Laboratory Exercise # 13: Disinfectants Purpose: The purpose of this exercise is to test the effectiveness of various disinfectants, antiseptics and mouthwashes on Gram + and Gram - bacteria. We will utilize scientific reasoning to evaluate the results. Introduction: There are many products on the market today that claim to kill bacteria, but how do you really know if they are doing the job they say they can do? To be a wise consumer first you need to be able to define several terms that may be found on the labels of these products. What does it mean to sterilize, disinfect or act as an antiseptic? For a surface to be sterile there must be destruction of all life forms: vegetative cells, spores and viruses. The microorganisms are either destroyed or physically removed from the objects or substances. Sterilization can be easily accomplished with the use of an autoclave, but do any chemicals available to use in the marketplace also have this ability? The destruction of pathogens on inanimate objects is the job of a disinfectant. These chemicals rarely achieve total killing of all life forms, but they should be able to eliminate vegetative forms of most potentially hazardous and pathogenic microorganisms. This does not include spores of tubercle bacteria and many viruses. The use of these chemicals is strictly limited to inanimate objects such as tabletops, tile walls, etc. If a chemical is given the term antiseptic it should be able to inhibit or destroy microorganisms on living tissue in an attempt to prevent infection. Therefore antiseptics can't harm the tissue and are less antimicrobial than other chemicals already discussed. 1
Materials: 2 Large Trypticase soy agar plates per pair of students Selected disinfectants, antiseptics and mouthwashes Sterile discs Forceps Sterile swabs Metric ruler Trypticase Soy Broth cultures of: Staphylococcus epidermidis Escherichia coli Laboratory Prep; to be completed by the student prior to the lab 1. Read all steps of the procedure of the experiment. 2. Research five products from one of the following categories to ascertain the manufacturer s stated effectivity rates. a. Antiseptic b. Disinfectants c. Mouthwashes Record your research findings for five products here, being sure to provide your URLs. 3. Develop a hypothesis for one of the products you will test during the experiment. Procedure: 1. Label the edge of each plate with a student name and the name of the microorganism. Evenly space the numbers 1-5 around the center portion of the plate. Record what each product number is in the lab manual. 2. Using a sterile swab seed the two TSA plates one with Staphylococcus epidermidis and the other with Escherichia coli. This is accomplished by inserting the sterile swab in the broth culture and then rolling it in three directions with overlapping strokes in order to produce a lawn of growth. Allow the plates to sit for 5 minutes after inoculation. 2
3. Dip a sterile blank in a small amount of the product, which has be placed in a small container. It is not recommended that the disk be immersed into the container of the product. 4. Using sterile forceps place the saturated disk on the agar and gently tap it down. Caution: when sterilizing the forceps, wave them through the flame a couple of times. Don't leave in the flame, as heat will be transferred up to the handle! 5. Incubate the plates for 24 hours at 37 C. Over incubating will often result in overgrowth of the microorganism and no zone of inhibition. 6. Recording your results: a. Note the area around the disk. Is there an area of no growth of the microorganism? b. Measure this area known as the "zone of inhibition" in millimeters. c. Record your results on your data chart as well as the one on the board so they can be shared with the class. d. Decide the effectiveness of each product and rate as most, moderately or least effective. 3
Data: Sample Tested Zone S.epidermidis Disinfectants Effectivity Rate Zone E. coli Effectivity Rate Antiseptics Mouthwashes Questions: 1. What is meant by seeding the plate? 2. What is a "zone of inhibition? Does this necessarily mean that the bacteria are dead within the zone? 3. For each of the categories of chemicals tested are they more effective against gram positive or gram negative microorganisms? 3
Final Lab Report. Type the report and turn in on the date designated by your instructor. (2 point) Purpose of the experiment: (3 points) Research results of manufacturer s effectivity rates (provide URLs): (2 points) Hypothesis: (4 points) Data chart to include: The products you tested Bacterial cultures used Measurement of zone of inhibition in mm Development of a rating scale for effectivity of the products tested (4 points) Conclusions to include: A summary of whether or not your experimental results support or contradict your original hypothesis. Key facts from your background research to help explain our results. If the results of your experiment did not support your hypothesis, don t change or manipulate your results to fit your original hypothesis, simply explain why things did not go as expected. Suggested changes in the experimental procedure and/or possibilities for further study. 4