Air-sampled Filter Analysis for Endotoxins and DNA Content

Note: A detailed list of all the materials and instruments used in this protocol is shown in the Materials section.

1. Air Sampling on Filters

1. Preparation of Filters
   1. For high volume sampling, use 20.3 x 25.4 cm² filters. Choose the specific type of filter that best fits the research needs, as well as the filter cut-off size, if applicable.¹ Here, use quartz microfiber filters.
   2. Pre-bake filters intended for organic and biological compound sampling to destroy organic residues. To pre-bake the filters, wrap them individually in aluminum foil and then bake them in a laboratory furnace at 450 °C for at least 5 hr.
   3. Store the baked filters at -20 °C until sampling.
2. Instrument Handling and Air Sampling
   1. Clean residual dust from the head of the high volume sampler. Wipe the parts with clean paper wipes and allow them to air-dry before reconnecting them to the sampler.
   2. Disinfect the filter cassette with ethanol, and work with gloves and a lab coat at all times.
   3. Place a pre-baked filter inside the clean filter cassette, and proceed according to the high volume sampler manual.
   4. Mark the date and any unusual weather conditions, if applicable (e.g. rain, dust storm) on the aluminum wrap, and save it in a clean place until the completion of sampling.
   5. Collect the sampled filter and fold it in half, with the sampled side facing inward.
   6. Wrap the filter in the original aluminum foil and store at -20 °C until analysis.
      Note: If the time lag between sampling and analysis is longer than 2 months, store the sample at -80 °C to avoid degradation of the organic and biological material.

2. Endotoxin Analysis

Note: Disinfect the work surface with 70% ethanol and work with pyrogen-free tubes, tips and reagents only. If glassware are used, pre-heating at 250 °C for 30 min, or 200 °C for 60 min is required.²⁵ Prepare all reagents in a class II biosafety cabinet and work with gloves and a lab coat at all times.

1. Reagent Preparation
   1. Follow the LAL kit manufacturer's protocol to rehydrate the lysate shortly before use.²⁶ Tap gently on the vial to dislodge LAL remaining on the bottle walls. Lift the stopper gently to break the vacuum. Using a needled syringe, add 5 ml of pyrogen free water (PFW) to the vial to rehydrate all the lysate content and mix gently to avoid foam formation.
   2. Seal the vial with plastic paraffin film when not in use and store at 4 °C for up to two days. Store any remaining lysate at -20 °C for up to three months.
      Note: This reagent can be frozen only once.
   3. Rehydrate the control standard endotoxin (CSE), which contains 0.5 µg E. coli, in accordance with the manufacturer's protocol.²⁷ Determine the specific amount of water to be added to the CSE vial from the manufacturer's website²⁸ by entering the lot number for the kit where specified on the website. The final concentration of E. coli in the vial, is measured in endotoxin units (EU; where 10 EU = 1 ng). Label this vial Ed0.
   4. Seal the CSE vial with plastic paraffin film when not in use and store at 4 °C for up to 4 weeks.
2. Standard Curve and Spiked-filter Preparation
   1. In a class II biosafety cabinet, prepare 22 pyrogen-free 2 ml centrifuge tubes containing decreasing amounts of CSE (tubes Ed1-Ed11) or no CSE (the blank tube containing only PFW) to produce a duplicated endotoxin dilution series, as shown in Table 1.
   2. In a biological cabinet with circular flow, cut 22 circular pieces of clean, pre-baked filter using a disinfected 1.12 cm diameter cork borer.
   3. Place the filter pairs into sterile petri dishes.
   4. Spike 50 µl from each member of endotoxin dilution series Ed2-Ed11 and from the blank tube onto a corresponding pair of filters.
   5. Leave the Petri dishes containing the spiked filters open under the hood to dry for 30 min.
   6. Place each filter piece in a pyrogen-free 2 ml tube.

Table 1: Endotoxin Standard Curve. Endotoxin concentration, volume of standard endotoxin and of pyrogen-free water to be added, and the total volume obtained are detailed for each dilution tube in the calibration curve.

