Detecting the Presence of Microbes: Instructor s Manual

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1 Detecting the Presence of Microbes: Instructor s Manual I. Purpose and Concepts Covered...1 II. Laboratory Set Up...4 III. Detecting Microbial Growth on Surfaces...5 A. ATP-Based Method...5 B. Culture Method...6 IV. Evaluating the Effectiveness of Quaternary Ammonium Compound Salts...7 V. Graphing Bacterial Growth Rates...8 VI. Supplier and Ordering Information...9 I. Purpose and Concepts Covered Microbes (fungi, bacteria, single-celled eukaryotes and other microscopic organisms) are everywhere. This lab will focus primarily on detecting bacteria; however, students will probably isolate fungi and possibly other microscopic organisms during the course of their explorations. Traditionally microbial contamination is detected on surfaces by using sterile technique to sample the surface and culture any organisms that might be present. This method has several limitations. First, in order to see any organisms, the culture medium may need to be incubated anywhere from 24 to 48 hours. Secondly, only those organisms that are well suited to the growth conditions that the researcher has chosen (incubation temperature, carbon source, ph, salinity, aeration) are likely to be detected in the culture. Another way to detect the presence of microbes is to assay for ATP, which is an indicator of metabolically active cells. Cells use ATP as energy currency to drive most of their cellular synthesis reactions. The BacTiter-Glo Microbial Cell Viability Assay measures ATP using a luminescent reaction.! This instructor s manual is available online only. This teaching resource is made available free of charge by Promega Corporation. Reproduction permitted for noncommerical educational purposes only. Copyright 2008 Promega Corporation. All rights reserved.! Important: Be sure to follow your institution s guidelines and rules for the proper disposal of biohazard waste. Important: Make sure! that you and your students follow your institution's laboratory safety rules and regulations at all times. The BacTiter-Glo Assay is designed for either single-tube or multiwell-plate formats for high-throughput screening (HTS). The homogeneous assay procedure involves adding a single reagent (BacTiter-Glo Reagent) directly to bacterial cells in medium and measuring luminescence. The formulation of the BacTiter-Glo Reagent supports bacterial cell lysis and generation of a luminescent signal in a homogeneous add, mix, measure format. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of cells in culture (Figure 1). The BacTiter-Glo Reagent relies on the properties of a proprietary thermostable luciferase (Ultra-Glo Recombinant Luciferase) and a proprietary formulation for extracting ATP from bacteria. The BacTiter-Glo Assay generates a glow-type luminescent signal, produced by the luciferase reaction shown in Figure 2, which has a signal half-life generally over 30 minutes depending on the bacterium and medium. The assay has been shown to detect a variety of bacteria, yeast and fungi (Table 1). The homogeneous format reduces pipetting errors that may be introduced during the multiple steps required by other methods of ATP measurement. 9/08 Page 1

2 Signal:Noise 7 10 E. coli S. aureus 10 6 P. aeruginosa 10 5 B. cereus Limit of detection Cells/Well Figure 1. Bacterial cell numbers correlate with luminescent signal. Four bacterial strains [Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923), Pseudomonas aeruginosa (ATCC27853) and Bacillus cereus (ATCC10987)] were grown in Mueller Hinton II (MH II) Broth (BD Cat.# ; see Technical Bulletin #TB337 for growth medium recommendations) at 37 C overnight. The overnight culture was diluted 50-fold in fresh MH II Broth and then incubated for several hours to reach log phase. Samples of the culture were serially diluted using MH II Broth in a 96-well plate. The assay was performed according to the protocol described in Technical Bulletin #TB337. The reconstituted BacTiter-Glo Reagent was equilibrated for 1.5 hours at room temperature to achieve better sensitivity. Luminescence was recorded on a GloMax 96 Microplate Luminometer (Promega Cat.# E6501). Signals represent the mean of three replicates for each measurement. Bacterial cell numbers were determined by plate counting of colony forming units on Luria-Bertani agar plates. The signal-to-noise ratio was calculated: S:N = [mean of signal mean of background]/standard deviation of background. There is a linear correlation between luminescent signal and the number of cells over five orders of magnitude. The limits of detection drawn from this experiment for E. coli, S. aureus, P. aeruginosa and B. cereus are approximately 40, 150, 70 and 10 cells, respectively. 4608MA HO S N COOH N S Beetle Luciferin + ATP + O 2 Recombinant Firefly Luciferase Mg 2+ O O S N N S Oxyluciferin + AMP + PP i + CO 2 + Light 5768MA Figure 2. The luciferase reaction. Mono-oxygenation of luciferin is catalyzed by luciferase in the presence of Mg 2+, ATP and molecular oxygen. Page 2 9/08

