Lab Ex 2-13-Effectiveness of Chemical Germicides

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May 4, 2024

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Effectiveness of Chemical Germicides: The Use-Dilution Test for Disinfectants and Antiseptics ¥ Theory Chemical germicides are substances designed to reduce the number of pathogens on a surface, in a liquid, or on or in living tissue. Germicides designed for use on surfaces (floors, tables, sinks, countertops, surgical instruments, etc.) or in liquids are called disinfectants. Germicides designed for use on or in living tissue are called antiseptics. Before a new substance can be registered by either the FDA or EPA and allowed on the market, it must be tested and classified according to its effectiveness against pathogens. The Use-Dilution Test, published by the Association of Official Analytical Chemists (AOAC), is one of many commonly used tests for this purpose. The Use-Dilution Test is a standard procedure used to measure the effectiveness of disinfectants specifically against Staphylococcus aureus, Salmonella enterica serovar Choleraesuis, and Pseudomonas aeruginosa. In the standard procedure, glass beads or stainless steel cylinders coated with living bacteria are exposed to varying concentrations (dilutions) of test germicides and then transferred to a growth medium. After a period of incubation, the medium is examined for growth. If a solution is sufficient to prevent microbial growth at least 95% of the time, it meets the required standards and is considered a usable dilution of that germicide for a specific application. Today’s exercise is an adaptation of this method. m Application This procedure is used to test the effectiveness of germicides against Staphylococcus aureus, Salmonella enterica serovar Choleraesuis, and Pseudomonas aeruginosa. ¥ In This Exercise Today you will examine the effectiveness of four germicides—two common household disinfectants and two over-the-counter antiseptics. The disinfectants selected for the exercise are household bleach and Lysol® Brand II Disinfectant. The antiseptics are hydrogen peroxide and isopropyl alcohol. The organisms used for the test are Staphylococcus epidermidis and Escherichia coli. (The usual test organisms are BSL-2 and we can demonstrate the protocol without using them.) You will first coat the beads with bacteria, expose them to three concentrations of your assigned germicide, and then use them to inoculate sterile nutrient broth. If all of the bacteria on the bead are killed during exposure to the germicide, the broth inoculated with that bead will remain clear. If any of the bacteria survive the germicide exposure, they will reproduce during incubation and make the broth turbid. You will use the results to determine the effective concentration (dilution) of your assigned germicide. The tasks for the exercise are divided among eight groups of students (or a convenient number for your lab size). Each group will be responsible for one organism and three dilutions of one germicide. Refer to Table 2-4 for your assignments. Finally, this is an interesting exercise with moderate amount of work involved. If you do not hurry and are careful to use aseptic technique, you will be rewarded with reliable data at the end. TABLE 2-4 Group Assignments Bleach Lysol® | | Hydrogen peroxide | | Isopropyl alcohol Vv Materials Per Student o Lab coat o Disposable gloves 0 Chemical eye protection Per Student Group 0 100 mL flask of sterile deionized water 0 Three concentrations of one germicide (listed above) 0 Five sterile 60 mm Petri dishes 0 One sterile glass 100 mm Petri dish containing filter or bibulous paper 0 One container of sterile ceramic or glass beads! 0 Sterile transfer pipette o Seven sterile nutrient broth tubes ! Sterilized #8 seed beads from a craft store will work for this purpose. SECTION 2 Microbial Growth H b