3. Endotoxin Extraction from the Experimental and Spiked Filters
   1. In a biological cabinet with circular flow, cut the sample filters (from Step 1.2) into circular pieces using a disinfected 1.12 cm diameter cork borer, and place them, together with the spiked standard filters (from Step 2.2), in pyrogen-free 2 ml tubes (i.e., the sample tubes).
   2. Add 1 ml PFW to the tubes and shake them for 60 min at room temperature, using a laboratory shaker.
   3. Centrifuge the sample tubes in a microcentrifuge at 375 x g for 10 min.²⁹
   4. Transfer the supernatant, which contains the endotoxins, into a new pyrogen-free 2 ml tube.
4. Limulus Amebocyte Lysate (LAL) Test
   1. Determine the endotoxin concentration in the samples by performing the LAL test in a pyrogen-free 96-well microplate with a flat bottom and a lid.
   2. Plan the plate array in advance, as shown in Figure 1. Include a standard curve prepared directly from endotoxin standard solutions Ed2-Ed11 (and thus covering the concentration range of 100-0.195 EU/ml (see section 2.2)) in every running plate. The standard curve should occupy wells 1-10 of rows A and B of the plate. Set aside wells 11-12 in these rows for blank samples (making a total of four blanks).
   3. Turn on the microplate reader and program it for a kinetic reaction involving incubation of the microplate at 37 °C and shaking every 5 min, followed by absorption measurements at 405 nm. Repeat 18 times for 1.5 hr.
   4. Determine the efficiency with which the endotoxins are extracted from the sample filters (i.e., the samples obtained from the spiked standard filters) via division of the calculated endotoxin amount by the original amount spiked.
   5. Start the assay by placing 50 µl of the standard endotoxin solutions (from step 2.4.2), or of the spiked filter extract and its blanks (from section 2.3), or of the experimental samples and its blanks (also from section 2.3), as per the planned plate-array (Figure 1) and close the cover.
   6. To each well, quickly add 50 µl of LAL solution, and gently shake the plate horizontally while it is placed on the table before lifting it into the reader.
   7. Place the plate in the plate reader and start the experimental run.

Figure 1: Endotoxin array plate. An example of an endotoxin analysis array in a 96-well microplate.

3. Genomic Analysis

Note: For DNA extraction, disinfect the work surface with 70% ethanol and work with sterile tubes, tips and reagents only. For q-PCR analysis of DNA, disinfect the work surface with surface DNA-decontaminant. Prepare all reagents in a class II biosafety cabinet and work with gloves and a lab coat at all times.

1. Preparation of the DNA Primers and Probes
   1. Prior to DNA extraction, either order a commercially-available set of primers and a probe (if the hydrolysis probe method is applied), or design a new set of primers and a probe using primer designing computational tools.³⁰
   2. Rehydrate the primers according to the manufacturer's protocol using either 10 mM Tris-0.1 mM Ethylenediaminetetraacetic acid (EDTA) (TE buffer) or PCR-grade water.
      Note: It is recommended to dissolve the initial PCR primer stock in TE buffer with low EDTA (0.1%) as it prevents primer degradation when kept for longer times. Use PCR-grade water for subsequent dilutions to reduce EDTA amounts that might inhibit PCR reactions.
   3. Prepare aliquots of 50 µl or 100 µl of the primer and probe in sterile 0.5 ml tubes and store at -20 °C until analysis.
2. Standard Cell Concentration Evaluation Prior to DNA Extraction
   1. In a suitably sized centrifuge tube, prepare a standard cell solution of the microbial species of interest (tube Od0, Table 2A). Mix the cell suspension by pipetting it up and down in the tube 7-10 times using a pipette with a small bore.
   2. If the cells are colored (e.g. certain fungal spores), do not perform a staining procedure. If cell staining is required, dilute the cell suspension in a suitable dye for microscopic detection of the cells of interest.³¹
   3. Clean the cover slip and hemocytometer with ethanol. Moisten and affix the cover slip to the hemocytometer.
   4. Load about 8 µl of the cell suspension into both hemocytometer chambers by carefully touching the edge of the cover slip with the tip of the pipette and filling the chamber by capillary action. Do not over/under fill the chamber.
   5. Determine the number of cells by viewing them under a microscope at 400X magnification (40X in objective and 10X in ocular). Nine squares measuring 1 x 1 mm² and arranged in a 3 x 3 grid should be seen. Focus the microscope on one of the four corner squares of the grid (a higher magnification can be used). The 1 x 1 mm² square should contain 16 smaller squares.
   6. Count all the cells in the four 1 x 1 mm² corner squares, as well as in the middle square of the 3 x 3 grid. If there are too many or too few cells to count, repeat the procedure, either concentrating or diluting the original cell suspension as appropriate (15-50 cells should overlay a singling 1 mm² area).
   7. Calculate the concentration of the cells (cells ml^-1) in the standard cell suspension Od0 from the cell count averaged across the five counted squares as follows: Cell count ml^-1 = Average cell count square^-1 Dilution Factor 10⁴.
3. Evaluation of DNA Extraction Efficiency
   1. Prepare nine sterile 0.5 ml tubes according to Table 2A.
   2. Dilute the standard cell solution (Od0) to contain about 10⁷ cells ml^-1 in a total volume of 20 µl (Od1).
   3. Add 18 µl of sterile nuclease-free PCR-grade water (NFW) to tubes Od2-Od8 and 20 µl to the blank tube, Od9.
   4. Transfer 2 µl of the cell solution from Od1 into Od2. Pipette gently to mix and transfer 2 µl from tube Od2 to Od3. Continue diluting the cells in the same manner along the tubes up to and including tube Od8 to obtain a serial dilution of 10⁷-10⁰ cells ml^-1.
   5. In a biological cabinet with circular flow, cut 18 circular pieces of clean, pre-baked filter, using a disinfected 1.12 cm diameter cork borer. Place the filters in pairs into sterile Petri dishes.
   6. Spike 10 µl from dilutions Od1 to Od8 and from the blank tube Od9 onto each of the paired filters, such that there is one filter pair corresponding to each standard cell concentration.
   7. Leave the Petri dishes open under the hood and let them dry for 30 min.
   8. Place each filter piece in a sterile screw-top 2 ml tube and extract the DNA according to the procedure detailed in section 3.4.
4. DNA Extraction from the Experimental and Spiked Filters
   Note: For DNA extraction from filters, use commercial kits designed for DNA extraction from soil according to the manufacturer's protocol with the following modifications.
   1. For each filter sample (Od1-Od8), prepare a mixture of acid-washed glass beads in a 0.5 ml sterile tube. Use 0.1 g beads having a diameter of 425-600 µm and 0.3 g beads having a diameter of ≤106 µm.
   2. In a biological cabinet with circular flow, cut three circular pieces from randomly-selected locations on each filter sample using a disinfected 1.12 cm diameter cork borer, and place them in screw-top 2 ml tubes.
   3. Add the glass bead mixture to the tubes.
   4. Add the cell-lysis buffer supplied with the extraction kit to each tube in the biological cabinet, with the amount indicated in the manufacturer’s protocol.
   5. Prepare a bucket of ice and disrupt the cells mechanically using a bead beater.
   6. Beat the beads for 1 min and then place them on ice for 1 min. Repeat five times.
   7. Proceed with the kit supplier's protocol from the post-lysis step.
   8. After elution with the supplied elution buffer (contains 10 mM Tris buffer) or NFW, reload the eluted sample through the column again to improve DNA yield.
      Note: If not analyzed on the same day as the DNA extraction, samples can be stored at -20 °C until analysis.

