Lab 7: Chemical Methods of Microbial Growth Control: Disinfectants
Learning Objectives:
• Identify how household cleaners affect the growth of bacteria
• Identify the effectiveness of various antibiotics against Gram-positive and Gram-negative bacteria
Exercise 1: Disinfectants
Disinfectants are widely used to control the growth of microorganisms on inanimate objects. These kill microbes by alteration of cell walls and
membranes or damaging proteins and nucleic acids. Depending upon the susceptibility of microbes to these chemicals they are classified into-
high, intermediate, or low level disinfectants.
In this exercise, we will test the susceptibility of E. coli (Gram negative) and S. epidermidis (Gram positive) bacteria to commonly used
disinfectants at home and compare to effectiveness of 10% bleach commonly used in labs.
Materials:
Broth cultures of S. epidermidis (Gram-positive bacteria), E. coli (Gram-negative
bacteria), TSA plates, sterile glass tubes, sterile swabs, household disinfectants or
cleaners (students may bring a cleaner from home), 10% bleach, ruler, marker
Additional recommended cleaners: 3% solution of H2O2, 95% Ethanol
Method:
1. Divide class in groups of 4 students. Each group will test one cleaner.
2. Each group will have 2 TSA plates- one plate per bacterial organism.
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BIOS242 Lab 7
Name: Group Protozoa-
3. Divide the plate as shown in the image by drawing the quadrants at the back of the plate. Write the number of minutes in each quadrant
as well as the name of bacteria and name of the cleaner along with initials of student to identify the plate.
4. In a clean tube, add 2 ml of overnight culture of bacteria and streak in 0 min space with cotton swab on TSA plate. Now add 1ml of
disinfectant, mix it well and start the timer.
Note: One group should use 10% bleach. Other groups may use household cleaner/disinfectant of their choice. 3% solution of H 2O2 and
95% Ethanol are recommended but not mandatory to use. Each group will decide to use a cleaner/disinfectant in consultation with their
instructor. Depending on the class size, use 4-6 different household cleaners for this experiment.
5. At the designated times (0, 1, 5, and 10 min), dip the sterile swab in the tube and swab over the surface of the plate in the appropriate
section.
6. Repeat the process for the second bacteria.
7. Incubate the plates at 37◦C for 24 hours.
8. Observe your plates and estimate growth of bacteria with 0 being no growth and 5 being maximum growth. Obtain results from other
groups in your class. Record the observations in lab report, complete tables for both organisms and answer all questions.
Exercise 2: Antimicrobial Agents
Antimicrobial agents are the drugs that treat infections. These can be antibiotics, antifungals, antiviral or antiprotozoal drugs. Natural antibiotics
are the chemicals produced by microorganisms that kill bacteria. Natural antibiotics can be modified to semisynthetic antibiotics in a lab to
improve their efficacy, stability and effectiveness. Successful chemotherapy requires selective toxicity. Selective toxicity takes advantage of
structural and metabolic differences between the host and microbial cells. These drugs kill the microorganisms by inhibition of cell wall synthesis,
protein synthesis, cell membrane, metabolic pathways, DNA/RNA synthesis, and host recognition. Efficacy of a drug is tested in a lab using the
Kirby- Bauer or disk diffusion test. If the drug inhibits growth of microorganism, a clear zone of no microbial growth is formed around the
antibiotic disc. This zone is called the zone of inhibition. In this exercise, we will test the susceptibility of Gram-negative (E. coli, S. marcescens)
and Gram-positive (S. epidermidis, B. subtilis) bacteria to three different antibiotics with different modes of action.
Materials:
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