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Bacteria Killing Assay using agarose plates Dan Ardia, Franklin & Marshall College: August 2008 Modified from protocols by Kirk Klasing and Anna Forsman Original method: Millet, S., Bennet, J., Lee, K. A., Hau, M. & Klasing, K. C. 2007 Quantifying and comparing constitutive immunity across avian species. Developmental And Comparative Immunology 31, 188-201. Overview: All animals have an innate ability to recognize and kill microorganisms. This is a coarse assay of innate immunity that assesses the ability of immune factors in the blood to kill bacteria/yeast. With whole blood, the assay integrates phagocytosis by macrophages, as well as complement and lysozyme activity. When just plasma is used, phagocytosis is thus not included. We mix whole blood or plasma with a known dilution of bacteria/yeast in a growth medium. If the individual has the ability to kill off the pathogen, then the resultant agar plates will show reduced bacterial growth relative to controls. The metric of interest is the percent bacteria killed relative to control plates. Because of this, it is important to pay close attention to the control plates. There should not be too many samples processed without running control plates. We generally run our samples in batches of about 10-24 individuals with control plates done at the beginning and end of each run. Because the bacteria/yeast are at a known dilution, it is important that they not be able to continue to grow or degrade. Thus, whenever the bacteria/yeast are out of the fridge, they must be kept on ice at all times. This table shows common microorganisms used in this assay. The incubation times given are for chickens and cockatiels from work done by Kirk Klasing (UC-Davis). Adjust dilutions and incubation as required by your species. Note: We’ve used E. coli (8739), S. aureus and Candida for tree swallows. E. coli gets an incubation time of 45 minutes for nestlings, 30 minutes for adults. The others need 3 hours incubation for adults and nestlings. Only E. coli (#8739) is killed by plasma alone; the others require whole blood. Strain Dilution E. coli ATCC # 8739 Microbiologics #0483E7 1:10 E. coli ATCC #51813 Microbiologics #0791E8 C. albicans ATCC #10231 Microbiologics #0443E7 S. aureus ATCC # 6538 Microbiologics #0485E7 S. epidermidis ATCC #12228 Microbiologics #0371E8 1:10 Bacteria Killing Assay 1:4 Incubation Comments time 15-30 min You can use serum or plasma for this strain – killing is complement dependent 90 min Mostly complement independent and requires phagocytosis 4 hr Killing mostly by phagocytosis 1:4 4 hr 1:4 4 hr Mostly complement independent and requires phagocytosis Mostly complement independent and requires phagocytosis 1 Materials: -Petri dishes. I prefer Fisher Media Miser plates -Tryptic soy agar (TSA) -CO2 independent media (Invitrogen; Gibco media #18045) -L-Glutamine (Sigma) -Microorganisms (Microbiologics) -Falcon tubes (50ml for stock solution, 15ml for working solution) -Microcentrifuge tubes (1.5ml) -Alcohol lamp or gas burner for sterilizing -Dish of 70% EtOH for sterilizing -Glass spreading rod Lab equipment needed: -Incubator (capable of 40ºC) -Laminar flow (cell culture) hood -Vortexer Do beforehand: - Make plates ahead of time or the day of the assay. Autoclave 1.5mL tubes, pipette tips, mix and autoclave 1x PBS. Day of the assay: - Make bacteria stock and CO-independent media on the first day of the assay. It is best to use the stock solution within 1 week. Make fresh working solution of bacteria everyday. Step 1: Pouring Plates (do up to one week beforehand) 1. Add 20g of tryptic soy agar to 500mL of tap water in a 1L bottle and mix by shaking. Loosen the cap slightly, cover with aluminum foil and autoclave for 20 min (no steam dry). This makes ~25-30 plates. 2. Remove from autoclave and tighten lid. Make sure the benchtop is sterile by wiping down with alcohol (or bleach), and laying down bench paper. 3. Put on fresh gloves, and in a sterile, area, open a bag of Petri dishes (SAVE BAG) and begin pouring the hot agar into each plate leaving the lids off while they cool. Pour enough agar to cover the bottom of the plate. If agar is too thick, reading the colonies is harder because the agar becomes cloudy. Always make sure that the plates cool down completely (e.g. leave for a few hours) before putting lid on – otherwise there will be condensation, which results in spreading of colonies. Bacteria Killing Assay 2 Swirl gently and begin pouring the agar immediately after autoclaving. Cooling even for ~10min would make the agar clumpy and the plates at the end uneven. Avoid bubbles. If bubbles are introduced, move them to the edge of the plate with a sterile pipette tip. Put the lids back on ~40-60 min after you start pouring (or until there is no more steam on the lids, plates should be cool to the touch). Place the plates lid-side up in the plastic bag they came in and store them in the refrigerator. 4. When the agar is hardened, place the lids back on and stack. Store the plates upside down inside the bag the Petri dishes originally came in. Step 2: Making media (30 mL/4 mM stock) 1) Pipet 29.4 mL of Co2 independent media into a sterile 50 mL falcon tube. 2) Add 0.6 mL (or 600 uL) of 200 mM L Glutamine. MIX WELL as it needs to resuspend. Step 2: Preparing Microorganisms (Epower microorganisms, Microbiologics) If the stock solution never gets warm, it should last a couple of weeks. The main concern is contamination which will show up as streaky or turbid solution. In general, the bacteria decrease over time, so proper control plates are essential. 