Eclipse te300 inverted fluorescent microscope

To visualize X. innexi within nematodes, we engineered it to express the green-fluorescent protein. pBSL118, a mini Tn5-GFP donor plasmid was used in combination with S17–1 λpir from pUX-BF13, a Tn5 helper strain, to perform GFP conjugations [30 , 107 (link), 108 (link)]. Briefly, donor, recipient, & helper strain were streaked for single colonies on LB + pyruvate agar plates and grown for 24–48 h at 30 °C without exposure to light. Single colonies were picked grown overnight at 30 °C in liquid LB, with supplementation with 300 μM diaminopimelic acid for the helper and donor strain. Cells were subcultured into fresh medium and grown for an additional 4 h after which 900 μl of X. innexi (HGB1681 or HGB1997) and 300 μl each of the helper and donors strains were pelleted separately, washed and re-suspended at their original volumes. The three strains were then mixed together, and plated as a single spot onto a permissive LB pyruvate + DAP plate. After 24 h incubation an inoculation loop was dragged through the spot and the collected cells were re-suspended in LB and plated onto a selective LB pyruvate with ampicillin and kanamycin. After 24–48 h incubation at 30 °C the resulting colonies were analyzed for the expression of GFP with a Nikon Eclipse TE300 inverted fluorescent microscope.

Cells were harvested at 72 h post induction of viral replication. All staining was performed on ice in PBS, 1% BSA, and 0.1% sodium azide. Cells were washed twice with PBS then stained with the K8.1 primary antibody at 1:100 in 5% FBS for 1 h. Cells were washed three times with PBS and stained with the secondary antibody anti-mouse Alexa 680 (Molecular Probes) at 1:1000 for 30 min then washed. Microscopy was performed with the Nikon Eclipse TE300 inverted fluorescent microscope equipped with filter sets TE300 FITC, TE300 Texas Red HYQ, and TE300 633. Images were acquired with a Photometrics CoolSnap cf digital camera and MetaVue imaging software at 20X amplification. Cells were analyzed by flow cytometry on an LSRII (Beckman-Coulter).