3 Table 1. BacTiter-Glo Reagent Works with a Variety of Microbial Organisms. Gram-Negative Bacteria Gram-Positive Bacteria Others Escherichia coli Pseudomonas aeruginosa Enterobacter cloacae Flavobacterium okeanokoites Haemophilus influenzae Proteus vulgaris Salmonella typhimurium Yerinia enterocolitica Fancisella philomiragia Staphylococcus aureus Enterococcus faecalis Streptococcus pneumoniae Bacillus subtilis Bacillus cereus Arthrobacter luteus Saccharomyces cerevisiae Candida albicans* Mycobacterium avium Mycobacterium paratuberculosis *For Candida albicans a 15-minute incubation time is required for optimal signal, and the limit of detection is around cells. Evaluating the Effectiveness of Quaternary Ammonium Compound Salts Staphylococcus aureus is a common Gram positive bacterium that can cause skin and invasive infections in a variety of host organisms, including humans. For years, strains of S. aureus that are resistant to multiple antibiotics including methicillin (methicillin-resistant Staphylococcus aureus, MRSA) have been associated with nosocomial (hospital acquired) infections. However, outbreaks of MRSA infections are becoming increasingly common in other environments including prisons, daycare centers, athletic centers, colleges and public schools. The emergence of MRSA outside of healthcare settings has led to the classification of two basic categories of MRSA infections: healthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA). Most of the recommendations on control involve stringent handwashing, recommendations for minimum temperatures for washing and drying shared laundry items such as towels, and use of surface disinfectants that are certified by the EPA as effective against MRSA. Importantly, handwashing is effective only if surfaces encountered after handwashing are appropriately disinfected. Quaternary ammonium cations are charged polyatomic ions that retain their charge regardless of ph. The salts of these compounds (quats) are commonly used as disinfectants and include benzalkonium chloride, a preservative found in many ophthalmic solutions. These compounds are usually membrane-active agents that disrupt the integrity of microbial cells. Several companies, including Clorox and 3M, manufacture quats that are EPA-certified MRSA disinfectants, but to be effective the directions for use must be followed. The students will have the opportunity to investigate the difference between following the directions for use for a quaternary disinfectant to the letter versus performing a cursory spray and wipe cleaning. Does following the specified protocol make a difference in disinfecting a surface? Measuring Bacterial Growth Using Conventional and ATP-Based Methods We examined the growth of E. coli using either the BacTiter-Glo Assay or optical density (O.D.) measurement. The results are shown in Figure 3. The extended sensitivity and range of the BacTiter-Glo Assay allows users to monitor E. coli growth immediately after inoculation. When monitoring growth by O.D., the first significant measurement (0.025) did not occur until 5 hours after inoculation. The growth curve determined by the BacTiter-Glo Assay has a dynamic range over six orders of magnitude compared to the growth curve determined by O.D. measurement, which has a range only of about two orders of magnitude. The increased dynamic range allows researchers to more easilymonitor slow-growing bacteria. 9/08 Page 3

