The overall goal of this procedure is to describe how to accurately analyze endotoxin and DNA content of extracted airborne particles sampled on filters. This method can help answer key questions in indoor and outdoor biological aerosol research, such as understanding health reasons. Demonstrating the filter sampling procedure will be Yinon Mazar, a graduate student from my laboratory.
For high volume sampling, use filters made of quartz fiber. Or choose the specific type of filter as well as a filter cutoff size, that best fits the research needs. Prebake the filters intended for organic and biological compound sampling by first wrapping them individually in aluminum foil.
And then baking them in a laboratory furnace at 450 degrees Celsius for at least five hours. This will destroy any preexisting organic residues. Store the baked filters at 20 degrees Celsius until they are to be used for sampling.
Next, clean and disinfect the high volume sampler such as the one described in the accompanying text protocol. Then, place the prebaked filter into the filter cassette and run the sampler in accordance with manufacturer's instructions to collect an appropriate sample. In a class two biosafety cabinet, cut 22 circular pieces of clean, prebaked filter material using a clean 1.12 centimeter diameter cork borer.
Use these pieces to prepare a range of endotoxin spiked filters to be used as controls. Next, cut the filters containing the collected sample using a clean 1.12 centimeter diameter cork borer and place each sample into its own pyrogen free two millileter tube. Also, transfer the dried endotoxin spiked filters into separate pyrogen free two millileter tubes.
Add one milliliter of pyrogen free water to each tube. And shake them for 60 minutes at room temperature using a laboratory shaker. Next, centrifuge the sample tubes in a microcentrifuge at 375 times G for 10 minutes.
Then, transfer the supernatant which contains the endotoxins into a new pyrogen free two milliliter tube. Prepare a pyrogen free 96 well flat bottom microplate with a lid for the endotoxin test. By first laying out the sample locations.
Next, turn on the microplate reader and program it for a kinetic reaction at 37 degrees Celsius with shaking every five minutes. Followed by measurements at 405 nanometers. Repeat the five minute shake and the plate reading 18 times.
Load the samples into the acate by placing 50 microliters of the standard endotoxin solutions previously used to spike the filters into rows A1 through A10 and B1 through B10. Place the blanks into A11, A12, B11 and B12. Next, add the supernatant from the spun spiked filter extract and its blanks into rows C and D.Then, add the supernatants from the experimental samples and blanks into the remaining wells as needed.
To activate the test, quickly add 50 microliters of the limulus amebocyte glycate solution to each well. Gently shake the plate horizontally. Before lifting it into the plate reader and starting the experimental run.
Once finished, remove the plate from the plate reader. Determine the endotoxin level in each sample using the standard curve, and also calculate the efficiency with which the endotoxins are extracted from the sample filters. First, prepare the DNA spiked filters as described in the accompanying text protocol.
For each filter sample, prepare a mixture of acid washed glass beads to 0.5 milliliter sterile tubes at 0.1 grams of beads that have a diameter of 425 to 600 micrometers and 0.3 grams of beads that have a diameter of less than or equal to 106 micrometers. Next, cut three circular pieces from randomly selected locations on each filter sample using a disinfected 1.12 centimeter diameter cork borer. Place the filter samples into individual screw-top two milliliter tubes.
Then, add the glass bead mixture to each tube along with 60 microliters of the cell lysis buffer supplied with the DNA extraction kit. Close the tubes and place the samples into the bead beater. Use the bead beater to disrupt the filtered cells mechanically for one minute and then cool the samples by placing them on ice for an additional minute.
Repeat this cycle fives times. Proceed to follow the kit supplier's protocol and extract the DNA from the lysed cells. After elution, reload the eluted sample through the extraction column a second time to improve the DNA yield.
Switch on the quantitative PCR instrument in advance to allow it time to warm up. In a new program file, insert the details for the quantitative PCR run in the instrument operating software. Next, disinfect the work surface with surface DNA decontaminant and work only with sterile tubes, tips and reagents.
Also, place a bucket of ice next to the working bench and place the tac polemoraris mix into the bucket. Prepare the quantitative PCR reaction mix as described in table four of the accompanying text protocol. And store the reaction mix in the dark and on ice until use.
Next, place a clean microplate into a plate holder to prevent it from touching the working surface and to keep it clean. Add one microliter of standard DNA, sample DNA or nuclease-free water, inside the wells of the quantitative PCR microplate in triplicate. Then, add nine microliters of the master reaction mix into each reaction well.
When finished, cover the plate with optical adhesive film and seal it tightly on all sides. Spin down the plate in a centrifuge equipped with plate buckets for one minute at 1000 times G.Before placing it in the thermocycler. Finally, activate and run the prepared quantitative PCR program.
During the endotoxin extraction, filter samples are spun on a centrifuge to extract the endotoxin from the filter. Different centrifugation speeds result in various extraction efficiencies. Especially at higher endotoxin concentrations.
A preliminary test for extraction efficiency should be conducted for the specific filter chosen and protocol performed. Higher extraction efficiencies are obtained from spiking clean filters compared with sampled quartz filters and both of these efficiencies are lower than that achieved via direct detection of the standard solution. Once mastered, the endotoxin analysis can be done in three and a half hours if it is performed properly.
Following this procedure, other methods like DNA sequencing can be performed in order to answer additional questions regarding the transport of organisms in the atmosphere. After watching this video, you should have a good understanding of how to efficiently extract endotoxins and DNA from air sample filters and analyze them accurately.
