 In this video, we will learn about Gram Staining Procedure. Gram Staining Technique is a differential staining technique which is used to differentiate two bacteria, Gram Posture Bacteria and Gram Negative Bacteria. Mainly there are three types of bacteria on the basis of their cell wall. Number one is the Gram Posture Bacteria which contains more than 60% Peptidoglycan in their cell wall. Number two is the Gram Negative Bacteria which contains less than 10% Peptidoglycan in their cell wall. And number third is the Acid Fast Basili which contains more than 60% Mycolic Acid in their cell wall. For Gram Posture Bacteria and Gram Negative Bacteria, we use Gram Staining Technique and for Acid Fast Basili, we use G.L. Nielsen Staining or Zaden Staining. But in this video, we will learn about how to perform Gram Staining Procedure. Let's move towards the procedure area. First step of the procedure is the preparation of the smear. So first of all, what is smear? Smear is the pasting of the sample on the glass light. So first of all, we take a disposable loop and take a sample with the loop and we take a slide and prepare a smear in the oval shape just like that and discard the loop in the discard box and pass the slide over the flame until it dries. So while doing this procedure, we have to take care that we have to pass it over the flame so we have to touch it on the back side of our hand and see if we feel too much heat then it's fine. If we feel too much heat then it means that our smear is damaged. After the fixation of the smear on the glass light with the help of heat, then we will move towards our first dye which is crystal violet. Flood the crystal violet on the slide. The first dye that we will use in Gram Staining is crystal violet and its color is purple. This is the crystal violet dye. As I told you earlier that in the cell wall of a gram-positive bacteria there is more than 60% peptidoglycain, so crystal violet retains in the cell wall of a gram-positive bacteria. After that we will wash it with tap water. After one minute we wash the glass light with tap water and apply the second dye which is Grams Avidine. Grams Avidine is also known as modern dye and it is used to fix the bonding between gram-positive bacteria cell wall and crystal violet. Flood the smear with the Grams Avidine for one minute. As I told you earlier that in the cell wall of a gram-positive bacteria there is more than 60% peptidoglycain, so when we apply crystal violet then crystal violet will make the bonding with the peptidoglycain and to strengthen and fix the bonding so that when we apply crystal violet then the color will not be faint, it will not be colorless, so we apply Grams Avidine. So after one minute we will wash with the tap water, now we will apply ethanol as a decolorizer for 10 to 15 seconds. After 10 seconds we wash again the glass light with tap water, after 10 seconds we again wash the glass light with tap water, after applying the decolorizer we will wash the slide with the tap water and now we will apply counter stain which is known as Saffronine. Apply the Saffronine for one minute. As I told you earlier that in the cell wall of a gram-negative bacteria there is less than 10% peptidoglycain, so gram-negative bacteria retains the Saffronine, that's why gram-negative seems pink in color during microscopy. After one minute we will wash the slide with tap water, now air dry the slide, after air dry any side we will see under the microscope, after air dry the slide we will take the slide and put a drop of the Cedarwood oil or olive oil to see under the 100x lens of the microscope. As we know very well that 100x lens is also known as oil immersion lens, just like that put a oil drop on the smear, see under the 100x lens of the microscope.