4 Luminescence (RLU) O.D Time (hours) Time (hours) 4616MA Figure 3. Evaluating bacterial growth using the BacTiter-Glo Assay. E. coli ATCC strain was grown in Mueller Hinton II (MH II) Broth (BD Cat.# ; see Technical Bulletin #TB337 for growth medium recommendations) at 37 C overnight. The overnight culture was diluted 1:106 in 50 ml of fresh MH II Broth and incubated at 37 C with shaking at 250 rpm. Samples were taken at various time points, and the BacTiter-Glo Assay was performed according to the protocol described Technical Bulletin #TB337. Luminescence was recorded on a GloMax 96 Microplate Luminometer. Optical density was measured at 600 nm (O.D. 600 ) using a Beckman DU650 spectrophotometer. Diluted samples were used when readings of RLU and O.D. exceeded 10 8 and 1, respectively. II. Laboratory Setup Important Note: Be! sure that you know, understand and follow your institution s laboratory guidelines for safety and biohazard waste disposal at all times. Note: This laboratory unit assumes that students have been taught basic aseptic techique. Note: We recommend using fresh, sterile nuclease-free water because it is unlikely to be contaminated with luciferin. Luciferin contamination will cause your results to be invalid. This laboratory is based on performing approximately 70 luminescent assays in a 96-well plate in a total volume of 200 µl (using 100 µl of BacTiter-Glo Reagent). The laboratory can be adapted for microcentrifuge tubes. If you wish, you can choose to have your students perform only certain laboratory investigations such as evaluating the effectiveness of quaternary ammonium compounds. Please adapt this laboratory to meet the needs of your teaching situation. Remember that you can store the prepared BacTiter-Glo Reagent at 4 C for 4 days or at 20 C for one week. We strongly recommend running the lab with four samples to validate the equipment, samples and reagents. Perform sampling the surface lab protocol for up to four swabs. Validation (Instructor: 1 positive control, 1 negative control, 2 samples = 4 wells) Detecting Microbial Growth on Surfaces: Each group of students will perform one negative control, one positive control, and two samples (5 groups = 20 wells) Evaluating Quat. Compounds: Each group of students will perform one negative control, one positive control, one cursory spray and wipe, one clean according to directions (5 groups = 20 wells) Growth Curves: Each group of students will make luminescence measurements at 0, 60, 120, 180, 240 and 300 minutes (5 groups = 30 wells) Preparing the BacTiter-Glo Reagent 1. Thaw the BacTiter-Glo Buffer and equilibrate to room temperature before use. Buffer can be thawed and stored at room temperature for up to 48 hours. 2. Prepare the BacTiter-Glo Reagent. Transfer 10 ml of BacTiter-Glo Buffer into the amber bottle containing the BacTiter-Glo Substrate to reconstitute the lyophilized enzyme/substrate mixture. Mix by gently swirling or inverting several times for a homogeneous solution. Store at room temperature for at least 15 minutes before use. Page 4 9/08

5 III. Detecting Microbial Growth on Surfaces III.A. ATP-Based Method Instructor: Be sure to remove the BacTiter-Glo Reagent from the freezer to thaw before the lab activity. All steps are carried out at room temperature. All students will require safety equipment (lab coats, safety glasses, gloves, etc.) as required by your institutional safety department. Each group of students will need: one 5.0 ml overnight culture of E. coli one 5.0 ml tube of sterile culture medium 4 Fisherbrand Sterile Polyester Applicators four 1.5 ml sterile microcentrifuge tubes pipet and tips capable of accurately dispensing 100 µl pipet and tips capable of accurately dispensing 250 µl Nuclease-Free Water BacTiter-Glo Reagent reservoir for dispensing reagent Note: Wear gloves to avoid contaminating surfaces and samples with ATP from your hands. 1. Wipe any surface with your swab. Try to find a location where you suspect bacterial contamination. You do not need to wet the swab. You will collect samples from two locations. 2. Place your swab in a 1.5 ml tube. Break off the stick. Add 250 µl of Nuclease-Free Water. Label your tube with your initials and location from which the sample was obtained. 3. Use your third swab for your positive control. Dip the swab into the overnight culture. Remove excess liquid from the swab by pressing it against the side of the culture tube. Place your swab in a 1.5 ml tube. Break off the stick. Add 250 µl Nuclease-Free Water. Label your tube with your initials and PC for positive control. 4. Use your fourth swab to prepare your negative control. Dip the swab into the sterile culture medium. Remove excess liquid from the swab by pressing it against the side of the culture tube. Place your swab in a 1.5 ml tube. Break off the stick. Add 250 µl Nuclease-Free Water. Label your tube with your initials and NC for negative control. 5. Remove 100 µl of solution from each tube containing a swab and place it in a well of a 96-well plate. 6. Pour the BacTiter-Glo Reagent into a reservoir. Add 100 µl of BacTiter-Glo Reagent to all wells containing sample. 7. Measure luminescence on a plate-reading luminometer. 8. View the results on a luminometer or in Excel. 9/08 Page 5