Table 2: DNA Standard Curve. Standard microorganism cell dilution series (A) detailed for the standard cell volume, NFW volume, and the total volume in each dilution tube. DNA standard curve preparation (B), detailed for the standard DNA volume, NFW volume, and the total volume in each dilution tube.

5. DNA Standard Curve Preparation
   1. Extract DNA directly from a standard sample of the microorganism of interest (Dd0) using the same procedure as described for the experimental filters in step 3.4.
   2. Use a spectrophotometer to determine the DNA concentration of the standard DNA sample (Dd0) at 260 nm.
   3. Prepare nine sterile 0.5 ml tubes from which to prepare a DNA standard curve, as shown in Table 2B.
   4. If necessary, dilute the standard DNA solution Dd0 to within a concentration range of 1-10 µg ml^-1 in the first tube (Dd1) in a total volume of 20 µl.
   5. Add 18 µl of NFW to tubes Dd2-Dd8 and 20 µl to the blank tube.
   6. Transfer 2 µl of the standard DNA solution from Dd1 into Dd2. Pipet gently to mix and then transfer 2 µl from Dd2 to Dd3. Continue diluting the DNA in the same manner along the tubes to achieve a serial dilution of 10⁰-10^-7 in tubes Dd2-Dd8 together with a blank tube containing only NFW.
      Note: If not analyzed on the same day of extraction, store samples at -20 °C until analysis.
6. Quantitative-PCR Analysis
   1. Switch on the q-PCR instrument in advance to warm up.
   2. In a new program file, insert the details for the q-PCR run in the instrument-operating software as detailed in Table 3.
      Note: Different instruments have different optimal timing for each thermal cycle step. This input is specified in the instrument's manual.
   3. Disinfect the work surface with surface DNA-decontaminant and work only with sterile tubes, tips, and reagents.
   4. Prepare a bucket of ice next to the working bench. Store Taq-polymerase mix on ice.
   5. Calculate the total number of reaction wells that will be occupied in the plate. Make theoretical allowance for extra reactions (5%) and multiply the enlarged number of reactions by the volume per reaction to calculate the total reaction volume.
   6. Prepare the reaction mixed pool, starting from NFW, primers, probe, and lastly the Taq polymerase mix. See example for the volume calculations for the mixed pool in Table 4.
   7. Store the reaction mix in the dark and on ice until use.
   8. Place 1 µl of standard DNA, sample DNA, or NFW (as a negative control) inside the wells in the q-PCR microplate in triplicates.
   9. Ensure that the plate is in a plate holder to prevent it from touching the working surface and to keep it clean.
   10. Add 9 µl of the mixed pool (Table 4) into each reaction well.
   11. Cover the plate with optical adhesive film and seal it tightly from all sides.
   12. Spin down the plate in a centrifuge with plate buckets for 1 min at 1,000 x g before placing it in the q-PCR device.
   13. Place the plate into the instrument and activate the running program.

Table 3: Details for q-PCR operating software. Details of the analysis parameters to be entered into the q-PCR program file.

Table 4: Quantitative-PCR Reaction Mix Calculation. Volume per reaction, number of reactions, allowance for error, and the total calculated volume to be added into the reaction mix per reagent are detailed.