To begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
Place the uncovered plate under a dissecting microscope and then observe the individual communities within each quadrant of the plate.
Then, repeat the observations for each of the other two bacterial species.
To perform Gram staining of your three bacterial species, first label the ends of three microscope slides with the initials of each bacteria species, your initials, and the date. HYPOTHESES: In this experiment, the experimental hypothesis might be that it is possible to elucidate different bacterial species and shapes using Gram staining, and that Gram-positive bacteria will stain purple with crystal violet whereas Gram-negative bacteria will appear pink due to the safranin counterstain. The null hypothesis could be that all of the bacterial types will retain both stains equally, and staining will not help elucidate their species or shape.
To begin the first stain, attach a clip to the slide for ease of handling. For each bacterial smear, remember to be sure to use a cleaned and flame-sterilized inoculating loop.
Place two loopfuls of sterile distilled water onto the center of the slide.
After sterilizing the loop again, cool the loop by touching an uninoculated portion of the agar several times.
Then, remove a small amount of culture from an isolated colony and mix it with the water on the slide.
Allow the slide to dry at room temperature and heat-fix the smear by quickly passing it through a flame.
Once dry, pipette several drops of crystal violet onto the slide and let it sit for two to three minutes.
Carefully rinse the slide with distilled water by aiming the direct flow towards the top of the slide, allowing the water to gently flow down. Do not aim the water flow directly at the smear.
Next, cover the slide with Gram's iodine, leave it to sit again for two minutes, and then gently rinse with distilled water.
Decolorize the slide with 95% ethanol until stain no longer washes away.
Immediately, rinse the slide with distilled water. This will limit over-decolorizing.
Then, add several drops of safranin counterstain and leave for 30 s. This will stain any Gram-negative cells present.
Gently rinse the slide with distilled water and blot dry with absorbent paper.
After staining the other two slides in the same manner (steps 6 – 17), observe each with a microscope.
Use a low power objective first to make coarse adjustments. When you find an ideal section, move on to the higher magnifications.
After further imaging and adjusting the smear with a medium power objective, add immersion oil directly to the smear. NOTE: The oil is needed for high power objectives that will provide the best micrographs.
Now, use colored pencils to draw the observed bacteria in the appropriate magnification circles and fill in the observed characteristics in the appropriate column, noting the bacteria's shape and whether it is Gram-positive, identified by darker purple staining, or Gram-negative, identified with pink staining. Click Here to download Table 2
Finally, repeat the observation, drawing, and characterization for the other two stained bacterial slides.