 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 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. After the fixation of the smear on the glass light with the help of heat then we will move towards over first dye which is crystal violet. Flood the crystal violet on the slide for 1 minute. So 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 a gram postive bacteria cell wall there is more than 60% of taptinoblique. So crystal violet retains in the cell wall of gram postive bacteria cell wall. Gram postive bacteria cell wall picks up the dye and retains it. So the reason for this is that gram postive bacteria has a purple color in the microscope. After that we will wash it with tap water. After 1 minute we wash the glass light with tap water and apply the second dye which is grams avodine. Grams avodine 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 grams avodine for 1 minute. As I told you earlier that in a gram postive bacteria cell wall there is more than 60% of taptinoblique. So when we apply crystal violet then crystal violet will make the bonding with taptinoblique. And to strengthen this bonding and to fix it so that when we apply the right color of crystal violet then the color of crystal violet will not be faint and the color of crystal violet will not be less. So we apply grams avodine. So after 1 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 applying the decolorizer we will wash the slide with the tap water and now we will apply counter stain which is known as saffronene. Apply the saffronene for 1 minute. As I told you earlier that in a gram negative bacteria cell wall there is less than 10% of taptinoblique. So the gram negative bacteria retains the saffronene that is why the gram negative seems pink in color during microscopy. After 1 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 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. So like that put a oil drop on the smear and see under the 100x lens of the microscope.