The most common way to screw up is to use the fine

Info icon This preview shows pages 7–10. Sign up to view the full content.

View Full Document Right Arrow Icon
The most common Way to screw-up is to use the Fine and Course Focus Knobs to focus the Image of the Field Diaphragm. No! Bad Student! B ad ! Always use the Condenser Focus Knob to focus the Image of the Field Diaphragm.
Image of page 7

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
Lab 3 Page 8 The Gram Stain (Atlas Figure 6-1, Page 45) Materials (per Student): • Slides • Loop • Bunsen Burners • Gram Stain Reagents (Crystal Violet, Iodine, Alcohol, Safranin) • DI Water Bottle (not your Bacdown Bottle) • The Bacterium to be Gram Stained • Gram Stain Controls (incredibly Optional): - Staphylococcus epidermidis is our Gram Positive Control. - E. coli is our Gram Negative Control. Fun With Stains Before you do your first Gram Stain you should do a brief Exercise that quite graphically shows what’s going-on during a Gram Stain: 1. Add a Drop of Crystal Violet to a clean Glass Slide. 2. Add a Drop of Iodine to the Crystal Violet on the Slide. You’ll see a metallic-looking Crystal Violet-Iodine Precipitate forming on the Slide, just like it forms inside the Bacterial Peptidoglycan Layer. 3. Add a Drop of Ethanol. Gadzooks! You will see the Crystal Violet-Iodine Precipitate dissolves. And Ethanol solubilizes the Crystal Violet-Iodine Precipitate in the Peptidoglycan Layer, too. Neat, huh? Gram Stain Procedure: 1. Make a Smear of the Bacterium you want to Gram Stain in the Center of a Slide (Use the Goldilocks Three Slide Method you learned last Week). You can also make Smears of your Controls on either Side of your Bacterium on this same Slide. But the Difficulty of properly destaining three Smears has proven to be such a Nuisance that most Students don ʼ t use our Controls. It ʼ s your Call. 2. Let the Smears Air Dry. To paraphrase the Coroner of Munchkin City be sure that: It’s not only merely dry, it’s really most sincerely dry. 3. Heat Fix your Smears by passing your Slide -- Smear-Side Up -- through the Flame 2 or 3 Times. 4. Place your Slide on your Staining Rack.
Image of page 8
Lab 3 Page 9 5. Flood the Smears with Crystal Violet Stain. Leave the Stain on your Slide for 1 (one) Minute. 6. At the End of the Minute, tip the Slide so most of the Crystal Violet drains into the Sink. 7. Rinse off the remaining Crystal Violet with DI Water. Remember, it ʼ s basically impossible to rinse for too long. 8. Flood the Smears with Iodine and let your Slide sit on the Staining Rack for 1 Minute. 9. At the End of the Minute, tip the Slide so most of the Iodine drains into the Sink. 10. Rinse off the remaining Iodine with DI Water. 11. While holding the Slide at a 45° Angle, drop 10 Drops of Ethanol over 10 Seconds onto your Smear. You should see Crystal Violet-Iodine Complex washing away (which is why this Step is sometimes called Decolorization). Go immediately to the next Step. 12. Stop the Decolorization by rinsing with beaucoup DI Water.
Image of page 9

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
Image of page 10
This is the end of the preview. Sign up to access the rest of the document.

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern