Controlling Microbial Growth

STUDENT HANDOUT:

• Controlling Microbial Growth Student Handout

LABORATORY REPORT FORM:

• Controlling Microbial Growth Report Form

INTRODUCTION:

Microbes, organisms too small to be seen with the naked eye, are all around us. They are in the air we breathe, the water we drink, and the ground we walk upon. Microbes are also in and on our bodies. Microbes exist in a parasitic, mutualistic, or commensal relationship with the human body, which acts as a host. Usually, these ubiquitous microorganisms are harmless. When an individual has an infectious disease, however, it is important that the transmission of the pathogenic microbes be halted. Food industries, pharmaceutical manufacturers, water treatment plants, health care facilities, and various others constantly monitor for the presence of microbes and maintain quality by controlling the microbes in their respective environments.

There are multiple ways to control microorganisms. Physical means to do so include irradiation (ultraviolet and gamma), extremes of osmotic pressure and pH, and temperature. Disinfectants, antiseptics, and antibiotics are chemical means to control microbes. 

A common technique to test the effect of chemical control on microbial growth is to use a disc-diffusion assay.  In this experiment, sterile discs are soaked in the chemical.  The discs are then placed onto a lawn of bacteria.  If the chemical inhibits growth, a zone of clearing will become visible (Figure 1).  

Figure 1.  Disc-diffusion assay.  To determine if an antibiotic is really effective, the diameter of zone of clearing is measured and then compared to the tables of sensitivity, intermediate or resistance according to the Clinical Laboratory Standards Institute's Performance Standards for Antimicrobial Disk Susceptibility Tests.  Image from Wikimedia Commons.

OBJECTIVES:

1. Learn how we can determine the antimicrobial effectiveness of temperature, disinfectants, antiseptics and/or antibiotics.

PRECAUTIONS:

1. Handle the bacterial cultures with care. Wash your hands with antimicrobial soap before and after handling cultures, and wash work surfaces with disinfectant.

2. Clean up spills using disposable plastic gloves, paper towels, and disinfectant. Dispose of all cleanup materials in the biohazard bag.

3. Do not put fingers or any objects near eyes or mouth while working.

EXPERIMENT #1:  Effect of boiling on microbial growth

MATERIALS (Work in pairs):

1. Permanent marker

2. Inoculating loop

3. Bunsen burner

4. Test tube rack

5. 2 sterile nutrient broth tubes

6. Boiling water bath

7. Tongs

8. Culture of Escherichia coli

9. 37°C incubator

10. Biohazard bag

PROCEDURE:

1. Mark both nutrient broth tubes with your initials and date. Label them #1 and #2. Keep them in the test tube rack.

2. Aseptically inoculate both broth tubes with E. coli using the inoculating loop and Bunsen burner.

3. Keep the inoculated tube #1 in a test tube rack at room temperature.

4. Place the inoculated test tube #2 in a boiling water bath for 15 minutes (Figure 2).  After 15 minutes, remove the tube (using tongs) to your test tube rack.  Allow the tube to cool to room temperature.  

5. Store the newly inoculated tubes in the test tube rack at room temperature for up to a week (or incubate at 37°C for 48 hours). Your instructor should include sterile nutrient broth tubes as controls. 

Figure 2.  Water bath incubation. Image made using ChatGPT (AI).

OBSERVATIONS/CONCLUSIONS:

1. Is tube 1 cloudy or clear? Is tube 2 cloudy or clear?  

2. What do these results tell you?

EXPERIMENT #2:  Comparison of the antibacterial effects of different substances

MATERIALS (Work in pairs):

1. Permanent marker

2. Culture of Escherichia coli

3. Mueller-Hinton agar plate

4. Sterile swabs

5. Metal & plastic paper clips

6. Plastic or plastic-coated paper clips

Make sure the paper clips are placed in 70% ethanol for 10 minutes before beginning the experiment.  Allow clips to air dry before placing them on the agar plate.

7. Antibiotic discs

8. Sterile filter paper disks soaked in disinfectants

9. Inquiry item (or teacher-preferred item)

10. Forceps

11. Beakers of ethyl alcohol

12. Bunsen burner

13. Masking tape

14. 37°C incubator

15. Ruler

16. Biohazard bag

2.  Aseptically place a sterile swab into the broth culture of E. coli. Twirl the swab to saturate the cotton and then press the swab against the side of the tube to squeeze out any excess fluid.

3.  Create a spread plate by lifting the petri plate slightly like a clam shell. Using the cotton swab, spread the bacteria in a straight line down the plate as shown in Figure 4.  Then in a zig-zag fashion, spread out your inoculation.  Rotate the plate 60° and continue swabbing the plate.  Rotate the plate again 60° and continue swabbing the plate.  Make sure to swab the entire plate leaving no space uninoculated.  This will create a lawn of bacteria. 

Figure 4.  Creating a lawn of bacteria.

4.  Dispose of the used swab in the beaker of disinfectant or as instructed. 

5. Choose one of the following Item pairs (A and B of same item) of test substances for use on the spread plate.

6.  Using flame-sterilized forceps (your teacher will demonstrate), place the "A" unit of your pair in the center of the "A" side of the lawn plate. Tap the object lightly so that it adheres to the agar surface.

7.  Reflame the forceps and allow them to cool for a few seconds.

8.  Using the flame-sterilized forceps, place the "B" unit of your pair in the center of the "B" side of the lawn plate. Tap the object lightly so that it adheres to the agar surface.

9.  Using two small pieces of masking tape, tape the plates together and incubate in an upside-down position at room temperature for  up to a week (or incubate at 37°C for 48 hours).

You can watch a video on how to alcohol sterilize tools.

OBSERVATIONS/CONCLUSIONS:

1. After the plates have incubated and the lawn of bacteria has grown, examine the plates for zones of inhibition.  Using a ruler, measure the diameter of the zone of clearing (in millimeters), as shown in Figure 5. 

Figure 5.  Measuring zones of clearing.  Image from: Kirby-Bauer Disk Diffusion Susceptibility Test Protocol.  Written by Jan Hudzicki (ASM MicrobeLibrary).

2.  Record your results and the results from plates inoculated by the other students in the Microbiology Laboratory Report Form.

3.  Did bacteria grow to the edge of object A?

4.  Did bacteria grow to the edge of object B?

5.  What does a zone of “no growth” around one of the test objects mean?

KEY TERMS:

• antibiotic 

• antiseptic 

• aseptic 

• commensalism 

• disinfectant 

• inoculate

• lawn

• microbe 

• mutualism 

• osmotic pressure 

• parasitism 

• pathogen

• pH

• ubiquitous

SUGGESTED READINGS:

One of the most famous antibiotics is penicillin, which is produced by the fungus Penicillium.  Penicillin was discovered by Alexander Fleming.  Learn more about Fleming and his discovery:

Tan SY, Tatsumura Y. (2015).  Alexander Fleming (1881-1955): Discoverer of penicillin. Singapore Med J. 56(7):366-7.