The Lab Exercises have been created over many years as a team effort by the Molecular Biology and Microbiology faculty.
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簞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簞 C.
BEFORE LEAVING, PUT AWAY EVERYTHING AND CLEAN
BENCHES AND THEN YOUR HANDS!!!