6 III.B. Culture Method Each group of students will need: four sterile LB plates four Fisherbrand Sterile Polyester Applicators sterile inoculating loops 1. Use the additional swabs to obtain samples from the two surfaces you sampled in the ATP-based method in Section III.A. Use the third and fourth swabs to obtain samples from your hands before and after hand washing (Steps 6 and 7). 2. Immediately after sampling the first surface, obtain a sterile LB plate. Hold the swab comfortably in one hand and lift the lid of the LB plate with the other hand. Use the lid as a shield to protect the agar from aerial contamination. Lightly run the swab back and forth in a zig-zag pattern across one quadrant of the plate. 3. Turn the plate one quarter turn, and use a sterile inoculating loop to streak a second quadrant. 4. Turn the plate a second quarter turn, and use another sterile inoculating loop to streak a third quadrant. 5. Repeat the process for the second surface with a second, sterile LB plate. 6. For the handwashing sample, have one student sample the palm of his or her hand and inoculate an LB plate as above. 7. After inoculating the plate, have the student wash his or her hands according to the protocol recommended by your local hospital or community health department. A second student in the group should swab the clean hands and inoculate a second LB plate according to the steps described above. 8. Be sure the plates are labeled on the bottom with the date, group name and sample source. 9. Incubate the plates upside down to prevent condensation from collecting on the agar surface. 10. Incubate the room surface samples on the lab benches at room temperature. Incubate the samples from the students hands in a 37 C incubator. For Discussion 1. Compare and contrast the ATP-based method and the culture method in terms of advantages and disadvantages. Think about how the experiments were performed and the kind of data you obtained from each method. Describe scenarios or environments in which one method might be more useful than the other. Can you describe a situation when you would benefit from useing both methods? Page 6 9/08

7 IV. Evaluating the Effectiveness of Quaternary Ammonium Compound Salts In this laboratory the students will investigate whether how much of a difference appropriate use of disinfectant makes in eliminating bacterial contaminants from a surface. Each group of students will need: two surfaces that can be contaminated with Staphylococcus aureus (e.g., stainless steel trays that can be sterilized by autoclaving after the laboratory). two overnight cultures of Staphylococcus aureus Fisherbrand polyester applicators sterile 1.5 ml microcentrifuge tubes (alternatively contamination can be detected using a culture method similar to that described in Section III.B or by using surface contact plates) Nuclease-Free Water sterile culture medium BacTiter-Glo Reagent pipettor and pipet tips capable of accurately dispensing 100 µl pipettor and pipet tips capable of accurately dispensing 250 µl reservoir for dispensing reagent 96-well plate 3M TB Quat Disinfectant Ready-To-Use Cleaner (or other EPA-certified disinfectant that can kill Staphylococcus aureus) plate-reading luminometer appropriate safety equipment 1. Use a sterile applicator to spread cells from your overnight cultures of S. aureus over the entire surface of each sterile, stainless steel tray. Be careful not to contaminate the surrounding lab bench surface. 2. Allow any excess liquid to dry. 3. Label four 1.5 ml tubes (PC for positive control, NC for negative control, SW for spray and wipe, and 3-min for 3-minute protocol). 4. For your positive control, use a Fisherbrand polyester applicator to sample one of the trays that you have seeded with S. aureus. Place the applicator into a 1.5 tube and break off the stick. Add 250 µl Nuclease-Free Water. 5. For your negative control, use a Fisherbrand polyester applicator dipped in sterile culture medium. Place the applicator into a 1.5 ml tube and break off the stick. Add 250 µl Nuclease-Free Water. 6. For for the first tray, you will perform the spray and wipe protocol. Spray the 3M TB Quat Disinfectant Ready-To-Use Cleaner on the tray surface and immediately wipe the surface of the tray with a paper towel. Discard the towel in the laboratory biohazard waste. 7. Use a Fisherbrand polyester applicator to swab the surface of the tray. Place the applicator into a 1.5 ml tube and break off the stick. Add 250 µl of Nuclease-Free Water. 8. For the second tray, follow the directions for the proper use of the 3M TB Quat Disinfectant Ready-To-Use Cleaner. The technical data for the cleaner is available at: Commercial/Care/Solutions-for/Infection-Control/3M-TB-Quat-Disinfectant- Cleaner-Ready-to-Use/ Instructor: Be sure to remove BacTiter-Glo Reagent from the freezer to thaw before the lab activity. All steps are carried out at room temperature. Note: Staphylococcus epidermidis may be substituted for S. aureus. Important: Be sure to know, understand and follow your institution s procedures and guidelines for laboratory safety and disposal of biohazard waste. Be sure students follow all safety procedures. 9/08 Page 7

