# 3.2: Procedures

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## Preparation of a Bacterial Smear and Gram Staining

This semester you will be performing three staining procedures: Gram stain, acid-fast stain, and endospore stain. All three of these staining procedures begin with the preparation of a bacterial smear.

Materials

• Clean microscope slides
• Staining trays and newspaper
• Gram stain reagents: crystal violet, Gram’s iodine, safranin, 95% ethanol
• Water bottle (for rinsing)
• Bacterial cultures: Escherichia coli, Staphylococcus aureus, Micrococcus luteus, Pseudomonas aeruginosa, Corynebacterium xerosis, and Neisseria sicca

## How to make a Bacterial Smear

1. Label a clean glass slide as demonstrated by your instructor.

2. Add a small drop of saline to the slide (you will usually put two bacteria on one microscope slide- Follow your instructor’s specific instructions). This can be done by placing a drop of saline onto your inoculation loop and then transferring it to the slide. If you use the saline dropper directly on the slide, do not release a full drop.

3. With an inoculation loop or needle, pick up a small amount of bacteria. Mix it well with the saline and spread the mixture over a wider area of the slide.

• Be careful not to have the two smears run into each other.

4. Air dry the bacterial specimen on the slide (slide warmers may also be used).

5. When slides are completely air-dry, heat fix the bacterial specimen by passing the slide slowly over the flame twice (your instructor will demonstrate this).

• Heat fixing kills cells, and adheres them to the slide.
• Cells will be rinsed off the slides if they are not heat fixed properly.
• Be careful not to overheat the slides in this procedure

After heat-fixing is complete, you are ready to gram stain your slide.

## Gram Staining Procedure

• For all steps in the gram staining procedure, add enough of the solution to cover the areas of the slide that have bacteria on them. You do not need to flood the entire slide.
• All staining should be done over a staining tray. Be sure to put newspaper under the tray in case of spillage.
• Gloves should be worn while staining and removed before working with the microscope.
STEPS EXPLANATION
1. Add a few drops of crystal violet (primary stain) to the smear and let it sit for 60 seconds.
2. Rinse the slide with water. All cells are purple after this step. Stopping here would be a simple stain.
3. Add a few drops of Gram's iodine (mordant) to the smear and let it sit for 60 seconds. Gram’s iodine forms a complex with crystal violet
4. Rinse the slide with water. All cells are purple after this step.
5. Decolorize with 95% ethanol: let the alcohol run over surface of slide until no more crystal violet color comes out of the smear (time varies—no more than 5-10 seconds). Gram positive cells retain crystal violet and remain purple. Gram negative cells lose crystal violet and are now colorless
6. Rinse with water. Water rinse stops the decolorization process
7. Add a few drops of safranin (counterstain) and let it sit for 60 seconds. Safranin is a pink/red dye
8. Rinse with water. Blot dry on bibulous paper. Be careful not to wipe off the bacteria. Gram positive cells remain purple; gram negative cells are now pink/red
9. Observe your slide under the microscope. For each organism, determine morphology, arrangement and Gram reaction

3.2: Procedures is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Joan Petersen & Susan McLaughlin.