Exercise 11 - Controlling the Growth of Microorganisms

Exercise 11 - Controlling the Growth of Microorganisms Exercise 11 - Controlling the Growth of Microorganisms Objectives: Following this exercise the student should be able to: 1. describe factors that contribute to effective control of microbial growth. 2. test the effectiveness of selected disinfectants by aseptically preparing a standard concentration stock culture, performing a lawn culture, and creating disinfectant test discs. 3. interpret the data from the experiment. 4. evaluate the role of time, dose, concentration and load in effective microbial control. The control of microorganisms by the use of various physical methods or chemicals is a common feature of everyday life. From our homes to the hospital, many of our daily tasks target controlling the growth of unwanted microbes. Physical methods include: temperature, radiation, ultraviolet light, filtration, and mechanical removal. Bleach, ammonia, and alcohol are common chemicals we use to target and destroy potentially harmful microorganisms. Many people use antiseptic soap to wash their hands and mouthwash to counteract mouth odor produced by microbes. How effective are these products and what organisms are they active against? This is your opportunity to test the effectiveness of your own household products and activities. Select 2-4 products from mouthwash to soap and cleansers and bring them to school the day of the lab. Physical Control of Microorganisms - Using your text complete the chart below: Physical Control Mode of Action Common Use Method (How does it work?) Temperature – Freezing Temperature – Cold Temperature – Heat Temperature- Pasteurization Autoclaving –

Radiation – such as gamma radiation Ultraviolet Light

Filtration

Microwave

Desiccation – drying and salting Physical Removal 2

Chemical Control of Microorganisms Chemicals that control the growth of microorganisms are generally divided into 2 main groups; antiseptics are chemicals used on living tissue in an attempt to reduce the population of microbes present, disinfectants are chemicals used on inanimate objects and are potentially harmful to living tissue. The effectiveness of these chemicals depend upon six main factors;  the target organism  contact time  concentration  organic material present  toxicity  pH  whether the action is lethal (-cidal) or inhibiting (-static)

The activity of these chemicals may be limited in their effect and the particular type of microbe; hence we have bactericidal or bacteriostatic agents, viricidal or fungicidal agents. If the chemical kills all living organisms it is referred to as a sterilant, these substances may be useful but highly toxic to humans as well as microbes. Some chemicals just reduce the rate of growth or destroy only specific harmful organisms. In labs the chemicals are tested for effectiveness under a variety of conditions, dilution, and contact times. This experiment will involve a more simple test using sterilized filter discs impregnated with a given chemical, placed upon a lawn culture of 3 different types of bacteria.

Materials:

For each group Sterile filter discs 3 Kirby Bauer plates Sterile forceps 8 beakers 8 test substances from home disinfectant discard beaker (bathroom cleaner, soap, Clorox etc) 3 sterile cotton swabs STOCK CULTURES: 3 sterile water tubes Staphylococcus aureus 1 MacFarland standard Pseudomonas aeruginosa E. coli First Lab Session 1. Put on your PPG and wear eye protection for this lab exercise. 2. Begin by labeling a tube of sterile water, a Kirby Bauer plate (the large plates for each of the stock cultures - E.coli, Staphylococcus epidermidis, and Pseudomonas aeruginosa. 3. Assign a number to each disinfectant e.g. Clorox =1, ammonia = 2, Lysol = 3, etc. 3

4. Prepare a standard concentration of each of the stock cultures using the correctly labeled sterile water tube, a sterile swab, and aseptic technique. Lightly touch the stock colony with the swab and then agitate the swab in the sterile water. 5. Hold the tubes up to the light in front of a lined card. Compare the cloudiness

in the inoculated water to the t F l a a

a o e l c u t t d d r r McFarland tube to create a standard k d M a n S n r concentration. You may increase the S c C u concentration by dipping the original swab in the water again, but do not touch the stock culture a second time. If the concentration is too great (the water tube is cloudier than the McFarland standard) you must start over. When the concentrations are equivalent, dispose of the swab in a beaker of disinfectant. 6. Using a fresh sterile swab, dipped once in the standardized concentration of stock culture, streak the entire surface of a plate in three directions to produce confluent growth; this is called a LAWN culture. It is important to get your streaks close together so that the entire surface is evenly inoculated. The instructor will demonstrate this. Ultimately you will have a lawn culture for each stock organism (E.coli, Staphylococcus epidermidis, and Pseudomonas aeruginosa) 7. Pour a small amount of each test chemical into 4 different beakers. Next a sterile disc will be prepared that is soaked in each of the 8 chemicals the lab group has decided to test. Pick up a single sterile disc, using a sterilized pair of forceps, and touch the edge of it to the chemical. DO NOT place the disc more than halfway into the solution. Allow the solution to absorb into entire disc. Each test chemical will be tested against each stock organism (E.coli, Staphylococcus epidermidis, and Pseudomonas aeruginosa). 8. After you have prepared a disc with #2 the first chemical place it on the E.coli #1 lawn culture, then repeat the

procedure for Staphylococcus #4 epidermidis and Pseudomonas aeruginosa lawn cultures. Number #3 each disinfectant disc on the bottom of E.coli #5 the plates immediately after applying #6 Lab 1 date the discs, so you can identify them later. #7 9. Continue this preparation for all of 8 #8 chemicals being tested arranging them as seen in the diagram. 10. Prepare all three lawn plates, with the same 8 disinfectant discs, Incubate @ 35C for 48 hours. 4

11. Indicate the name of the disinfectant, the expiration date, the manufacturer’s claim for effectiveness, and the active ingredients.

Disinfectant Expiration Manufacturer’s Hypothesize Active ingredients date Claims the effectiveness 1.

12. Considering the manufacturer’s claim make a hypothesis as to whether you think this disinfectant will be effective against Gram positive and/or Gram negative organisms prior to reading the results. Use rationale from the book and your knowledge of microbes to make this hypothesis.

13. Replace equipment and discard stock cultures and contaminated materials in the appropriate biohazard location. 5 Results Exercise 11 - Controlling the Growth of Microorganisms

Name______Lab session two: 1. Collect your plates from the incubator and record the results. 2. Disinfectant results. Measure the DIAMETER of the zone around the disc using a millimeter ruler. The instructor will demonstrate this.

Disinfectant E.coli Staphylococcus Pseudomonas epidermidis aeruginosa 1.

3. What conclusions can you make from the data on the disinfectant tested?

4. Often physical controls and chemical controls are combined to increase effectiveness. Can you name two instances where this is the case? 6

5. How accurate were your hypotheses? If you were incorrect on any explain the reason that you may have been wrong.

6. In the chart below consider the situation and list the physical or chemical or combined methods most commonly used for each example. Situation requiring Common Methods Used microbial control Milk

Surgical tools

Operating Room

Drinking Water

Swimming Pool

Kitchen Counter

Hospital Room

Injection site on skin