L17. Gram staining

This was the second experiment that we did on Monday 2nd of March. Gram staining is a method of differentiating bacterial species into two large groups: Gram positive and Gram negative. This differentiation is based by the chemical and physical properties of their cell walls by detecting a peptidoglycan, which is presents in a thick layer in gram-positive bacteria. The objectives of this experiment were to differentiate yogurt bacteria and realte the staining procedure with the structure of the cells.

MATERIALS

-Hot plate
-1 slide
-1 coverslip
-Tongs
-Needle
-Gram stain (crystal violet, iodine and safranin)
-Ethanol
-Microscope
-Yogurt

PROCEDURE 

 1- First we prepared a heat fixed sample of the bacteria by spreading somre yogurt on a slide and drying it on the hot plate.
2-Then we covered the smear with crystal violet and waited for 1 min. After that we rinsed it with distilled water.
3-We applied iodine solution for another 1 min and again rinsed it with distilled water.
4-Then we decolorized using ethaol. Drop by drop until the purple stops flowing and washed immediately with distilled water.
5-Lastly we covered the sample with safranin stain for and exposure time of 45 seconds and rinsed the sample with tap water.
6-Finally we dried the under part of the slide with paper and viewed it on the microscope.

Results and observations: We saw some bacteria red and other purple. Why?

Gram Positive Cell Wall:

Gram-positive bacteria have a thick cell wall which is made up of peptidoglycan (50-90% of cell wall), which stains purple.  Peptidoglycan is mainly a polysaccharide composed of two subunits.  The thick peptidoglycan layer of Gram-positive organisms allows these organisms to retain the crystal violet-iodine complex and stains the cells as  purple.

Gram Negative Cell Wall:

Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall) and lose the crystal violet-iodine complex during decolorization with the alcohol rinse, but retain the counter stain Safranin, thus appearing reddish or pink. They also have an additional outer membrane which contains lipids,  which is separated from the cell wall by means of  periplasmic space.