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The Lab Exercises have been created over many years as a team effort by the Molecular Biology and Microbiology faculty.

1. Date and Time

Fall 2004, 1.5 hours

2. Orientation Exercise:

To teach you basic microbiology techniques, we will begin with a brief orientation exercise.

3. Special Materials:

mixed culture, Gram stain dyes, example Gram stain slide, example streak plate

4. Procedure:

On a single slide, smear the mixed culture and Gram stain it.

Gram Stain: We will use a modified procedure for Gram staining called the Bacto 3-step Gram stain procedure.

Start with an air-dried, heat-fixed smear (Don’t overdo the heat-fixing!).

BactoGram Crystal Violet 1 min. Wash gently with tap water. Drain well so as not to dilute the iodine in the next step.
BactoGram Iodine 1 min. Wash gently with tap water
Bacto 3-Step Safranin 10 sec. Wash and blot dry gently taking care not to rub the smear off the slide.

Bacteria that stain red are Gram negative; those that stain purple (or blue) are gram positive. The Gram positive bacteria retain the purple stain in their cell wall (crystal violet). Gram’s iodine is a mordant that binds the crystal violet to the cell wall. Gram negative bacteria do not retain the purple dye in their cell wall and must be counter-stained with a pink colored dye, safranin, to visualize them under the microscope.

Examine the morphology and Gram reaction of the organisms. Note the shape of the organisms, round or rod-like, and the color, purple for Gram positive and red for Gram negative. Repeat the procedures until you are proficient in both the preparation of films and in the use of the Gram stain method. Ask an instructor to check your slides.

You will be using this staining method throughout this laboratory and in your clinical career. Because of the significant differences in antibiotic susceptibilities of Gram negative and Gram positive bacteria, it is important to be able to rapidly distinguish these two types. Gram staining is one of the most basic, yet most important steps in the identification of bacteria. Do not underestimate the usefulness of the Gram stain.

4.1. Part A. Throat flora exercise: Special Materials: Blood agar (BA) plate, tongue depressor, sterile swab

Using a tongue depressor and a sterile swab, have a colleague swab your throat. Use the side of the swab to touch the tonsillar crypt, remembering that poking around back there can cause tissue damage as well as activating the gag reflex. Use the swab to inoculate blood agar (BA) plates. Use the swab for the first streak, and a loop for subsequent ones. The blood agar is a very rich medium that will permit growth of most of the aerobic normal flora.

Your plates will be incubated at 37°C under increased CO2 tension for 24 hours; they will then be transferred to the refrigerator for you.

4.2. Part B. Neisseria - Special Materials: 1 chocolate agar plate, sterile swab

Use Chocolate agar for isolation of Neisseria species and the specific oxidase test.

Make a fresh throat swab and inoculate a chocolate agar plate. These plates will be incubated for 24 hours at 37° C.