Uplan fln 40 0.75 ph2

To investigate saliva generated bacterial aggregation, 7×10⁸ cfu of DL1 were resuspended in 1 ml human saliva (processing of saliva was performed as described above) and incubated in a 2 ml reaction tube at 37°C on a rocking table, promoting horizontal movement from an angle of ±15° at 20 rpm (Barnstead Thermolyne Vari-Mix) which yielded an efficient mixing of the bacteria in saliva. At various time points 50 μl aliquots were sampled and saliva induced cellular aggregation was determined by comparing the counts of detectable cfu with or without 5 s low-power ultrasonic treatment (VibraCell VC-50; SONICS^®).
Microscopic examination was performed directly on 10 μl aliquots using UPlan FLN 100×/1.30 Oil Ph3 on BX51 equipped with DP72 CCD camera (Olympus). Bacterial aggregate diameter was determined with UPlan FLN 40×/0.75 Ph2 and calibrated cellSens^® digital imaging software Version 1.3 (Olympus) calculating the arithmetic mean of a minimum and a maximum measurement for each observed object.

To visualize the planktonic oral microbial flora 1h after toothbrushing, 1ml of fresh human saliva was incubated with LIVE/DEAD^® BacLight^™ Bacterial Viability Kit containing SYTO^®9 and propidium iodide in a final concentration of 1.67μM (Invitrogen^™) and 1μg/ml Wheat Germ Agglutinin Alexa Fluor^® 350 conjugated (Invitrogen^™) for 10min at 37°C. Mucous fraction obtained by 10min centrifugation at 4500×g was examined using UPlan FLN 40×/0.75 Ph2 and UPlan FLN 100×/1.30 Oil Ph3 on BX51 equipped with BX-URA2 reflected fluorescence system and X-Cite^® 120W metal halide lamp (Olympus). Separate images were taken with DAPI-1160A, FITC-3540B, and TRITC-A BrightLine^® fluorescence filters (Semrock), as well as U-PCD2 phase contrast condenser using DP72 CCD camera and merged with cellSens^® digital imaging software Version 1.3 (Olympus).