Microbiology Lab 2 Practical

What is the goal of the Gram stain?

o classify bacteria as Gram-positive or Gram-negative based on cell wall structure.

What is the first step before staining?

Prepare a thin bacterial smear on a clean slide and let it air dry.

Why is heat fixing important?

It kills bacteria and firmly attaches them to the slide so they don’t wash off during staining.

What is the first reagent in Gram staining and its purpose?

Crystal violet (1 min) — stains all cells purple (primary stain).

What is the second reagent and its role?

Gram’s iodine (1 min) — acts as a mordant to form a dye–iodine complex inside Gram + cells.

What happens during the alcohol step?

Decolorizer (95% ethanol or acetone-alcohol) dissolves outer membranes of Gram – cells and removes dye complex; Gram + stay purple.

What’s the biggest mistake during decolorizing?

Leaving it too long — even Gram + cells will lose color and give false negatives.

After staining, what should you do before viewing?

Gently rinse, blot dry with bibulous paper (do NOT rub), then add a drop of immersion oil.

What is the color result of Gram + vs Gram –?

Gram + = purple | Gram – = pink/red.

What should you always do first before using the 100× objective?

Start at the 4× scanning objective to locate and center the sample.

After finding your specimen at 4×, what’s next?

Switch to 10×, use coarse focus to sharpen the image, center the specimen again.

What do you do at 40× objective?

Refine using the fine focus knob only, and center your specimen for oil immersion.

Before switching to 100×, what should you apply?

lace one small drop of immersion oil directly on the area of interest on the slide.

How do you bring the 100× objective into place safely?

Rotate the nosepiece until the 100× lens clicks into the oil (don’t drag through air first).

What knob should you use at 100×?

Fine focus only — never use coarse focus under oil.

How can you adjust clarity if image looks dark or hazy?

Open the iris diaphragm and adjust the light intensity.

What’s the total magnification at 100×?

100× objective × 10× ocular = 1000× total magnification.

After viewing under oil, what must you do?

Clean the 100× lens gently with lens paper and lens cleaner.