Experiment 1: Staining

Simple staining quickly assesses bacterial shape and size. This experiment uses crystal violet to help visualize and understand microbial structures more accurately.

Procedure

Prepare the Agar Plates:

1. Loosen or remove the cap on the Nutrient agar bottle. Place the bottle in the microwave (if you do not have a microwave, place the bottle in a heat-safe bowl and pour boiling water around the bottle) and heat until the entire agar bottle is liquefied. You will need to remove the bottle and swirl every 10 seconds to distribute the heat.
   Note: If you notice the agar boiling over, STOP the microwave and let the bottle cool down before handling. Hot agar can violently explode out of the bottle if heated too quickly and/or shaken. After boiling has stopped, use a hot pad protecting your hands and remove the bottle from the microwave. Use caution when removing the bottle from the microwave as it will be HOT!
2. Gently swirl the bottle to mix the solution a final time.
3. Slowly pour the liquefied agar solution into the bottom half of five petri dishes so that it covers the entire bottom of the dish. It is important that the entire bottom is coated and that the agar is given time to spread out over the dish.
4. Place the lids onto the petri dishes and let them sit at room temperature until the plates are solidified and cooled. The plates can be used immediately or stored in the refrigerator for later use.
5. If you have stored the plates in the refrigerator, remove them and allow them to sit at room temperature for at least one hour before use.
6. Turn the petri dish over and use a permanent marker to draw a line down the center to divide the plate in half.

Inoculate the Prepared Plates:

7. Look around for three surfaces to swab microorganisms from. Things such as shoes, a table, teeth, mouth or throat, bathroom doors, and shopping carts can be great sources. Try to avoid surfaces that are frequently cleaned with antibacterial detergents and soaps.
8. Label each side of the bottom of your petri dish with the titles of the surfaces you select (e.g., if you choose a table, label the dish as “Table”).
9. Put on a pair of gloves, and begin collecting your bacteria samples by rubbing a cotton swab on the location you select. Be sure to get good coverage on the cotton swab; and, use a new cotton swab for each sample.
10. Remove the lid from the agar plate but hold it closely over the top of the plate to use as a shield to prevent airborne contamination of the plate.
11. Carefully streak the cotton swab onto the gelled medium (agar). To do this, start at the top and work down in a zigzag motion. Ensure that you do not press too hard when streaking the swab on the agar to prevent cutting into the agar surface.
12. Repeat Steps 10 and 11 with your second cotton swab on the second half of the agar plate.
13. Place the lid onto the agar plate and seal it with a strip of Parafilm™ (hold one end of the Parafilm™ firmly against the side of the petri dish and stretch the other side to cover the entire perimeter).
14. Let the plate incubate in a warm location for three days, or until visible colony growth has formed.

Smear the Slide

**You will be preparing three slides in this experiment. One of these three slides will be used in each of the three following staining procedures: simple, negative, and Gram.**

15. Use the wax pencil to make two circles on three different microscope slides.
16. Use the wax pencil to label each slide as either “Simple”, “Negative”, or “Gram”.
17. Use a pipette to transfer one drop of sterile saline into each circle (6 drops total).
18. Find 6, well-developed and isolated bacterial colonies from your petri dish growth. Try to find three colonies from each surface type.
19. Use a sterile inoculating loop to transfer a small amount of one of the bacterial colonies to a circle on the first slide. Mix gently.
20. Repeat Step 19 for 5 more colonies using the remaining 5 circles that you drew on the slides. Allow the samples to air dry. Each slide should have one colony sample from each surface type.
21. Sterilize the forceps with your candle. To do this

    Figure 7: Grasp the slide with the forceps and pass the slide through the flame

    1. Pour isopropyl alcohol into a 100 mL beaker until you have a depth of 2 - 4 cm. Place the cap back on the bottle and position it clearly out of the way.
    2. Light your tea-candle and set it aside. Be very cautious that your tea-candle is safely positioned and away from the beaker of isopropyl alcohol.
    3. Dip the forceps into the isopropyl alcohol for 10 seconds.
    4. Without touching the forceps to anything, carefully pass the end of the forceps through the flame several times.
22. Pick up one slide with the forceps and flame it by passing the slide, smear side up, through the candle flame 4 - 5 times. Repeat this process until all of the slides have been flamed. Extinguish your flame when complete.
23. You will use the Simple slide for the rest of this procedure. Place the Negative and Gram slides in each half of the 9 cm. petri dish, and safely store for later use.

Figure 8: Pour crystal violet over the smear. Image courtesy of the CDC: Public Health Image Library.

Stain the Slide (Simple Technique)

24. Over a sink or disposable plastic container, saturate the bacterial sample with crystal violet and wait 1 minute.
25. Rinse the slide with deionized water for 30 seconds.
26. Cut the bibulous paper into a small strip and use one piece to blot the excess water from the slide. Take caution not to disturb the sample. Reserve your second half for Experiment 3.
27. If you have a microscope available, observe the stained slide under increasing the magnification and record what you see at each magnification in Table 1. If there is no microscope is available, refer to Figures 9 and 10.
28. Place your slide in a disposable plastic container, and pour bleach over the surface until the sample is completely covered/saturated. Allow the bleach to soak for approximately 20 minutes and then rinse the bleach down the sink with running water.
29. Wrap the slide in Parafilm™ and dispose of it in the trash.

Figure 9: Simple stains use one dye only. Crystal violet was applied to this slide to show chains and solitary Gram negative Legionella pneumophila bacteria. Image courtesy of the CDC: Public Health Image Library.	Figure 10: The crystal violet stained bacteria above are Vibrio comma, the cause of Asiatic cholera. Note the flagella structures. Image courtesy of the CDC: Public Health Image Library.