# 11.2: Procedures

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Note

Materials that come in contact with our bodily fluids are treated as biohazardous waste and must be autoclaved before discarded. Place these items (swabs, tongue depressors, and empty urine cups into the orange biohazard bag in the front of the room. Wrappers may be placed in regular garbage unless they also come in contact with body fluids.

## A. Throat cultures

One blood agar plate/student

1. Obtain a sterile tongue depressor and cotton swab.

2. Take a throat culture from your lab partner by swabbing the back of their throat in the region of the uvula (be careful not to touch the sides of the mouth or the tongue).

3. Use the cotton swab to inoculate the entire surface of a Blood agar plate.

4. Discard the swab and tongue depressor in the biohazard waste container provided.

5. Plates will be incubated at 37˚C for 48 hours and then refrigerated until the next lab period.

## B. Urine cultures

One blood agar (BA) plate and one Eosin Methylene Blue (EMB) plate/ student

1. Divide your plates into 2 areas- one labeled “simulated” and one labeled “mine”.

2. Obtain a sterile container and cap, and bring it with you to the bathroom to collect a urine sample (do a “clean-catch”). You do not need a large amount of urine- just enough to moisten a cotton swab!

3. Bring your sample back to the lab, and use it to inoculate the side of the EMB and BA plates labeled “mine.”

4. Discard the cotton swab in the biohazard waste container provided.

5. Take another trip to the bathroom to discard the remaining urine from the cup, and then discard the EMPTY urine cup in the biohazard waste container provided.

6. Use one of the four simulated urine samples at your table to inoculate the other side of the BA and EMB plates (you may use your inoculation loop for this).

7. Plates will be incubated at 37˚C for 48 hours and then refrigerated until the next lab period.

## C. Anaerobic cultures

Per pair of students: 1 TSA plate

1. Divide your plate into two areas—one labeled BS (for Bacillus subtilis) and the other CS (for Clostridium sporogenes).

2. Inoculate your plate with these two bacteria (use inoculation loop).

3. Your instructor will divide the plates from your class—half will be incubated aerobically, the other half will be placed into an anaerobic jar. Your instructor will also demonstrate the proper use of the anaerobic jar.

4. Plates will be incubated until the next lab period.

## D. Examination of prepared slides of aerobic and anaerobic pathogens

When you have finished setting up your cultures, examine the following prepared slides under the microscope. Sketch what you see in the spaces provided, and try to look for distinguishing characteristics to help you remember what these organisms look like.

Note

Not all slides have been gram stained, and you should not assume anything from the color of the organism unless you know how the slide was stained (Remember: E. coli would be purple if simple stained with crystal violet!)

Slides to view:

1. Streptococcus pyogenes

2. Streptococcus pneumoniae

3. Clostridium perfringens

4. Clostridium tetani

## E. Vectors of infectious disease: Demonstration of a mechanical vector-the sowbug

Sowbugs (also called pillbugs) belong to Phylum Arthropoda and Class Crustacea (and are therefore related to crabs, lobsters, etc.) There are 4000 described species of pillbugs. They live humid areas in leaf litter, in rock piles or at the base of buildings. They primarily act as decomposers. THEY DO NOT BITE AND THEY DO NOT CAUSE DISEASES. We are using them today as an example of a mechanical vector.

One TSA plate/table

TSA Plate Number: ____________________

1. Number the 4 TSA plates (1/table) 1 – 4. Bring all of the TSA plates to the front of the room at the same time.

2. Obtain a sowbug from the container at the instructor’s table. (Please treat them gently—they will be released into the wild when we are done with them.)

3. Allow it to walk across the surface of TSA # 1 plate for 2 minutes.

4. Pass the sowbug on to TSA plate #2, and repeat the above process. Continue until the sowbug has walked across each of the four plates.

5. Incubate plates until the next lab period.

## F. Vectors and Pathogens

The following slides have been set up on demonstration scopes. Where possible, the vector slide is set up next to the slide of the pathogen that it transmits (in bold). Observe these slides and sketch the organisms in the spaces provided. Then fill out the table included in the results section.

1. Xenopsylla cheopis (rat flea): transmits 2. Yersinia pestis (bubonic plague)

3. Ixodes scapularis (tick): transmits 4. Borrelia burgdorferi (Lyme disease)

5. Anopheles gambiae (mosquito): transmits 6. Plasmodium vivax (malaria)

7. Pediculus corporis (body louse): transmits 8. Borrelia recurrentis (relapsing fever)

This page titled 11.2: Procedures is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Joan Petersen & Susan McLaughlin.