Compound microscope

5 dpf zebrafish larvae were embedded in 1.5% low gelling agarose (Sigma) in a dorsoventral position. The Purkinje cell layer was observed in these larvae under the 63× water immersion objective of a Nikon compound microscope. Patch pipettes (OD: 1.5 mm, ID: 0.86 mm) were pulled using borosilicate glass capillaries and a P-97 pipette puller (Sutter Instruments). A single pipette was backfilled with a mixture of tetramethylrhodamine dextran (TMR-dextran) and serotonin/neurobiotin and inserted through the skin of the larva. The pipette tip was positioned near a cell body in the Purkinje cell layer and 3–5 electric pulses (30 V, 30 ms) were administered, until the neuron was completely filled with the dye. The larva was then released from the agarose and fixed in 4% paraformaldehyde after 30 min and processed for visualizing the injected serotonin or neurobiotin.

Embryos were collected in agar containing plates for 12 hours and incubated for another 36 hours at25°C. Viable first instar larvae were removed from cultures. The cuticles of unhatched (dead) embryos were dechorionated and mounted in Hoyer's medium and incubated for 24 hours at 50°C to digest soft tissues. Resulting cuticles were then viewed and photographed with dark field optics in a compound microscope (Nikon, Japan). For X-Gal staining embryos were fixed and stained with X-Gal using standard procedures. Both controls and experimental embryos were incubated in parallel for the same amount of time to allow for direct comparisons.

The different isolates from the lignin‐containing minimal medium were re‐streaked in different Petri dishes with the same medium to isolate one colony morphology. These new cultures were incubated for 5 days until medium clarification around the colonies was observed. Each colony was initially observed under a light microscope at 100× magnification (Nikon compound microscope) to confirm yeast morphology (i.e., oval/circular apiculate, elongated with a diameter of about 10 µm; see Figure A1 in Appendix A). Once the morphology of the different strains was confirmed, they were reintroduced into sterilized medium, either M9 or Sabouraud dextrose agar (SDA, Bioxon®) plus kanamycin (10 μg/mL), to obtain pure cultures. All yeast strains were grown overnight in liquid lignin degradation medium (LDM) at 35°C in a shaker bath (Lab‐Line) (150 rpm), and then mixed with 20% glycerol and stored at −80°C (Panasonic VIP PLUS ultralow‐freezer).

One to two milliliters of each sample (living, RNAlater preserved or Lugol's preserved) was placed on a large microscope slide. The sample was then diluted with ~1 mL of sterile, nuclease free water (Bioshop Canada Inc.) and examined under an inverted microscope (Leica) or a compound microscope (Nikon, with long working distance objectives) at 10x magnification. Individual cells were isolated through suction using 20–40 μl drawn-out disposable pipets, either with a Narishige micromanipulator or simple manual suction. This isolation procedure was modified from Throndsen (1978 ) by removing the use of Formvar film. The isolated cell with associated contaminants was transferred to a new water droplet of DNA nuclease free water. This isolation and transfer was repeated 2–5 times to remove any contaminants and/or preservative residue. Individual cells were then isolated for the final time and transferred to a 0.2 mL PCR tube containing 200 μL of 10% (w/v) Chelex® 100 solution (Richlen and Barber, 2005 (link)). The samples were stored from 1 to 51 days at 4°C in the dark until DNA extraction (Tables 1, 2; Supplement 1 in Appendix).

Red cell indices were determined by a complete blood count (CBC) analysis that was conducted using an automated cell counter (Beckman Coulter LH 780 Analyzer). Measured red cell indices included red cell count (RBC's), hemoglobin concentration ([Hb]), hematocrit (Hct), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW). Furthermore, morphological evaluation of erythrocytes was conducted by microscopic examination of Giemsa-stained blood films, using a compound microscope (Nikon, Japan).

The β-GLUCURONIDASE (GUS) expressing F. oxysporum 5176 strain was a kind gift from U. Schumann [32 (link)]. GUS staining was performed according to [33 (link)]. Analysis of GUS staining was performed on ten plants of both WT and med18, which were gently uprooted twelve days after infection and washed in distilled water before being vacuum infiltrated in staining solution and incubated at 37°C. GUS stained roots were imaged under a compound microscope (Nikon) and scored for the presence of GUS as a percentage of the total root length using ImageJ.