Although several methods of concentration of samples were initially evaluated, the adsorption-precipitation protocol with aluminum chloride (AlCl3), previously described in Randazzo et al. (2019) (link), was selected because of better quality and quantity of RNA obtained. Briefly, 150 mL of water was transferred into a beaker of 200 mL and 75 μL of the enveloped rhabdovirus SVCV (105 TCID50/mL) (an enveloped RNA virus) was inoculated to each water sample as a concentration control. The SVCV virus was selected because it is an enveloped virus like the SARS-CoV-2, and it is frequently used in our research group. The pH of each sample was adjusted to 6.0 and 0.9 N AlCl3 solution was added to the sample at 1:100 ratio. The pH was again readjusted to 6.0 and samples were mixed at room temperature in an orbital shaker at 150 rpm during 15 min. Then, samples were centrifuged at 1700g for 20 min. in a Sorval ST Plus Series centrifuge (Thermo Scientific, USA) and the pellet was resuspended in 10 mL of 3% beef extract at pH 7.5 and transferred to 15 mL centrifuge tubes (Falcon). Samples were mixed again at room temperature in an orbital shaker at 200 rpm for 10 min. and centrifuged at 1900g for 30 min. in the Allegra™ X-22R Centrifuge (Beckman Coulter). Finally, the pellet was resuspended in 1 mL 1× PBS and samples were stored at −20 °C until RNA isolation.
15 ml centrifuge tubes
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15 ml centrifuge tubes are laboratory equipment designed for sample separation and concentration through centrifugation. These tubes have a capacity of 15 milliliters and are typically made of durable, sterile materials suitable for a variety of scientific applications.
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4 protocols using 15 ml centrifuge tubes
1
Enveloped Virus Concentration Protocol
2
Trophozoite DNA Extraction Protocol
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In preparation for DNA extraction, trophozoites were harvested by placing culture tubes on ice for 10 min. Tubes were then gently inverted to mix the contents, and 2 ml of each suspension were pipetted into 15 ml centrifuge tubes (Falcon), and PBS pH 7.4 was added to a final volume of 10 ml. Tubes were centrifuged at 1,000g for 5 min. The supernatants were decanted and the pellets were washed twice more by repeating the centrifugation. Trophozoites were suspended in a final volume of 1 ml PBS. Total DNA was extracted from the trophozoites using the DNeasy Tissue Kit (Qiagen Inc., Mississauga, ON), using a modified protocol. Two hundred microliters of the suspended trophozoites were transferred to 1.5 ml microcentrifuge tubes, and lysed overnight at 56°C using 180 μl of lysis buffer and 20 μl of proteinase K (20 mg/ml) supplied with the DNeasy Tissue Kit. The manufacturer’s instructions were then followed to purify the DNA. Nucleic acid was eluted with 100 μl of elution buffer. Positive control, G. duodenalis cysts (Waterborne, Inc., New Orleans, LA), and negative control, DNase-free water (Sigma-Aldrich Canada Co., Oakville, ON), were also extracted in parallel.
3
Quantifying Dermal Microfilariae Loads
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After extirpation of nodules from the skin tissues, portions of 20 cm2 (5 cm × 4 cm), of the upper epidermal layer of the skin were then measured and cut out using a scalpel and incubated in Petri-dishes containing RPMI medium + PSN antibiotic, at 37 °C, 5% CO2 for 4 h. This is to enable the mf to migrate out of the dermal tissue into the medium. The medium containing the emerged mf was then passed through a fine gauze into 15 mL centrifuge tubes (falcon, USA) in aliquots of 10 mL and centrifuged (Humax 14 k human, Germany) at 2000 rpm for 10 min for the mf to settle. For mf counts, the supernatant was reduced to 5 mL, thoroughly mixed and four drops of the suspension, 10 μL each, were placed on a clean slide and the mf counted under an inverted light microscope at 40× objective (Motic). Dilution or further concentration were performed whenever the mf load was too high or too low respectively. The average number of mf thus obtained was then used to compute the total number of mfs in the 5 mL of suspension. The cattle were then grouped as microfilaraemic or amicrofilaraemic, based on the presence or absence of dermal mf.
4
Stress Responses of Planktonic Cultures
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Planktonic cultures were cultivated in 15 ml centrifuge tubes (Falcon) in M9 complete medium and incubated overnight at 37°C with shaking at 200 rpm for 24 h. For stress conditions, 1 mL was collected, centrifuged at 10,000 × g for 3 min and the supernatant was discarded. Starvation was induced by replacing M9 complete medium with a solution of M9 salts without glucose (carbon starvation) or without ammonium chloride (nitrogen starvation) as medium for 3 days. Cultures were exposed to nitric oxide (NO) by supplementing the M9 complete medium with the NO donor sodium nitroprusside (SNP; Sigma Aldrich) at 10 μM, 100 μM, and 1 mM for 3 days. Oxidative stress was induced by supplementing M9 complete medium with 100 μM, 1 mM and 10 mM H2O2 (Biorad) for 3 days. Mitomycin C (Sigma Aldrich) was added to M9 complete medium at 3, 30 and 150 μM to induce DNA damage and cultures were treated for 3 days. All treatments were performed as biological triplicates and were compared to cultures maintained in M9 complete medium. The samples were collected daily to determine the phage titre.
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