Antibiotics in Action

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    Culturing Bacteria
    Appendix

    Appendix

    This appendix contains procedures for preparing smears and Gram stains, procedures which you may need in order to carry out the Culturing Bacteria activities. Be sure to read the General Safety Guidelines and the Microbiology Safety Guidelines before carrying out either of these procedures.
    		Culturing Bacteria Menu

    Introduction
    Activity #1: Bacterial Enrichment
    Using a Carrot Medium
    Activity #2: Enrichment of Microbes
    Using a Milk Medium
    Appendix
    General Safety Guidelines
    Microbiology Safety Guidelines

    Materials and Apparatus

    • Microscope slide
    • Bunsen burner
    • Transfer loop or needle
    • Beral® pipette
    • Gram's iodine solution
    • Hucker's ammonium oxalate crystal violet stain
    • 95% ethyl alcohol
    • Safranine
    • 6% aqueous malachite green
    • 0.25% aqueous safranine

    Smear Preparation Technique

    1. Prepare a bacterial smear on a clean glass slide. To do this, first clean a glass slide to remove any fatty materials (as from one's fingers) from the slide. The slide is washed in soapy water, dried, then gently heated on one side in the flame of a Bunsen burner.

    2. Handle the slide by grasping the EDGES and placing it on the desk, FLAMED SIDE UP.

    3. Flame a transfer loop and use it to place a drop of water in the center of the slide.

    4. Flame the transfer loop or needle again and use it to remove a MINUTE amount of material from your bacteria culture (tube, plate, or flask).

    5. Spread the material on the needle in the drop of water on the glass slide, spreading the liquid over an area of about 1 square inch.

    6. Sterilize the needle before proceeding.

    7. Dry the film on the slide by holding it high over a low Bunsen burner or alcohol flame. DO NOT OVERHEAT TO PRODUCE STEAM!

    8. FIX the film by passing the slide five or six times THROUGH the upper portion of the Bunsen flame. The smear is now ready for viewing and staining.

    Gram-Staining Techniques

    (Note that one can purchase a set of stains and wash liquids from commercial biological supply companies such as Carolina Biological and Science Kit/Boreal.)

    1. Prepare a smear of a bacterial sample (see separate instructions).

    2. Cover the smear with Hucker's ammonium oxalate crystal violet stain and allow to remain for 1 minute.

    3. Drain the excess stain and gently wash the slide in tap water.

    4. Flood with Gram's iodine solution and leave for 1 minute.

    5. Rinse with 95% ethyl alcohol until no more dye flows away from the smear (about 30 seconds to 1 minute).

    6. Counterstain with safranine for 10-30 seconds.

    7. Wash in tap water, drain off the excess water, blot, and allow to air dry.

    Spore Staining Techniques

    Certain bacteria produce resistant cells, called spores, that are characterized by their heavy wall and their lack of affinity for stains. As in the case of acid-fast organisms, special techniques are required to stain these cells.

    1. Prepare a smear in the usual way EXCEPT that the slide should be passed through the flame 20 times.

    2. Immediately flood the warm slide with 6% aqueous malachite green and stain for 10 minutes.

    3. Rinse with tap water for 10 seconds.

    4. Counterstain with 0.25% aqueous safranine for 10 seconds.

    5. Rinse with tap water, gently blot, and air dry.

     

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