Materials Included In Kit

Additional Materials Required

Prelab Preparation

Prepare agar by first heating 400 mL of distilled or deionized water. Once water is near boiling (90–95 °C), add 10 g of agar powder to the water. Stir constantly for several minutes to dissolve the agar. The resulting solution should be lightly amber colored with little undissolved material. Fill the bases of the Petri plates approximately half full (about 12–15 mL). Cover the plates with the lids and allow the agar to solidify for several hours or overnight. If the plates will not be used the next day, they must be kept refrigerated until the lab.

Prepare a 10% bleach solution no more than 2 days in advance by mixing the 80 mL of bleach to 720 mL of water.

Safety Precautions

After use, agar plates will contain viable microbes. For this reason, a known culture is used in Activity 2. (The plates used to culture skin and surface bacteria should not be reopened. Use sterile techniques at all times while handling bacterial cultures. Before disposal, plates should be soaked in a 10% bleach solution or autoclaved. The plates should then be disposed of directly into the garbage (see Disposal section). Set a good example for sterile technique of students when handling culture plates. Be sure to sterilize work areas with a 10% bleach or Lysol^® solution and remind students to wash their hands with soap after handling microbes and before leaving the laboratory area. Bleach solution is a corrosive liquid and may cause skin burns. Avoid contact with heat, acids and organic materials; chlorine gas will evolve. Isopropyl alcohol is a flammable liquid, slightly toxic by ingestion and inhalation. Methylene blue will stain skin and clothing. Please consult current Safety Data Sheets for additional safety, handling and disposal information. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The agar plates, cotton swabs, inoculating loops and pipets may be disposed of according to Flinn Suggested Biological Disposal Method Type I by sterilizing the agar plates with a freshly prepared 10% bleach solution or autoclaved at the culmination of the activity (left over bleach from this activity may be used). Isopropyl alcohol may be stored for future use or disposed of according to Flinn Suggested Disposal Method #18a. Methylene blue may also be stored for future used or disposed of according to Flinn Suggested Disposal Method #10.

Lab Hints

• Best results are obtained if students have not washed their hands for a few hours prior to lab. If students have very clean hands or have used antibacterial products the day of the lab, they may not achieve any bacterial growth in the “skin control” section of the Petri plate.
• A known culture of Bacillus megaterium is grown in Activity 1 and observed in Activity 2 to avoid the use of pathogenic microorganisms. Do not allow student to reopen the Petri plates used in the Skin and Surface tests as they may contain pathogenic organisms.
• Place the antibacterial soap, isopropyl alcohol and bleach solution in a central location as all students will be using the same bottles.
• Bacterial growth is usually observed within 48 hours; however, it tends to be optimal after 72–96 hours.
• Announce to the class any surfaces you do not want treated with bleach before beginning the activity.
• Some teachers prefer to heat-fix the bacteria to the slide in Activity 2, step 4 themselves rather than having students do it. That way only one Bunsen burner setup is required and ensures that it is done properly.
• Preferably distilled water should be sterile. Boiling will suffice as a sterilization method for this activity. Place an uncovered, unchipped/unscratched beaker of distilled water in an autoclave for 15 minutes at 15 psi. Use a container much larger than the volume of liquid added to prevent boiling over.
• Remind students to wipe the oil off the 100X objectives with lens paper at the end of the activity.

Teacher Tips

• The Prelab Preparation, including making the agar plates, will take approximately 20 minutes. The first activity of streaking the plates will typically require a 50-minute lab period. The follow-up activity should be completed 3–4 days following and will require a second 50-minute class period.

• Use purchased stock cultures, available from Flinn Scientific, to study how various bacterial strains react differently to antibacterial products.
• Generally students will see the most growth on the “control” sections, some growth on the “antibacterial soap” section and little to no growth on the “alcohol and bleach” sections.
• Some groups may observe bacterial growth in disinfected sections, although most groups will observe no growth. This may be due to contamination from breathing directly onto the plate, accidentally touching a surface which was not disinfected with the cotton swab, etc.
• You may want to include a short post-lab discussion tying together the observed results from this lab to the real world. For example, you may discuss how bleach is often used to disinfect surfaces and bacterial cultures in laboratories and hospitals rather than household cleaners due to the relative effectiveness of these chemicals, and the importance of prepping skin with alcohol or iodine before breaking the skin in a medical environment. This may lead into discussions of complications (e.g., nosocomial infections in hospitals, post-surgery infections).

