Name ______Due Date ______Period _____

PAP BIOLOGY : COMPARING THE EFFECTS OF COMMON ANTIMICROBIALS ON BACTERIAL GROWTH

BACKGROUND:

Most bacterial (and other microorganisms) are harmless. In fact, many bacteria are beneficial. Cheesemaking, decay, and soil building are a few of the important processes that depend on the action of decomposing bacteria, which thrive on decaying organic matter. However, some bacteria are pathogens (disease causers). Tuberculosis, tetanus, strep, syphilis, and some pneumonias are a few of the serious diseases caused by bacteria.

Alcohol and some mouthwashes are antiseptics, substances that stop the growth of infectious agents, and are used on cuts or wounds to stop bacterial infection. Others, like chlorine bleach, are too toxic for use on living tissue. These are called disinfectants and are used on clothes, surfaces, or other non-living objects. These agents generally work by causing the destruction of the cell membrane (causing the cell membrane to lyse). Even though antiseptics and disinfectants are very useful in helping to prevent infections, we cannot use them internally to treat an infection. If bacteria enter our bodies, we rely on another class of chemicals called antibiotics to kill them. Although their use is now commonplace, antibiotics were only discovered about 50 years ago. The antibiotics were the first of the “miracle drugs” and they have permanently altered the course of history. Unfortunately overuse and misuse of antibiotics has allowed certain types of bacteria to become resistant to treatment.

The procedure of placing bacteria on agar plates is called inoculation. Organisms so small that they can only be seen with a microscope are living all around, on, and in us. They include bacteria, viruses, molds, and yeasts.

Pre-Lab

1.  Use your pen or a highlighter to mark the definitions of the 3 bold-face words in the second paragraph.

2.  Apply these 3 terms (show that you know what they mean) by matching the common examples given to the correct term.

A.  ______antibiotics 1. Lysol wipes

B.  ______antiseptics 2. penicillin and tetracycline

C.  ______disinfectants 3. Alcohol, iodine and peroxide

3.  Label the following diagrams with the structures shape (coccus, bacillus, or spirillum) and its arrangement (staphylo or strepto)

Shape: A. ______B. ______C. ______

Arrangement: D. ______E. ______F. ______

Using your prior knowledge of bacteria, answer the following questions:

4.  Common bacteria, like the kind we will grow in this lab, are classified in:

A.  Domain ______B. Kingdom ______

5.  Bacteria are considered to be prokaryotes / eukaryotes. (circle one)

6.  Explain your answer to question 5: ______

7.  Are most bacteria unicellular or multicellular? (circle one)

PURPOSE

In this lab you will compare the effectiveness of disinfectants, antiseptics and/or antibiotics in preventing the growth of bacteria from your environment to the effectiveness of water (a control).

MATERIALS:

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sterile agar plates

antiseptics

disinfectant solutions

antibiotic discs

permanent marker

water

forceps

cotton swabs

tape

metric ruler

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PROCEDURE DAY 1

***Keep Petri dish closed at all times except when inoculating and placing disks in quadrants*****

1.  On the outside of the bottom of each Petri dish, close to the edge of the dish, write your lab group name and class period in small letters with the marker. Refer to Figure A.

2. On the outside bottom, draw lines to divide the dish into quadrants and number them 1, 2, 3, and 4.

3.  To inoculate your Petri dishes (to put bacteria on your agar plate):

a.  Dampen (not dripping wet) one cotton swab in the tap water,

b.  Rub the cotton swab over the area you would like to sample (desk top, pencil sharpener, etc),

c.  Gently rub the cotton swab over the entire surface of the agar of your dish (NOTE: Agar is like jello. If you press too hard on the agar with the cotton swab, the agar will tear. Do not puncture the agar!) Refer to Figure B below.

i.  Raise the top of your Petri dish cover slightly. Starting at one edge, rub the cotton swab back and forth across the entire surface.

ii. Turn the Petri dish a quarter of a turn and re-streak the swab across the agar surface to ensure that bacteria are on the entire surface. (Remember, bacteria are microscopic so you won’t be able to see it

4.  Replace the cover of the petri dish.

5.  Dispose of the cotton swab in the container of bleach. Be careful not to place the swab on any surface or allow the swab to come into contact with anyone.

6.  To assay bacterial effectiveness, you will conduct a sensitivity test. You will place 3 small disks soaked in different bactericides and one disk soaked in sterile water on your plate, one per quadrant.

Use a paper towel to drain off excess drippings BEFORE you put it over the plate so that the agar plate is not contaminated.

Raise the cover of the petri dish slightly and place one disk that has been soaked in sterile water in quadrant #1 and record it in the data table below. Follow the same procedure of the other 3 disks placing them in quadrants 2, 3, and 4. Record this information in the data table below.

7.  Tape both Petri dishes shut and place them in the incubator. You will not open them again.

Quadrant / Antimicrobial Reagent
1. / Water (control)
2.
3.
4.

Location Swabbed______

Name ______

Period ______

DUE DATE: ______

Analysis:

ANALYSIS -Day 1 – Answer the following questions. Refer to the background section of this lab, as well as your notes, text, and other available references for help.

1. Predict what will happen in each quadrant.

2. How the zone of inhibition is produced.

4. Why is it important to use sterile techniques while inoculating the agar plate?

5. a. What was the control used in this experiment?

b. Why is a control needed?

6. Why would an antibiotic disk have no zone of inhibition around it?

7. What does it mean in step 7 when it says “To assay bacterial effectiveness…” (HINT: use a dictionary!)

8. Why is nutrient agar used in this experiment?

ANALYSIS - Day 2 – Answer the following questions

1.  Based on you data, rank your test reagents in order from the most effective to least effective at killing the bacteria in your petri dish.

2.  Which of your test reagents would be the best to clean up a contamination of this bacteria in the lab?

Explain your answer:

PROCEDURE DAY 2

Zone of Inhibition

When you swab bacteria over the surface of the agar in the pattern shown above and allow the bacteria to grow, it will grow uniformly on the surface of the agar (called a bacterial lawn). When an antiseptic or disinfectant is introduced, the area where no growth occurs is called the zone of inhibition. The larger the diameter of the zone of inhibition, the more effective the substance is at preventing bacterial growth.

***DO NOT OPEN YOUR PETRI DISH!!!!

1. Observe your petri dish after the incubation period. Hold your dish up to the light to see the zones of inhibition more clearly.

2. Use a metric ruler to measure the zone of inhibition (the diameter of the clear zone). DO NOT OPEN YOUR PETRI DISH!!!!

3. Draw, label, and color a diagram of your petri dish in the space below. Use a petri dish and a ruler to draw the dish. DO NOT OPEN YOUR PETRI DISH!!!!. Label each quadrant with the appropriate number. Color the drawing the correct colors that you observed. The zones of inhibition should be the correct size (use a ruler!) and should be labeled. Put a title for your figure on the line provided. DO NOT OPEN YOUR PETRI DISH!!!!

4. DO NOT OPEN YOUR PETRI DISH!!!! Return the taped petri dish to your teacher for disposal. DO NOT OPEN YOUR PETRI DISH!!!!

Data:

Table 2. ______

Quadrant / Antimicrobial Reagent / Zone of Inhibition (mm)

Figure 1. ______

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