Gram staining

Question 1

Primary Stain

Answer 1

Crystal Violet

Question 2

Mordant

Answer 2

Gram’s Iodine

Question 3

Decolorizer

Answer 3

Alcohol/acetone

Question 4

Secondary stain

Answer 4

Safranin

Question 5

Primary stain

Answer 5

Colored dye, usually purple that impairs its color to all cell

Question 6

Mordant solution

Answer 6

Substance that fixes a stain or dye

Question 7

Counterstain

Answer 7

Stain with a contrasting color to the primary stain.

Question 8

Gram’s Positive bacteria stain

Answer 8

◘ Result depends on the bacteria cell wall structure
◘ Have a single thick cell wall
◘ Allows retention of the primary stains
◘ Prevents the decolorizer from penentrating and washing out the primary.

Question 9

Gram’s Negative bacteria stain

Answer 9

◘ Have multiple thin layer walls
◘ Allow decolorizer to penetrate the cell walland wash out the primary stain
◘ Retains the last stain imparted in the cell walls

Question 10

Crystal violet

Answer 10

◘ Primary stain
◘ Will stain all gram positive microrganisms a purple color

Question 11

Gram’s iodine

Answer 11

◘ Fixer or mordant
◘ Is a substance which fixes a stain or a dye
◘ Fixes to crystal violet permanently to the gram positive micoorganisms

Question 12

Ethyl alcohol (95%) and (3%) acetine

Answer 12

◘ Decolorizer
◘ Is applied momentarily to the cells on the slide and rinse with the decolorizer until clear
◘ It removes the crystal violet from the non- gram positive

Question 13

Safranin

Answer 13

◘ Secondary stain
◘ Stain all gram negative microorganism a bright pink or red color

Question 14

Gram’s stain procedure purpose

Answer 14

◘ To direct examination of the specimens and subcultures
◘ To deternine the bacteria gram stain reactivity to aid in diagnosis or for further organisms work up and identification

Question 15

What is the safety measure that you always need to observe?

Answer 15

Always observe appropriate safety and health precautions when working with infectious materials

Question 16

Which are the Gram’s stains specimens?

Answer 16

◘ swab
◘ solid media
◘ liquid media

Question 17

Gram’s stain procedure

Answer 17

1. label the slide
2. Prepare the specimen
3. Heat fix the slide; taking care not to burn the organism
4. Place slide on a staining rack and overly the smear with Crystal violet solution. Let stand for one minute
5. Hold the slide at a 45 degree angle and rinse throughly with water
6. Overlay the slide with Gram’s iodine and let stand for one minute
7. Hold the slide at a 45 degree angle and rinse throughly with water
8. Flood the slide with the decolorizer until no violet color washed off
9. Immediately rinse throughly with water, ensuring the decolorizer is totally removed to prevent over decolorization
10. Overlay with Gram’s Safranin -O 30-60 seconds
11. Rinse with water
12. Gently blot the slide dry with bilbous paper towels. Take care not to wipe away the specimen
13. View the smear at 100x Oil Immersion Objective

Question 18

How do you prepare the swab specimen?

Answer 18

◘ Roll the swab on the slide
◘ Air dry the slide, do not blow or fan

Question 19

How do you prepare the solid media specimen

Answer 19

◘ Place a drop of sterile water in the slide
◘ Emulsify one colony of the suspected organism
◘ Distribute evenly

Question 20

How do you prepare the liquid media specimen

Answer 20

◘ Mix specimen well
◘ Use the loop to transfer the organism to the silde

Question 21

What is a gram stain?

Answer 21

It’s a DIFFERENTIAL stain that allows for classification of a bacteria as either gram-positive or gram-negative. Discovered by Hans Christian Gram.

Question 22

The most important determining factor in this procedure is:

Answer 22

The fact that bacteria differ in their RATE of decolorization.
-Gram (+) decolorize slowly and maintain the primary stain.
-Gram (-) decolorize easily.

Question 23

What were the three species of bacteria observed? What was their morphology & arrangement? What was the final color, and what does that mean on whether it was gram (+) or gram (-)?

Answer 23

1. Staphylococcus epidermis: sphere, clustered; purple= gram positive.
2. Bacillus subtilis: chains of rods; purple= gram positive.
3. Escherichia coli: rods, no specific arrangement; pink= gram negative.

Question 24

Why will old gram-positive cells stain gram-negative?

Answer 24

The cell can only retain primary stain for so long, so the cell wall degenerates and the stain can move to the outside of the cell wall, making it appear gram-negative.

Question 25

Can iodine be added before the primary stain in a Gram stain?

Answer 25

No. Iodine causes crystal violet to bond to the peptidoglycan in the cell walls, preventing the crystal violet from being washed off.

Question 26

List the steps of staining and the color of each in gram pos. and gram neg.:

Answer 26

1. Crystal violet: GP cells= purple, GN cells= purple.
2. Iodine: GP= purple, GN= purple.
3. Alcohol: GP= purple, GN= colorless.
4. Safranin: GP= purple, GN= pink.

Question 27

Which step can be omitted without affecting the gram reaction?

Answer 27

safranin.

Question 28

Suppose you Gram stained a sample from a pure culture of bacteria and observed a field of red and purple cocci. Adjacent cells were not always the same color. WhY?

Answer 28

The culture may be old

Question 29

Suppose you are viewing a Gram stained field of red rods and purple cocci through the microscope. Why can this happen?

Answer 29

It is probably a mixed culture.

Question 30

Explain the effects of the alcohol decolorizer on Gram+ and Gram- cells:

Answer 30

-Gram+: stays stained purple because there are lots of thick layers of peptidoglycan in the cell wall that traps the stain, making it hard for it to escape to the outside.
-Gram-: has a small layer of peptidoglycan in the cell wall that allows for movement of the stain to the outside of the cell.