Gram Stain

Objective:

To test a sample of laboratory prepared bacteria and categorise it according to Christian's gram positive and gram negative classes and also by viewing it under a high powered microscope and oil immersions; classify its shape and note any special characteristics.

Introduction:

Bacteria was categorised into two groups in 1884 by the Danish Bacteriologist Christian, gram positive and gram negative by a staining technique where the ability to avoid de-coloration of Crystal Violet solution by alcohol would render the category of gram positive, and gram negative if the bacteria is de-coloured. This could be noted by the final colour of the bacteria: a violet colour where Gram positive and a pink colour of the Safranin added pending the de-colouring process.

Materials:

1. Bacteria Sample 2. Microscope Slide 3. Gram Staining Kit and Wash Bottles a. Crystal Violet Solution b. Iodine Solution c. 95% Ethyl Alcohol d. Safranin e. Distilled Water 4. Bibulous Blotting Paper 5. Microscope 6. Oil

Procedure:

A. Preparation :

1. Bacteria is cultivated on agar jelly in an incubator at 25°C for 24 hours. 2. Obtain a microscope slide and with a toothpick, smear a thin coat of the bacteria sample onto the slide 3. Cover the smear with a drop Crystal Violet and leave standing for 20 seconds 4. Wash off the stain with distilled water; drain and blot off the excess with bibulous paper. 5. Apply Gram's Iodine on the smear and leave to stand for 1 minute. 6. Drain the excess iodine and apply 95% Ethyl alcohol for 20 second duration or till the alcohol runs clearly from the slide. 7. The smear should rinsed for a few seconds with distilled water to stop the action of the alcohol. 8. Drain and blot off the excess with...