Ringer s solution

Larvae with micro-tumors detected by fluorescence microscopy were enzymatically digested at different hpi to be analyzed by flow cytometry. Larvae were first anesthetized with 0.004% tricaine, transferred to 1.5 ml vials with calcium-free Ringer's solution 116 mM NaCl, 2.9 mM KCl, 5 mM HEPES, pH 7.2 (all reagents from Sigma-Aldrich) and incubated at room temperature for 15 min. Larvae were transferred to a 35 mm culture dish (Falcon, Franking Lake, NJ, USA) with 2 ml of trypsin 0.25% EDTA 1x solution (Gibco), incubated at 37°C and dissociation was mechanically assisted by performing up and down pipetting with a 200 µl tip every 3–4 min until a single cell suspension was visualized under the microscope (∼15 min). Trypsin was neutralized using twice the volume of 5% FCS in PBS 1×. Cell suspension was centrifuged at 3250×g for 10 min. Cell pellet was re-suspended in 100 µl of FACSFlow and run on a FACS canto II cytometer. Human cells were detected at 660/20 nm with excitation of the He-Ne 630 nm laser, to be differentiated from larvae cells. Fluorescent positive cell population was gated to carry out the proliferation analysis with FlowJo^® 10.3 software (Tree Star, Inc. Ashland, Oregon, USA) to estimate the number of cell generations.

Milk samples were directly serially diluted (1:10) in Ringer’s solution (Sigma-Aldrich, Milan, Italy) into 10-mL/volume test tubes, while the curd and cheese samples (10 g) were first, homogenized in sodium citrate (2% w/v) solution by means of BagMixer^® 400 (Interscience, Saint Nom, France) at the highest speed for 2 min and then serially diluted in Ringer’s solution. The dilutions of raw milk and pasteurized milk were plated on agar media to analyze the total mesophilic microorganisms (TMM), and the main pro-technological (lactic acid bacteria (LAB), including enterococci); spoilage (Pseudomonas spp.) and pathogenic (members of the Enterobacteriaceae family, coagulase-positive staphylococci (CPS), Listeria monocytogenes, Escherichia coli and Salmonella spp.) bacterial groups following the approach of Gaglio et al. [28 (link)].

A coupon was placed per well, and 1.5 ml of the respective inoculum was added. Controls with non-inoculated coupons were present in each P24 microplate. Incubation was performed at 25°C (room temperature) for 48 h or at 11°C (low temperature) for 7 days, in both cases without agitation. After this period, in order to remove planktonic cells, each coupon was rinsed through pippeting 1 ml of Ringer’s solution (Sigma–Aldrich, St. Louis, MO, United States) on each side of the coupon, which was then placed in a new P24 microplate that contained a layer of 20 sterile glass beads (ø = 3 mm). Thirty sterile glass beads and 1 ml of Ringer’s solution were then added to each well. The microplate was stirred in a Microplate vortex (Tittertek DSG, Flowlabs, Germany) for 1 min at maximum speed (position 10) in order to remove the biofilm from both sides of the coupons. From each well, 100 μl of the suspension was withdrawn in order to perform serial decimal dilutions to spot inoculate (25 μl) TSA-YE plates. Colony forming units (CFUs) were counted after overnight incubation at 37°C. The assays were replicated using four replicates: two biologically independent cultures on distinct days (two biological replicates), each with two repetitions under identical conditions (two technical replicates).

Bacterial quantification was done through serial dilution plating. Whole stems collected ~1 cm above the lowest inoculation point and up to the apical meristem were sampled at three time points; 4 dpi, 7 dpi and 14 dpi. Stems were pulverized and homogenized in Ringer’s solution (Sigma Aldrich). 50 μl of serial dilutions of the homogenate (10¹ –10⁶) were plated on SCM-F selective plates (Duchefa Biochemie). The medium was supplemented with 1.9 g L^-1 yeast extract, 20 μL L^-1 nalidixic acid (100 mg mL^-1), 8 mL L^-1 trimetroprim in MetOH 100% (10 mg/mL), 1 ml L^-1 cyclohexamide in MetOH 100% (100 mg mL^-1), 1 mL potassium tellurite (1%), 50 ml L^-1 nicotinic acid (2 mg/mL). Plates were supplemented with appropriate antibiotics when necessary (25 μl/mL rifampicin). Plates were incubated at 25°C for 7 days. Colonies on the plates were counted 7 days post plating and the log₁₀(cfu+1/g fresh tissue) per plate was calculated. Five biological replicates of RNAi::SlWAT1_1 plants and the susceptible cv. MM were used per time point. Two technical replicates per sample were plated. The same procedure was used for the quantification of in planta bacterial titers of strain NCPBB382 in slwat1 mutants. Five biological replicates per time point were used.

Infected (‘source’) colonies were used to make Crithidia inoculum. The original Crithidia cells infecting colonies came from three wild B. impatiens workers collected from Stone Soup Farm (Hadley, MA, USA: 42.363911 N, −72.567747 W) unless otherwise noted. To make inoculum, bees were dissected from the source colony daily using an established protocol^{20 (link)}. Bee digestive tracts (excluding the honey crop) were removed, placed into a 1.5 mL microcentrifuge tube with 300 μL of 25% strength Ringer’s solution (Sigma-Aldrich, St. Louis, MO, USA), finely ground, and vortexed for 5 seconds. Each sample was allowed to rest at room temperature for 4–5 hours. Crithidia cells were counted from a 0.02 μL sample per bee with a Neubauer hemacytometer^{20 (link)}. We mixed 150 μL of the supernatant with 25% strength Ringer’s solution to achieve a concentration of 1200 cells μL⁻¹. The inoculum was then mixed with an equal volume of 50% sucrose solution to yield inoculum with 600 cells μL⁻¹ and 25% sucrose. Experimentally infected bees were starved for 4–6 hours and then fed a 10 μL drop of inoculum with 6,000 Crithidia cells, which is within the range of concentrations bees are exposed to when foraging on flowers in the wild^{52 (link)}. Only bees that consumed the entire droplet were used in experiments.