil immersion will look for any bacteria.

Gram staining

Gram staining

References