8 IV. Evaluating the Effectiveness of Quaternary Ammonium Compound Salts (continued) a. Hold the container six to eight inches from the surface and spray until the tray is covered with the solution. b. Allow the tray to remain wet for three minutes. c. After three minutes, wipe with a clean paper towel. 9. Use a Fisherbrand polyester applicator to swab the surface of the tray. Place the applicator into a 1.5 ml tube and break off the stick Add 250 µl of Luciferin-Free Water. 10. Remove 100 µl of solution from each tube containing a swab and place it in a well of a 96-well plate. 11. Pour the BacTiter-Glo Reagent into a reservoir. Add 100 µl of BacTiter-Glo Reagent to all wells containing sample. 12. Measure luminescence on a plate-reading luminometer. 13. View the results on a luminometer or in Excel. For Discussion 1. CA-MRSA infections have been in the news with outbreaks occurring in public schools and college athletic departments. What are the current recommendations from the CDC for preventing CA-MRSA? Do you think that even if quats are provided in settings like locker rooms or gyms, they are effective? Why or why not? 2. Bacterial strains obviously develop genetically based resistance to antibiotics. Can bacteria develop genetically based resistance to disinfectants like quats? If so, what are the mechanisms of this resistance? V. Graphing Bacterial Growth Rates Instructor: Be sure to remove BacTiter-Glo Reagent from the freezer to thaw before the laboratory activity. Note: Be sure to know, understand and follow all of your institution s laboratory safety and biohazard waste disposal guidelines and procedures. Each group of students will need: shaking incubator two 250 ml culture flasks (one with a side arm for O.D. readings; each flask should contain 50 ml fresh, sterile Mueller Hinton II (MH II) Broth pipet and tips for delivering 1 10 µl of liquid pipet and tips capable of accurately delivering 100 µl pipet and tips capable of accurately delivering 200 µl BacTiter-Glo Reagent Nuclease-Free Water reservoir for dispensing reagent 96-well plate plate-reading luminometer spectrophotometer that can accommodate a sidearm flask (alternatively use a cuvette) overnight culture of E. coli Important! Aseptic technique is absolutely critical for this experiment. Do not contaminate your cultures when you are removing cells for your luminescence measurements. Page 8 9/08

9 1. Dilute overnight cultures by putting 1µl of the overnight culture into each of the flasks containing 50 ml of fresh MH II broth. 2. Immediately remove a 100 µl of culture medium and place it in a well of a 96-well plate. Add 100 µl of BacTiter-Glo Reagent and record luminescence. This is your zero time point. 3. Immediately take an O.D. reading at 600 nm of the culture in the sidearm flask. This is your zero time point. 4. Place the flasks into a shaking incubator set to 37 C and 250 rpm. 5. At 120, 180, 240 and 300 minutes, you will need to take both luminescent and O.D. readings of your cultures. For your luminescent readings, you may want to include a negative control of sterile culture medium along with each reading for good measure. Your O.D. measurements may not yield results until 300 minutes. 6. To see the plateau of the growth curve, will need to take measurements for up to 15 hours. Ask your instructor if additional measurements are needed. For Discussion VI. What is the advantage of an ATP-based assay like BacTiter-Glo Assay when working with extremely slow growing bacteria like Mycobacterium tuberculosis? Supplier and Ordering Information Product Cat. # BacTiter-Glo Microbial Cell Viability Assay* (100 reactions) G8230 Nuclease-Free Water* (50 ml) P ml tubes (1000/bag) V1231 *For laboratory use. Product Supplier Mueller Hinton II Broth Becton Dickinson Cat.# Costar Solid white 96-well plate Fisher Cat.# Costar Sterile Disposable Reservoirs Fisher Cat.# Fisherbrand Sterile Polyester Applicators Fisher Cat.# M TB Quat Disinfectant Available from local commercial vendors. Ready-To-Use Cleaner Visit the 3M web site: Note: Promega products used by educators in the US for teaching laboratories may be available at significant discount. Visit trainingsupport/ for more information. Bacterial Species ATCC Strain Number Escherichia coli Staphylococcus aureus Promega Corporation. All Rights Reserved. GloMax is a registered trademark of Promega Corporation. BacTiter-Glo and Ultra-Glo are trademarks of Promega Corporation. Costar is a registered trademark of Corning Incorporated. Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information. 9/08 Page 9

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