How to Identify Gram Positive Rods

Secure the use of a clean, aseptic laboratory space to conduct the Gram stain test. Wear clean lab clothing and shoe coverings to prevent contamination. Use gloves and a mask when handling bacterial cultures. Isolate a colony of bacilli for your test and grow it out on nutrient-rich agar in petri dishes. Sterilize the petri dishes and agar with a pressure cooker before using.

Add a few drops of clean water to the culture dish. Distribute it evenly over the agar. Drag an inoculation loop lightly across the agar's surface. Transfer the sample onto a clean glass slide and smear it evenly in circular area the size of a dime. Keep the size of the sample minimal; you should be barely able to see it with the naked eye.

Apply 95 percent methanol over the slide smear for two minutes to fix the cells and prevent distortion of their morphology. Air dry the slide or heat it gently over a low Bunsen burner flame. Move the slide back-and-forth to heat evenly and avoid localized hot spots. Apply the heat uniformly and consistently. Avoid taking the slide out of the flame until you're finished heating it.

Pour crystal violet stain reagent over the fixed slide. Let it stand for 30 to 60 seconds. Wash off the stain with tap water for a few seconds. Drain excess water from the slide. Cover the slide with iodine stain reagent for 30 to 60 seconds. Wash off the iodine with tap water. Tilt the slide and add a few drops of decolorizer. Apply it until the runoff is clean. Wash the slide.

Soak the slide with pink-colored safranin counter-staining reagent for 30 seconds. You may substitute basic fuchsin solution for the safranin. Gently wash the slide and shake of excess water. Blot it dry with absorbent paper and then air-dry the slide. Examine the prepared slide under a microscope equipped with a light source. Look for and identify gram positive bacteria rods with blue or violet coloration from the Gram stain test.