5. Remove a vial of lyophilized sample from the refrigerator and allow it to equilibrate to room temp. 6. Warm hydrating solution (1X PBS) to 337 C. To make a stock solution, you will need to dilute one pellet containing 107 bacteria in 40mL of sterile PBS. If the pellet contains 108 bacteria, make an additional 10-fold dilution. 7. Transfer a pellet using sterile (generally flamed) forceps into the hydrating fluid. 8. Place the hydrating microorganism suspension into a 35-37 C incubator for 30 min. 9. Following incubation, vortex or thoroughly mix the hydrated material. Store this Stock suspension in the refrigerator. Keep on ice whenever it is removed from the refrigerator. 10. To use the solution, vortex and then transfer the appropriate volume into a working solution tube. Bacteria Killing Assay 3 Step 3: Getting the proper dilution for the working solution The goal is to add an appropriate amount of bacteria or yeast to get about 100 to 150 colonies per 50 µL of diluted blood (i.e. per plate). Over time the appropriate concentration varies, but it normally runs from a low of 0.1ml microbe to 9.9 ml PBS to a high 1ml microbe to 9.0ml PBS. The key is to get it right, which means to plate out a range of dilutions the day before running the test. As a start I usually use duplicates of 0.1:9.9, 0.5:9.5, 1:9, 1.5:8.5. This allows me to titrate the right dilution. Fresh working solution should be made daily and kept on ice at all times. 1. Fill and label tubes with the needed amount of PBS before removing the stock solution from the fridge. You can use 15ml Falcon tubes with the exact amounts above or reduce the volumes to use 1.5ml centrifuge tubes (i.e. 10ul microbe to 990ul PBS). The danger of using smaller volumes is not getting a consistent sample of the microbes, thus you will see more variation among your duplicates. 2. Remove stock solution from fridge and put on ice. 3. VORTEX STOCK SOLUTION BEFORE EACH USE!! 4. Add the appropriate volume of stock to the working solution tubes. 5. Return working stock tubes to ice or refrigerator 6. Prepare cell culture media (as noted below) 7. Follow ratios as listed below (I.e. Mix 20ul of each working volume of E. coli with 100ul of cell culture media in each centrifuge tube.) 8. Incubate at 40deg for the same time period as blood samples will be used (i.e. 45 minutes for tree swallow nestlings vs. E. coli). 9. Plate out as indicated in protocol below. Step 4: Making cell culture media Media = CO2 independent media (Invitrogen Inc; Gibco media #18045) + 4mM LGlutamine. Don’t make the entire bottle of media, use a 50ml Falcon tube to make about 40mls worth. It will become contaminated quickly (that’s why it is sold – L-glutamine) For a 50ml Falcon tube: Pipet 29.4 mL of Co2 independent media into a sterile 50 mL falcon tube, then add 0.6 mL (or 600 uL) of 200 mM L Glutamine. MIX WELL as it needs to resuspend. Bacteria Killing Assay 4 Step 5: Processing samples Things to keep in mind: - Assay up to 10 samples at a time with media control at the beginning and end. - Keep sample tubes on ice at all times except during the 41C incubation. - For stock and working bacteria solution as well as samples containing bacteria, vortex thoroughly and aliquot immediately as bacteria settles very quickly. - Keep the stock and working solution of bacteria on ice at all times. - Use the offset method of placing the centrifuge tubes in a different row in the rack after using to avoid using wrong tube twice. Please note: volume and incubation times below here represent dilution used for testing tree swallow blood against E. coli—changes dilutions as needed for 1:4 dilution vs. Staph or Candida. To do duplicates, we would mix 5ul whole blood: 95ul media: 20ul microbe. If you are running controls (or testing dilutions) increase media to 100ul to make up for not having whole blood in mix. Turn on incubator prior to starting! How to calculate working bacteria solution: 1. Put 95ul of cell culture media in each centrifuge tube (20-200ul Pipettor will read 095). 2. Place 5ul of whole blood or plasma in each tube 3. Once plasma is in tubes, remove proper working stock dilution from fridge and place on ice. VORTEX before using. 4. Add 20ul of bacteria from the working dilution into the proper tubes. E. coli into E tubes (including controls), S. aureus into S tubes (including controls), Candida albicans in C tubes. 5. Close tube tops tightly and VORTEX 6. Place tubes in incubator at 40deg for appropriate time (45 minutes E. coli, 3 hrs Staph or Candida) Step 6: Plating the samples To avoid contamination plate under a laminar flow (cell culture) hood. 7. While tubes are incubating, remove and label Petri dishes on the underside (agar side). When labeling, be sure to include sample number, microorganism and date. If doing duplicates, then indicate this.. For example, a plate might say 94331-E-1 (bird 94331, vs. E. coli, duplicate 1. Always do two plates for each control. To make things easier, we generally label plates in reverse order of how they will be plated. 8. Once tubes are done in incubator, remove and VORTEX. Bacteria Killing Assay 5 9. Plating bacteria -Light burner and open alcohol dish. Be sure that the dish is not in the path of the flaming spreader -Flame sterilize spreader -Remove 50 ul from the tube and place in the center of appropriate Petri dish. DO A CONTROL PLATE FIRST AND THEN LAST -Using the flame-sterilized spreader spread the liquid evenly over plate by turning the plate. -Place the plate under the hood to dry with the top next to it. Plates should stay in hood for about 1-2 minutes. -Flame sterilize spreader -Repeat process (on other plates) and every couple of minutes, cover dried dishes and stack to side AGAR SIDE UP. 10. When entire run is completed places plates AGAR SIDE UP in incubator. Incubate the plates for min. 12 hours (overnight) at 40deg. 11. CLEAN WORK SURFACE AND HOOD SURFACE WITH BLEACH SOLUTION. Bacteria Killing Assay 6