Hi, I am Praveen Kambalkar from TNAO and I am going to present the video on Gram Staining. Gram Staining is one of the important techniques in microbiology and it is highly useful to identify the unknown bacteria whether it is gram positive or gram negative. The Gram staining method is developed by Hans Christian Gram.
It is the differential staining method of differentiating bacterial species into gram positive and gram negative, mostly on the basis of the cell wall contained, as gram positive contain more peptidoglycan than gram negative. These are all the materials required for the gram staining procedure. first one, Cerelyze inoculating loop, clean glass slide, a petri plate having the bacterial culture, crystal violet, it act as a primary stain, Gram-Cyodine, it act as a mordant, 95% ethanol, it act as a decolorizer and the last one, Saffronene, which act as a counter stain. First of all take a clean glass slide and wash it with 75% ethanol in order to remove all the contamination on the slide.
Then mark the slide with your initial in order to identify your slide. Then place a drop of water on the slide. sterilize the inoculating loop by heating it over the flame until it gets red hot then take the colonies of bacteria from the bacterial culture with the help of inoculating loop and place it over the water drop. By slight mixing form a smear on the slide.
Then allow the slide to air dry. After some time heat fix the slide. By just passing the slide over the flame for 2 to 3 seconds. Crystal violet.
It is blue in color. It acts as a primary stain. It stains both gram positive as well as gram negative bacteria. Apply it on the smear and leave it for the 30 seconds. After 30 seconds just rinse the slide in the sterile water in a such a way that most of the dye gets off.
Grams iodine it act as a mordant it reacts with crystal violet to form a complex apply this Grams iodine on the smear and Leave it for the one minute After one minute rinse the slide in sterile water so that most of the dye gets off. 95% Ethanol, it act as a decolorizer. Apply the 95% ethanol as run it over the stain area at 45 degree angle until the flow is clear.
Check the flow at the base or end of the slide. After running the slide with ethyl alcohol, rinse the slide in the sterile water in order to remove the remaining dye. Saffronine, it is a counter stain. It is red in color.
By the application of the saffronine, gram positive becomes purple color and gram negative becomes red or pink in color. Apply the saffronine. on the smear and leave it for one minute. After one minute rinse the slide in sterile water to remove the excess dye.
Now your slide is ready to view under the microscope. Place the slide under the microscope at 100x Oil Emulsion Objective Lens. It will produce the image showing gram positive as purple and gram negative as pink color bacteria.