Gram Stain Tutor

Question 1

The 4 gram stain reagents are:

Answer 1

1. crystal violet
2. Gram’s iodine
3. acetone-alcohol
4. Safranin (counterstain)

Question 2

Direct smear preparation:

Answer 2

1. Allow specimen to air dry

2. Fix specimen by passing slide over flame a few times (heat fixation)

Question 3

Step 1 of Gram stain procedure

Answer 3

Flood the slide with crystal violet. All bacteria will be stained purple. Rinse with water.

Question 4

Step 2 of Gram stain procedure

Answer 4

Flood the slide with Gram’s iodine. Then rinse with water. This does not cause a color change, but it allows the formation of crystal violet - iodine complex so that violet stain is not so easily removed.

Question 5

Step 3 of Gram stain procedure

Answer 5

Flood the slide with acetone-alcohol. Then rinse with water. This will remove violet staining from Gram-negative bacteria. Gram positive bacteria will remain stained with violet.

Question 6

Step 4 of Gram stain procedure

Answer 6

Flood the slide with Safranin counterstain. Rinse with water and air dry. Gram-negative bacteria will show up as pink.

Question 7

Why do gram positive and gram negative bacteria stain differently?

Answer 7

Due to structural differences in their cell wall. Namely, due to differences in amount of peptidoglycan and teichoic acids that help retain the Gram stain.

Question 8

True or false: It is possible to determine the species of bacteria from Gram staining results alone.

Answer 8

FALSE: definitive identification requires culture and biochemical testing.

Question 9

What kinds of white blood cells can you find in a gram stain?

Answer 9

PMN’s (polymorphonuclear neutrophils), lymphocytes, macrophages

Question 10

What do PMN’s look like?

Answer 10

segmented nucleus (often shows up as a WBC with multiple nuclei)

Question 11

Function of PMN’s

Answer 11

short-lived, phagocytic, usually appear in the acute stages of bacterial infection

Question 12

Lymphocytes

Answer 12

Usually rare to find them in a gram stain, except when looking at CSF (small # of lymphocytes in CSF is normal.

They are smaller than PMN’s and have a thin cytoplasm

Question 13

Macrophages

Answer 13

Slightly larger than PMN’s. Nucleus is large, non-segmented, slightly indented.

Question 14

Function of macrophages

Answer 14

long-lived, phagocytic, more prevalent in chronic infections