Differential staining

MBK – Lab Report

Differential Staining

Questions:

A. What is a differential stain? How is it different from a simple stain?
1. A differential stain is a stain that differentiates between gram-positive and gram-negative bacteria. Bacteria that stain purple are considered gram-positive and those that stain pink are considered gram-negative.
2. A simple stain is a stain that reacts with all microbes in and identical fashion. They are used to increase contrast.
3. They are different from one another because Differential stains affect specific microbes differently depending on the differences in their cell walls, whereas a simple stain affects all microbes the same.

B. Describe the differences between gram-positive bacteria and gram-negative bacteria cell walls?
Gram-positive cell walls are thick and have many interconnecting layers of peptidoglycan. Gram-negative cell walls are thinner and only have a layer of peptidoglycans that are two or three layers thick.

C. What is the purpose of crystal violet in the Gram’s stain procedure?
The crystal violet increases the contrast of both gram-negative and gram-positive bacteria making them appear purple.

D. What is the purpose of iodine in the Gram’s stain procedure? (What is a mordant?)
The iodine helps set the stain by forming an insoluble crystal violet-iodine complex. Basically helps retain the crystal violet to the cell.

E. What is the purpose of acetone-alcohol in the Gram’s stain procedure?
It is a decolorizer that differentiates between gram-negative and gram-positive. Only the gram-positive bacteria will retain the crystal violet-iodine complex. Gram-negative bacteria will not retain the color and the decolorizer will wash out the stain.

F. What is the purpose of safranin in the Gram’s stain procedure?
This is a basic dye that directly stains the gram-negative bacteria that have become decolorized. The gram-positive bacteria are already stained and are not affected by this. It gives gram-negative bacteria a pinkish red color

G. Why do gram-positive cells stain purple? Be specific
Gram-positive cells stain purple because they retain the crystal violet-iodine complex in their cell walls. Also because the crystal violet is a positive charge dye which is attracted to the negative charge in the cell and also the thicker peplidoglycan layer helps retain the dye more.

H. Why do gram-negative cells stain pink? Be specific
Gram-negative cells stain pink because they are not able to retain the crystal violet-iodine complex and become decolorized. Also because of the thinner peplidoglycan layer which had the dye removed by the acetone-alcohol then when the safranin was added and not rinsed off the cell it allowed the cell wall to retain the pinkish red dye.

I. Which of your organisms stained gram-negative? Do your observations correspond with what is known about the organism(s)? If not, explain reasons why the specimen may have appeared gram negative under the microscope
E.Coli and yeast.
J. Which of your organisms stained gram positive? Do your observations correspond with what is known about the organism(s)? If not, explain reasons why the specimen may have appeared gram positive under the microscope
Yeast, S.Epidermidis and L.Acidopholis.