Gram Stain

Question 1

what are the 4 differential stain

Answer 1

1. Primary Stain (Crystal violet)
   - stains all cells purple.
2. Mordant (Gram’s iodine)
   - forms CV-I complex → intensifies stain, makes it harder to wash out.
   - all cells still appear purple-black.
3. Decolorizer (95% Ethanol)
   - gram-positive: thick peptidoglycan shrinks pores → CV-I stays, cells remain purple.
   - gram-negative: lipids dissolved, thin peptidoglycan loses CV-I → cells become colorless.

⚠️ Most critical step:
over-decolorization → false Gram-negatives
under-decolorization → false Gram-positives.

4. Counterstain (Safranin)
   - stains decolorized cells pink (gram-negatives).
   - gram-positive stay purple.

Question 2

what are the expected results for
Staphylococcus epidermidis
Streptococcus pyogenes
Escherichia coli

Answer 2

Staphylococcus epidermidis
→ Gram-positive cocci in clusters, purple.

Streptococcus pyogenes
→ Gram-positive cocci in chains, purple.

Escherichia coli
→ Gram-negative bacilli, singles/pairs, pink.

Question 3

what is the function of 95% ethanol in Gram stain?

Answer 3

Decolorizer – removes CV-I complex from Gram-negative cells, but not from Gram-positive.
Alcohol dissolves lipids → CV-I complex washes out → cells take up safranin.

*Gram-positive bacteria remain purple after decolorization as thick peptidoglycan shrinks and traps the CV-I complex.

Question 4

this requires the use of at least four chemical reagents that are applied sequentially to a heat-fixed smear. The first reagent is called the primary stain.

Answer 4

differential staining
- function is to impart its color to all cells
- b Dr. Hans Christian Gram

Question 5

Which step in Gram staining is the most critical?

Answer 5

Decolorization step.

Question 6

Why do Gram-positive bacteria remain purple after decolorization?

Answer 6

Thick peptidoglycan shrinks and traps the CV-I complex.

Question 7

why do we have to use fresh culture/ or cultures prepared less than 24 hours

Answer 7

as older Gram-positive bacteria may not retain the primary stain well → become Gram-variable (some purple, some pink).

Question 8

why do we have to wash slides between reagents

Answer 8

Removes excess dye/chemicals.
Prepares slide for the next reagent.

Question 9

procedures for gram stain

Answer 9

1. Flood smear with crystal violet (1 min).
   → All cells purple.
2. Wash with water.
3. Flood with Gram’s iodine (1 min).
   → CV-I complex forms, all cells purple-black.
4. Wash with water.
5. Decolorize with 95% ethanol (drop by drop until runoff is almost clear, bluish tinge).

• Gram-positive: remain purple.
• Gram-negative: lose color.

6. Wash with water.
7. Flood with safranin (45 sec).
   -Gram-positive: stay purple.
   - Gram-negative: become pink.
8. Wash with water.
9. Blot dry with bibulous paper.
10. Observe under oil immersion and record:
    - Shape (coccus, bacillus, spirillum).
    - Arrangement (chains, clusters, pairs, etc.).
    - Color (purple vs pink).
    - Gram reaction (positive/negative).