0 Needle-nose forceps (or appropriate device for aseptically picking up beads) 0 Small screw-cap jar with alcohol (for flaming forceps) 0 Small beaker with 10 mL of disinfectant (for disposal of the broth culture) 0 Fresh broth cultures of these recommended organisms (only one per group): » Escherichia coli = Staphylococcus epidermidis Per Class (see Table 2-4) Disinfectants 0 0.01%, 0.1%, and 1% household bleach 0 25%, 50%, and 100% Lysol® Brand II Disinfectant Antiseptics 1 0.03%, 0.3%, and 3% hydrogen peroxide (3% is full strength as purchased at the pharmacy) 010%, 30%, and 50% isopropyl alcohol (70% or 90% is full strength as purchased at the pharmacy) AW > PROCEDURE? Timing is important in this procedure. Read through it and make a plan before you begin so your transfers and soaking times are done uniformly and are consistent with those of other groups. Lab One 1 Wear a lab coat, gloves, and chemical eye protection when performing this procedure. 2 Enter the name of your organism here: 3 Enter the name of your germicide here: 4 Obtain all of the necessary items for your group as listed in Materials. 5 Place the materials properly on your workspace as shown in Figure 2.48. Label all seven broths with your group name or number. Label three of the broths “Broth #1, #2, and #3” and label the last four broths “Control #1, #2, #3, and #4” as shown in the diagram in Figure 2.48. 6 Label three of the 60 mm plates with the name and concentration of your germicide. Label the other two plates “#4 sterile water” and “#5 sterile water.” 2 This procedure has been modified from its original form and is to be used for instructional purposes only. m MICROBIOLOGY: Laboratory Theory & Application, Brief 7 Add enough of each germicide concentration to its respective plate (Plates #1 through #3) to cover the beads (approximately 15 mL). Add an equal volume of sterile water into Plates #4 and #5. 8 Carefully mix your bacterial culture until uniform turbidity is achieved. Take care not to splash into the cap. 9 Aseptically transfer one loopful of culture broth to Control #2. 10 Alcohol-flame your forceps and aseptically drop six beads into the broth culture. (You will only use four beads; the other two are extras in case you drop one during the procedure.) Store your forceps in the alcohol jar between transfers. When it is time to make a transfer, pinch the forceps and remove them from the alcohol. Then pass them through the flame to burn off the alcohol, holding them away from the alcohol jar while doing so. If the alcohol jar catches on fire, smother the flame with the lid. When finished, return the forceps directly to the jar without flaming. 11 After 1 minute, decant the broth into a beaker of disinfectant. Remove as much of the broth as possible without losing the beads in the disinfectant. 12 Dispense the beads onto the sterile filter paper in the glass Petri dish. This can be done by tapping the mouth of the tube on the paper. If this doesn’t work, remove the beads with a sterile inoculating loop and flame it afterward 13 Using alcohol-flamed forceps spread the beads apart on the paper and allow them to dry for 10 minutes. Do not roll them around because this may remove bacteria. 14 After 10 minutes, place one bead in each of the three germicide plates using alcohol-flamed forceps. Mark the time here: 15 With alcohol-flamed forceps, immediately place the fourth bead in plate #4 (sterile water). 16 With alcohol-flamed forceps, immediately place a sterile bead in plate #5 (sterile water). 17 After 10 minutes from the time marked in step 14 (step 7 in the procedural diagram), remove the five beads from the solutions in the same order as they were added, and place them in their respective nutrient broths. Carefully mix the broths immediately to disperse any residual disinfectant on the beads. Do not splash the broth into the cap.

18 Incubate all seven broths at 35+2°C for 48 hours. 3 Again using Controls #1 and #2 as comparisons, 19 Save or dispose of the original culture as directed by examine Controls #3 and #4. Using “G” to indicate your instructor. growth and “NG” to indicate no growth, enter your results on the data sheet. Lab Two 4 Dispose of all plates in an appropriate autoclave 1 Remove all broth tubes from the incubator. Gently container when finished. mix the controls and examine them for evidence of 5 Your instructor will provide you with a means to Eg growth. Enter your results on the data sheet, page 139. Control #1 should have no growth and Control #2 should show turbidiy. If both of these conditions have been met, you may proceed. If not, see your instructor. share data obtained by all eight groups. Record these data on your data sheet. 6 Answer the questions on the data sheet. 2 Using Controls #1 and #2 as comparisons, examine broths containing beads exposed to germicide (Broths #1, #2, and #3). Using “G” to indicate growth and “NG” to indicate no growth, enter your results in both the individual data table and the class data table on the data sheet. SECTION 2 Microbial Growth H b

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