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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. 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 for 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. Germicide Bleach Lysol Hydrogen peroxide Isopropyl alcohol 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. TAILI 2-4 Group Assignments Materials Per Student EXERCISE 2-13 □Lab coat Staphylococcin epidermidis Group 1 Group 3 Group 5 Group 7 Disposable gloves □ Chemical eye protection Escherichia coll Group 2 Group 4 Per Student Group 100 ml. flask of sterile deionized water 2 Group 6 Group H □ Three concentrations of one germicide (listed above) Five sterile 60 mm Petri dishes □ One sterile glass 100 mm Petri dish containing filter or bibulous paper One container of sterile ceramic or glass beads Sterile transfer pipette □ Seven sterile nutrient broth tubes Steriland #seed beads from a craft store will work for this purpose. MCB2010Lab 141 135
□ Needle-nose forceps (or appropriate device for aseptically picking up beads) Small screw-cap jar with alcohol (for flaming forceps) □ Small beaker with 10 ml. of disinfectant (for disposal of the broth culture) □ Fresh broth cultures of these recommended organisms (only one per group): • Escherichia coli Staphylococcus epidermidis . Per Class (see Table 2-4) Disinfectants D0.01%, 0.1%, and 1% household bleach □ 25%, 50%, and 100% Lysol Brand Il Disinfectant Antiseptics □0.03%, 0.3%, and 3% hydrogen peroxide (3% is full strength as purchased at the pharmacy) 10%, 30%, and 50% isopropyl alcohol (70% or 90% is full strength as purchased at the pharmacy) 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." 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 85. 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.) Caution! 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. This procedure has been modified from its original form and is to be used for instructional purposes only 136 142 MICROBIOLOGY: Laboratory Theory & Application-Palm Beach State College
18 Incubate all seven broths at 35±2°C for 48 hours. 19 Save or dispose of the original culture as directed by your instructor. Lab Two 1 Remove all broth tubes from the incubator. Gently mix the controls and examine them for evidence of 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. 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. 3 Again using Controls #1 and #2 as comparisons, examine Controls #3 and #4. Using "G" to indicate growth and "NG" to indicate no growth, enter your results on the data sheet. 4 Dispose of all plates in an appropriate autoclave container when finished. 5 Your instructor will provide you with a means to 2 share data obtained by all eight groups. Record these data on your data sheet. 6 Answer the questions on the data sheet.
2 1. Please read the caption for general instructions and advice. 2. Label one uninoculated NB tube "Control #1 and set it aside, It is shown at the top right of this diagram) 3. Mix your original culture fabeled "OC in the diagram at the lower left until it is uniformly turbid. Then, transfer one loopful to control tube 2 shown at the tar nigh Original culture (mix well 4. Using alcohol-famed forceps. add six starlle beads to the original culture and let them soak for one minute 6. Using alcohol-amed forceps transfer the beads to the finer paper inside the 100 mm Patri dish to dry for 10 minutes 144 5. After one minute of soaking, decant the nutrient broth into a beaker of disinfectant Finh (storle) bead Disinfectant for broth disposal) Sterlie 100mm petri dish with filter paper 7a. After 10 minutes transfer three beads to the three 60 mm Petr plates containing diluted germicide Record the time. 7b. Immediately transfer the fourth bead to a 60 mm plate containing sterile water. 2.48 Procedural Diagram for Chemical Germicides At first glance, this procedural diagram looks pretty imposing Read it from left to right and follow the steps in sequence. With a little preparation and familiarity, it really isn't very complex. Germicide dilution Germicide dilution #2 8 Aer ten minutes from the recorded Sme, transfer th beads to their respective steri broths Germicide dilution 43 Plate 4 Sterile water 7c. Immediately transfer a sterle bead to the other 60 mm Petri plate containing storie water. Sterile water Plate 5 Broth #2 Control 63 Broth MICROBIOLOGY: Laboratory Theory & Application-Palm Beach State College -]] Control Broth 13 Incubate all broths at 35°C for 48 hours Control N Control 21 References McDonnell, Gerald E. Antisepsis, Disinfection, and Sterilisation Types Action, and Resistance Washington, DC ASM Press, American Society for Microbiologs, 2007, Widnet, Andreas E and Reno Frei Chap 7 in Manual of Clinical Microbiologs 9th ed. Patrick R. Murras, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, and Michael A. Pfales, ed Washington, DC ASM Pens, American Society for Microbiologs 2007.