Answers to Prelab Questions

1. Both disinfectants and antiseptics kill bacteria. What is the difference between these types of products?
   

   Antibacterial household products are considered disinfectants, whereas antibacterial products, such as mouthwash, antimicrobial soaps and other products mild enough for use on the body, are considered antiseptics.
   

2. What purpose do the “control” sections serve?
   

   The controls are samples which have not been treated with an antibacterial agent. They are present for comparison purposes.
   

3. Why are bacteria commonly found on the skin’s surface?
   

   The skin, being our outermost layer, comes into contact with bacteria in the air and on surfaces constantly. Also, non pathogenic bacteria colonize our skin, forming a balanced biotic community aiding in inhibition of pathogenic bacteria.

Answers to Questions

Activity 1

1. Draw the appearance of your plate on the figure below.

Student answers will vary, however the “skin control” and “surface control” sections should have more abundant growth than the disinfected sample sections.

2. Describe the amount of bacterial growth observed in each section using the following terms:

High—Bacterial colonies covering the section of the Petri plate
Moderate—There are several bacterial colonies in the plate section
Low—At least one distinct colony is present in the section
No growth—No colonies present in the section

3. Which section had the most bacterial growth? Which had the least? What factors may be responsible for these results?

Student answers may vary but it’s likely that “skin control” and “surface control” will have the most colonies present. It is likely students will find soap to be less effective than alcohol and bleach.

4. Did you notice any variations in appearance among the bacterial colonies? If so, describe these differences.

Differences may include color, texture and/or colony size.

5. What are some sources of error in this experiment that may have affected the results?

Sources of contamination include:
a. Airborne bacteria entering while streaking the plate.
b. Touching a surface other than the testing surface with the cotton swab accidentally or otherwise.
c. Agar was not sterile at the start of the activity.
d. Opening the plate after initial streaking.

6. When you have blood drawn at a doctor’s office, the skin is often wiped with an alcohol pad before inserting the needle. Why is this practice important?

To kill bacteria that may otherwise enter the body when the skin is punctured. Alcohol is generally more effective at killing bacteria than antibacterial soaps.

1. Sketch your view of Bacillus megaterium under the microscope at 100X magnification.

Student sketches should reflect large rods.

2. What shape bacteria were observed in your sample? How did they appear to be arranged? Use the proper terms from Figure 1 in the Background section.

Bacillus megaterium should be found as rods.

3. Why must the bacterial samples be heat-fixed to the slide?

The bacteria must be heat-fixed to the slide to keep them localized during the staining and washing procedure.

4. What purpose did the methylene blue stain serve in this activity?

Staining bacteria greatly eases viewing. An unstained sample would be nearly impossible to differentiate between bacteria.

Introduction

Antibacterial, antimicrobial, antibiotic, disinfectant and antiseptic—these are just a few familiar terms that are used to describe products which kill bacteria. This activity will explore the differences between these terms and test the efficiency of common antibacterial products.

Concepts

• Antimicrobial agents

• Inoculation
• Bacterial resistance
• Bacterial staining

Background

Bacteria are one of the simplest and most numerous life forms on Earth. Although they cannot be observed with the unaided eye, bacteria are present almost everywhere—inside and outside of the body, on surfaces in the classroom and home, in water, soil and even in extreme climates uninhabitable by other organisms. The presence of bacteria is vital to all life forms; good bacteria inhabit our airways, skin and gastrointestinal tract inhibiting the growth of pathogenic bacteria capable of causing infection.

Bacteria decompose and recycle organic materials such as dead plants and animals. Their importance is even present in medical and biotechnological advancements, such as vaccinations, genetic engineering and gene splicing. Bleach, isopropyl (rubbing) alcohol and antibacterial soaps are commonly used in the home and laboratory. All of these chemicals have the ability to kill both gram-positive and gram-negative bacteria as well as prevent bacterial growth for a short period of time. Antibacterial or antimicrobial are general terms used to describe a product that kills bacteria.

Bleach and other chemicals used on non-living objects are considered disinfectants, whereas antibacterial products, such as mouthwash or antimicrobial soaps, are used on the body and considered antiseptics. Isopropyl alcohol is suitable for use as both a disinfectant and antiseptic. Antibiotics, which will not be used in this activity, are chemical substances ingested by animals to inhibit bacterial growth.

