Bacterial Gram Stain Activity
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ward
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science
Recommended Grade Level(s):
Appropriate for: 12th Grade–College
Time Requirements:
Prep Time: 10 minutes
Activity Time: 10 minutes
Teaching Topics & Concepts:
In this procedure you will perform a stain commonly used in microbiology laboratories to differentiate Gram-positive and
Gram-negative organisms. The way an organism reacts to the Gram stain depends on its cellular structure. Gram staining is an
excellent way to categorize organisms with similar structures and is usually the first differential test performed on an unknown
organism during the identification process.
Materials:
• Pre-cleaned, glass microscope slides.
• Personal Protective Equipment (PPE): Gloves, lab coat and eye protection
• Bacterial Culture(s)
• Sterile Swabs or Loop
• Gram Stain Reagent Kit
• Plastic Transfer Pipettes (unless your stains are in dropper bottles)
Safety
• Wear safety glasses, gloves and lab coat or apron.
• Keep a beaker nearby to collect the liquid waste created by the staining process. Don't put the waste down
the drain.
• Always read the SDS for all chemicals before handling them.
• If using an open flame, always restrain loose clothing and hair. Keep flammable materials away.
!
Procedure:
1. Prepare Your Smear
• Place a small drop of sterile water on a blank slide (skip this step if you're using a liquid culture)
• Using the handle-end of a sterile cotton swab, or a loop, apply a smear of bacterial cells on the slide and allow to air-dry.
– Avoid clumps or debris; you don't need a lot of bacteria to be effective.
2. Fix your Smear
• Using slide-holders, orienting the smear upward, pass the entire slide through an open flame a few times.
– A slide dryer or micro-sterilizer with a slide-holder can also be used if an open flame isn't an option.
• Fixing will adhere the bacteria to the glass surface.
3. Primary Stain
• Flood slide with Crystal Violet stain. Allow to sit for 1 minute.
• Tip slide and rinse with water (a squeeze bottle is helpful, but be gentle!)
4. Mordant/Fixative Step
• Flood slide with Iodine. Allow to sit for no more than 1 minute.
• Rinse gently with water
5. Decolorizer Step
• Tilt slide and gently decolorize with solvent until purple color stops running.
– This step is easy to overdo; be very conservative with your ethanol and rinse with water immediately afterwards.
~ continued ~
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