Gram Staining

Biomedical Science Quiz 5/14/25 

1. What is the purpose of a streak plate? 

Isolate individual bacterial colonies from a mixed sample

2. What is the most common media used to grow bacteria for a streak plate?

The most common medium is nutrient agar

 3. What are the 4 main steps of Gram staining? 

1.Crystal violet – primary stain

2. Iodine – mordant (helps the dye set/adhere better)

3.Ethanol or alcohol-acetone – decolorizer

4. Safranin – counterstain

a. What is the primary stain? Counterstain?

Primary stain: Crystal violet

Counterstain: Safranin

b. What does iodine do? What does ethanol do?

Iodine binds with crystal violet to form a large complex that gets trapped in Gram-positive cells.

Ethanol (the decolorizer) removes the crystal violet-iodine complex from Gram-negative cells, which have a thinner cell wall, making them colorless until counterstained.

4. Why do we use a bunsen burner for Gram staining? 

The Bunsen burner creates an updraft of air that helps keep airborne contaminants away and sterilizes tools (like inoculating loops) by flaming them before and after use. It also helps fix the bacteria onto the slide during heat-fixing.

5. What is “clam-shell” technique, and how does it help avoid contamination? 

The clam-shell technique involves slightly opening the lid of a petri dish like a clam shell, rather than removing it entirely. This minimizes exposure to airborne contaminants

a. Gram-positive cells have a thick peptidoglycan cell wall, stain purple, and are generally treatable with antibiotics 

b. Gram-negative cells have a thin peptidoglycan cell wall + outer 

lipopolysaccharide layer, stain pink, and are more difficult to treat with antibiotics