Disinfectant products that claim to possess antibacterial properties often receive an associated EPA (Environmental Protection Agency) number code on the disinfectant product label. The presence of this number verifies that the safety and effectiveness information printed on the label have gone through appropriate testing and have been approved according to EPA standards and will not adversely affect human health or the environment when used as directed. Products lacking such a number code have not been tested and therefore the antibacterial effectiveness may be questionable. All antiseptic products must be tested and regulated by the FDA (Food and Drug Administration).

Recently, scientists have become concerned with bacterial resistance leading to the development of resistant strains. This means bacteria mutate to form new strains which are unharmed by the antibacterial/antibiotic products. Overuse of disinfectants and other antibacterial products may kill not only pathogenic bacteria, but also the bacteria that make up our natural flora. By occupying space and using resources that pathogens require to flourish, bacteria normally found on our bodies help inhibit growth of parasitic strains. If antibacterial products are constantly being used, it is easier for resistant strains to thrive when the natural flora population is low.

A common misconception is that antibacterial products may prevent or reduce the severity of viral infections—antibacterial agents are not effective against viruses.

Bacteria are often classified and named according to their physical shape and colony arrangements (see Figures 1 and 2).

Therefore, a long chair made of circular bacteria would be known as streptococcus (see Figure 3).

A clump of rod shaped bacteria would be called staphylobacillus (see Figure 4).

Experiment Overview

In this activity, the effectiveness of disinfectants and antiseptics including bleach, antibacterial soap and isopropyl alcohol will be tested and compared. Bacteria will be inoculated from a known culture on a Petri plate containing nutrient agar, stained and observed under a microscope.

Materials

Prelab Questions

1. Both disinfectants and antiseptics kill bacteria. What is the difference between these types of products?
2. What purpose do the “control” sections in this experiment serve?
3. Explain how or why bacteria normally present on the skin’s surface are beneficial to our health.

Safety Precautions

After use, agar plates may contain viable microbes. Although the bacteria are not likely to be pathogenic, do not open the plates unnecessarily. Use sterile techniques at all times when handling bacterial cultures. Bleach solution is a corrosive liquid, which may discolor clothing and may cause skin burns. Avoid contact with heat, acids and organic materials; chlorine gas will be generated. Isopropyl alcohol is a flammable liquid, slightly toxic by ingestion and inhalation. Methylene blue will stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Procedure

Activity 1

1. Using a wax pencil or permanent marker, mark and label sections 1–6 on the bottom of the Petri plate, as shown in Figure 5. Also, using a small font, write the group members’ initials on the plate.

2. Decide which partner is going to test the antiseptics on skin and which is going to test the disinfectants on the surface of the classroom. Follow the appropriate procedure below. Note: It is best to use a “dirty” finger, that is, one that has not been washed for several hours. If both partners have washed their hands recently, the selected “skin” partner should run their fingers through their hair and scalp before taking the control sample.
3. Obtain approximately 10 mL of sterile distilled water in a beaker. The water will be shared by both partners. Label the beaker H₂O.
4. Obtain approximately 10 mL of the 10% bleach solution in the second beaker. Label the beaker bleach.

Skin

1. Obtain a sterile cotton swab and dip the cotton tip into the distilled water.
2. Take the moistened tip and roll it around on the surface of an index finger so that it comes into contact with all sides of the cotton swab.
3. Raise the lid of the Petri plate about an inch.
4. Gently streak the agar in the section labeled “1” on the prepared Petri plate by zigzagging the cotton swab across the surface of that section only. Take care not to gouge the agar. Place the cover back on the plate. Note: Try not to breath directly onto the plate (see Figure 6). Place the used cotton swab into the bleach beaker or beaker containing the bleach solution.
5. Obtain a small amount of isopropyl alcohol from the bottle using a disposable pipet. Moisten a cotton ball using the isopropyl alcohol in the pipet.
6. Wipe an area on a different finger of the same hand used in the previous step with the cotton ball. Throw the used cotton ball into the trash.
7. Repeat steps 1–4, streaking section “2” this time. Remember to use a new sterile cotton swab!
8. Wash your hands with the antibacterial soap provided. Ensure you wash for 2 minutes. Do not dry the hand being swabbed.
9. Obtain a dry sterile cotton swab and roll it around on the surface of a third finger on the same hand.
10. Repeat step 4 with the cotton swab but streak section “3.”

