 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 postive bacteria and Gram negative bacteria. Mainly, there are three types of bacteria on the basis of their cell wall. Number one is the Gram postive 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 bacilli which contains more than 60% mycolic acid in their cell wall. For Gram postive bacteria and Gram negative bacteria, we use Gram staining technique and for Acid fast bacilli, we use G.L. Nielsen staining or Zedden 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 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 I discard the loop in the discard box and pass the slide over the flame until it dries. While doing this procedure, we have to take care of this. Whenever we have to pass it over the flame, we have to touch it on the back side of our hand. If we don't feel the heat, then it's fine. If we feel the heat more, then it means 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 for one minute. The first dye 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 Gram postive bacteria, more than 60% of the ptidoglycan is present. So crystal violet retains in the cell wall of Gram postive bacteria. The cell wall of Gram postive bacteria picks up the dye and retains it. This is why Gram postive bacteria has a purple color in the microscope. 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 Gram zavodine. Gram zavodine is also known as modern dye and it is used to fix the bonding between Gram postive bacteria cell wall and crystal violet. Flood the smear with the Gram zavodine for one minute. As I told you earlier that in Gram postive bacteria cell wall, more than 60% of ptidoglycan is present. So when we apply crystal violet, then crystal violet will make bonding with ptidoglycan. And to fix this bonding, when we apply decolorizer, the color will not fade. So we apply Gram zavodine. 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 again wash 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 suffranine. Apply the suffranine for one minute. As I told you earlier that in Gram negative bacteria cell wall, less than 10% of ptidoglycan is present. So Gram negative bacteria retains the suffranine. 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 drying the slide, we will see under the microscope. After air drying the slide, we will take the slide and put a drop of 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 an oil drop on the smear and see under the 100x lens of the microscope.