1、Gram stain,Principals,Materials and method,Results,Analysis,Department of immunology and microbiology Qingxin Chen,History of the Gram stain,The Gram staining method: the Danish bacteriologist , Hans Christian Gram, devised it in 1882 (published 1884), It is one of the most important staining techni

2、ques in microbiology. It is almost always the first test performed for the identification of bacteria.,Purpose of this experiment,To learn the Gram Stain technique To understand its value in the study of bacterial morphology,Principal of Gram Stain,Gram staining is based on the ability of bacteria c

3、ell wall to retain the crystal violet dye during decoloring solvent (alcohol ) treatment. The cell walls for Gram-positive microorganisms have a higher peptidoglycan and lower lipid content than gram-negative bacteria.,The microorganisms that retain the crystal violet dye particles are referred to a

4、s Gram positive , and appear purple under microscopic examination Others that do not retain the crystal violet dye particles are referred to as Gram negative, and appear red.,materials : P31,Plate culture alcohol lamp , microscope slide, inoculating loop normal saline absorbent paper Reagents : Crys

5、tal violet, Grams iodine solution, 95% alcohol , carbolfuchsin solution,Procedures,Preparation of the smear Using a sterile loop emulsify a little bacteria in normal saline (one loop) on a slide to make a thin film of the smear, then dry thin film by passing it several times through a flame then hea

6、t fix the slide by heating the thin film for a while (the slide should not become too hot to touch). Smears that are not properly fixed tend to be washed away during staining and washing resulting in the absence of stained bacteria.,Staining procedure,Primary stain: Smear flood with crystal violet 1

7、 min, gently wash with running tap water. Mordant: Flood with iodine solution 1 min, gently wash with water. Decolorization: Carefully decolorize with 95% alcohol for about 30 sec, gently shake slide , wash with water. Once again (This is the most critical step , be careful not to overdecolorize it

8、, otherwise, the result would be changed ). Time should be controled exactly Counterstain: Flood with carbolfuchsin 30 sec, wash with water.,observation of stained bacteria,Blot with absorbent paper (dont rub it) , observe under an oil-immersion objective.,Results,As shown below, bacteria that retai

9、n the violet-iodine complexes after washing in alcohol stain purple and are termed Gram-positive those that lose this violet-iodine complex and stain red from the carbolfuchsin counterstain are termed Gram-negative.,Typical Gram stain,Gram positive,Gram negative,Why did G+ bacteria and G- bacteria appear difference by Gram