Surface

1. Dip the tip of a sterile cotton swab into the distilled water.
2. Roll the moistened tip around on the surface (e.g., tabletop, doorknob, floor) so all sides of the cotton swab comes into contact with the object.
3. Carefully raise the lid of the Petri plate about one inch.
4. Gently streak the cotton swab in section “4” on the Petri plate by zigzagging the cotton swab across the surface of that section only. Take care not to gouge the agar. Replace the lid on the plate. Place the used cotton swab into the bleach beaker.
5. Obtain a small amount of isopropyl alcohol from the bottle using a disposable pipet. Moisten a cotton ball with the isopropyl alcohol in the pipet.
6. Wipe half of the area on the test surface used in step 2 with the alcohol. Discard the cotton ball in the trash.
7. Repeat steps 1–4, using a new sterile cotton swab rolling it across the area wiped in step 6. Streak section “5” of the Petri dish. Remember to use a new sterile cotton swab!
8. Obtain a small amount of bleach solution from the bottle using a disposable pipet. Moisten a new cotton ball with the bleach in the pipet. Note: Bleach will discolor clothing, use with care.
9. Wipe a section on the test surface used in step 2 with the bleach. Do NOT wipe the same section that was treated with alcohol. Discard the cotton ball in the trash.
10. Repeat steps 1–3 using a new sterile cotton swab rolling it across the area wiped in step 9. Streak section 6 of the Petri dish.
11. Seal the Petri dish shut with tape or Parafilm. Store the plate upside down so that the agar is on top of the plate, facing down, according to your teacher’s instructions.

Preparing Known Culture

1. Obtain a stock culture of Bacillus megaterium, inoculating loop and a fresh nutrient agar plate.
2. Thoroughly disinfect the lab table by applying a disinfectant such as Lysol and allow it to air dry.
3. Light the Bunsen burner and adjust to obtain a blue cone flame.
4. Loosen the culture tube cap but do not remove it.
5. Remove the tube cap and hold it between your little finger and ring finger of the hand holding the loop (see Figure 7).

6. Briefly flame the lip of the tube while rotating the tube to ensure no microorganisms remain on the lip (see Figure 8).

7. Dip the inoculating loop into the culture.
8. Briefly reflame the culture tube lip again, rotating the tube; recap and set aside.
9. Lift the lid of the nutrient agar plate, as little as possible, and spread the bacteria on the inoculating loop over the surface of the agar (see Figure 9). Rotate the Petri dish 90° and repeat.

10. Place the inoculating loop into the bleach beaker.
11. Label the bottom of the nutrient agar plate with your groups’ initials and the date. Seal the plate closed with tape or Petrifilm and store upside down according to your teacher’s instructions.

Activity 2

1. Carefully remove the tape from the nutrient agar plate containing the known culture of Bacillus megaterium.
2. Using a sterile inoculating loop, gently run it across the surface of a selected bacterial colony. Recover the Petri plate.
3. Sweep the loop in a dime-sized circle on the right side of a clean microscope slide. This will leave the opposite side free for holding the slide during staining (see Figure 10). Place the inoculating loop into the bleach beaker.

4. Bacteria must be heat fixed so they do not wash off the slide during staining. If you will be performing the heat fix, proceed to step 4a. If your teacher will be performing the heat fix, see your instructor then proceed to step 5.

1. Attach a clothespin to the slide on the opposite side from the bacteria. Pass the region of the slide with the bacteria sample through the Bunsen burner flame 3–5 times.

5. Allow the slide to cool approximately one minute.
6. Continue to hold the slide with the clothespin and suspend it above a waste container or sink.
7. Obtain a small amount of methylene blue stain in a disposable pipet.
8. Holding the slide at a downward angle towards the waste container, flood the bacteria on the slide with methylene blue stain. Place the slide with methylene blue stain on a clean paper towel and allow the stain to set for 1–2 minutes.
9. Hold the slide over the waste container and rinse with distilled water from a wash bottle by aiming the gentle stream of water above the stain area allowing the stain to drip into the waste container.
10. Gently pat the bottom of the slide dry with a paper towel. Exercise caution not to wipe the area containing the bacterial sample.
11. Set up the slide on the microscope stage and focus the image on low power. Once the image is focused on low power, increase to medium power.
12. Turn the nosepiece so that none of the lenses are in line with the sample.
13. Place one drop of immersion oil on the center of the sample.
14. Carefully turn the nosepiece of the microscope so that the 100X lens is in place for the viewing sample. If the sample is out of focus, use only the fine focus adjustment knob to bring the sample into focus.
15. Sketch observations on the worksheet.
16. Clean materials and workspace according to your teacher’s instructions.