Gram Staining

Purpose

To determine the gram stain of your bacteria sample

Gram

Staining

Gram Staining

Step 1: Crystal Violet

Flood for 1 minute with Crystal Violet. This stains all cells purple.

Step 2: Mordant--Gram’s Iodine

Flood with Grams Iodine for 1 minute. The mordant fixes the crystal violet in the cell, making it more intensely stained and harder to destain.

Step 3: Decolorizing Agent

Decolorize with 95% ethyl alcohol. In gram – cells, the alcohol dissolves the lipids in the outer layers making the cell wall more porous. Thus, the CV-I complex can be more easily removed from the thinner peptidoglycan layer leaving the the cells colorless. In gram + positive cells, the alcohol makes the pores smaller due to the dehydrating effect of the alcohol. Thus, the stain is more difficult to remove and the cells remain purple.

Step 4: Counterstain

Counterstain with safranin for 45 seconds. This is used to stain the cells that have been decolorized.

Purpose

Differentiate between gram – and gram + bacteria.

Determine the shape of the bacteria.

Procedure

place a loopful of tap water on a slide

Sterilize the inoculating loop in the flame of a gas burner.

Lift the lid of the culture plate slightly and stab the loop into the agar away from any growth to cool the loop.

Scrape off a small amount of the

organisms and close the lid Transfer a small sample of the

colony to the drop, and emulsify.

Allow the film to air dry.

1

3

2

Fix the dried film by passing it briefly through the Bunsen flame two or three times without exposing the dried film directly to the flame.

The slide should not be so hot as to be uncomfortable to the touch.

Flood smears with crystal violet. Let stand for 1 minute

Gently wash off with tap water.

Flood smears with Gram’s iodine. Let stand for 1 minute.

Gently wash off with tap water.

Add 95% alcohol drop by drop until the alcohol runs almost clear.

Gently wash off with tap water

Counterstain with safranin for 45 seconds.

Gently wash with tap water.

Blot dry and examine with oil immersion lens.

Gram + Cocci

1 2

Gram Negative

1 2

Shape

Cocci

round

Rod

Bacillus

spiral 1

2

3

Cocci Morphology

Gram-Negative Diplococcus

1 2

Gram-Positive Coccus Chains

1

2

Gram Positive TetraCoccus

1 2

Gram Negative Sarcinae

Gram Positive Cocci Clusters

1

2

Rod or Bacillus

Gram-Negative Rod

1 2

Gram Positive Club Shaped Rod

1 2

Branching Rods

1 2

Gram negative Comma form rods

1 2

Gram Positive Spore-forming Bacilli

Bacillus megaterium

Gram Positive Spore-forming Bacilli

Bacillus megaterium

Spirillum

1

2

3

4

Spirillum volutans

What shape are these

bacteria?

Are they Gram + or

Gram - ?

Quiz

•Write answers on separate sheet of paper to be turned in at the end of the quiz

Enterococcus faecalis

Lactobacillus

acidophilus

Micrococcus luteus

Sarcina aurantiaca

S. epidermidis

1. Cite the primary purpose of each of the following reagents.

1. Primary stain

2. Counterstain

3. Decolorizer

4. Mordant

2. Why is it essential that the primary stain and the counterstain be of contrasting colors?

3. Which is the most crucial step in the performance of the Gram staining procedures? Explain.

4. What are the advantages of differential(use of at least 3 reagents) staining procedures over the simple staining technique?