Office of Research and Development Photos Aerobic Endospore Procedure Laura A. Boczek Microbiologist US EPA – Office of Research and Development Aerobic Endospore Background Aerobic endospores are protective structures that some genera of bacteria have the ability to produce in response to a stressful environment. These genera are in general harmless to humans and are saprophytic organisms that are ubiquitous in many soil and water environments. These organisms can stay in the endospore form until conditions are favorable for them to germinate out of the endospore state to a vegetative state. This allows them to persist in their environment and resist many environmental stressors such as heat, desiccation, disinfection, and irradiation. 2 Aerobic Endospores Procedure Standard Methods 9218 A & B – outlines how to culture and measure aerobic endospores. Basic steps : 1. Obtaining a representative sample 2. Killing off any vegetative cells that could be in the sample by heat treatment 3. Using membrane filtration to concentrate endopsores in sample and then allow them to grow to enumerate Representative sample Samples should be collected using sterile wide mouth containers and care should be taken not to contaminate the samples during collection by using aseptic technique. Samples that contain a disinfectant residual such as chlorine should have sodium thiosulfate added to the sample bottle prior to collection in order to quench the chlorine. Samples should include a sufficient volume that can be processed to adequately determine treatment efficacy. • Sample waters will contain various amounts of endospores. Therefore if treatment efficacy is the reason why aerobic endospores are being measured a larger volume of sample may need to be collected and processed to determine efficacy. Samples should be shipped to laboratory in a cooler held at <10°C, but not freezing within 24 hours of collection. Aerobic Endospore Procedure Materials needed for Aerobic Endospore Analysis 1. Bottles or Erlenmeyer flasks with closures that are able to withstand high heat immediately followed by chilling in an ice bath. Glass is the preferred material due to its ability to transfer heat efficiently and care must be taken to ensure bottles won’t shatter during processing. 2. Membrane filtration apparatus as listed in SM 9222B.1.F 3. Water bath – preferably a shaking water bath, capable of heating samples to 80°C. 4. Thermometer – capable of measuring 25°C to 90°C. 5. Container for ice bath slurry. 6. Media – nutrient agar plus trypan blue dye / Incubator. 7. Wide field dissecting microscope, or other optical device. Aerobic Endospore Procedure First step in processing a sample for aerobic endospores is heat treatment. • Aseptically pour the sample into a sterile vessel that is able to adequately transfer heat in a water bath as well as withstand hot and cold temperature changes without breaking. Turn on water bath temperature 10 degrees higher temperature than heat treatment will be processed. If water bath is set to same value as the treatment temperature, the sample bottles will not be able to reach the appropriate temperature. • Samples should be processed at either 75°C for 15 minutes or 80°C for 10 minutes. Use a temperature control bottle. Temperature Control Bottle Important to have thermometer that will reach target temperature. Don’t place thermometer so that it touches the bottom of the bottle, you only want to measure the temperature of the water! Water in the control bottle should be the same level as in the sample bottles. Sample and Temperature Control Bottles Temperature and sample bottles should be the same type of bottle, and should be able to withstand temperature shock without breaking. Care should be taken to label bottle where it will not be submerged in the water bath (the label can come off if under water). I like to label both the bottle and the foil. Bottles should have a weight placed on them prior to being put in water bath to minimize the chance of flipping over in water bath. This will cause contamination and the sample must be discarded. Aerobic Endospore Procedure Place all bottles including the temperature control bottle into the water bath and start shaking (60 to 80 rpm). You may cover the samples with aluminum foil to hold in the heat and allow for bottles to come up to temperature faster. Take care when placing bottles into the water bath, make sure that the water level in the water bath is not too high so that the bottles would be contaminated. If shaking water bath is not accessible bottles need to be manually stirred periodically. When the temperature in the temperature control bottle reaches 73°C (if treatment is 75°C for 15min, or 78°C if treatment is 80°C for 10 min) then remove foil and turn down temperature on water bath setting. Start timer for either 10 or 15 minutes to correspond with correct temperature. Aerobic Endospore Procedure Water Water not above above sample sample level level Ice Bath Slurry Prepare ice bath slurry, by adding ice and water, but make sure that ice water level is not too high that it would contaminate the sample. Cool sample on ice water until it has reached at least room temperature or lower. Sample should then be safe to start membrane filtration. Membrane Filtration Follow SM 9222B for membrane filtration procedure, use a .45µm gridded filter to concentrate sample. Aseptically place filter onto nutrient agar plates supplemented with Trypan blue dye. Incubate plates at 35°C for 24 ± 2hrs, observe plates for bacterial growth. Use a wide-field dissecting scope, or other optical device to assist in counting bacterial colonies. Any colony growth is considered positive for aerobic endospore forming bacteria. Aerobic Endospore Procedure Nutrient Agar with trypan Nutrient Agar blue dye Nutrient Agar with trypan blue dye (color Nutrient Agar imparted onto filter) Aerobic Endospore Procedure Laboratory Testing Commercial Labs do exist to perform aerobic endospore testing! EMSL Analytical – many labs located throughout the US. Sound Microbiology Lab – located in Pacific northwest CH Diagnostic & Consulting – Located in Colorado This list is not exhaustive, and the quality of these labs has not been verified by EPA. Questions? Laura Boczek Microbiologist [email protected] 513-569-7282.