Bodo saltans culture protocol v1

Medium recipe: ATCC medium: 802 Sonneborn's Paramecium medium Solution 1 Rye grass Cerophyll: Cerophyll*...................2.5 g Distilled water..............1.0 L Add cerophyll to distilled water and boil for 5 minutes. Add 100 ml distilled water to compensate for evaporation. Filter through Whatman #1 filter paper and add 0.5 g Na2HPO4. Autoclave for 15 minutes at 121C. saltans food (K. pneumoniae, or E. coli). I used only the K. pneumoniae so far Agar Medium for Klebsiella pneumoniae ATCC-BAA-1705: Agar........................20.0 g Yeast extract................4.0 g Glucose......................0.16 g Distilled water............800.0 ml Dispense in 5 ml amounts. Autoclave for 25 minutes at 121C. Slant. Bacterium, grown on solution 2, is added to solution 1 (Just add very little, few colonies) and incubated at 30C for 24 hours prior to inoculation with Bodo saltans. Cerophyl powder that works best for the saltans is the powder from Pines. CultureMaintenance: Preparethe bacterizedBodo saltans medium as described above. InoculateaT25tissuecultureflask(50ml) containing20 to 25 ml of fresh medium with 1 to 2 ml from Bodo culture thatisatornearpeakdensity Incubatehorizontally at18 to 22°C(room temperature can work fine) withcapscrewedonnot very tightly. 4. Subcultureevery7 to 10days. I usually subculture 3 to 4 flasks every week Cryopreservation: HarvestandPreservation: Harvestcellsfromaculturethatisatpeakdensitybycentrifugationat800xgfor5min. 2. Adjusttheconcentrationofcellsto2x106to107 /mLinfreshmedium (Important step, even for transfection). Preparea20%(v/v)solutionofsterileDMSOinfresh Bodomedium. Add2.0mLofDMSOtoanicecoldtube PlacethetubeoniceandallowtheDMSOtosolidify(~5min)andthenadd8.0mLoficecold medium. InvertseveraltimestodissolvetheDMSO. Allowtowarmtoroomtemperature MixthecellpreparationandtheDMSOinequalportions.Thus,thefinalconcentrationwillbe106 to107and10%(v/v)DMSO.ThetimefromthemixingofthecellpreparationandDMSOstocksolution before thefreezingprocessisbegunshouldbenolessthan15minandnolongerthan30min. Dispensein0.5mLaliquotsinto1.0mLto2.0mLsterileplasticscrewcappedcryules(specialplastic vialsforcryopreservation). Placethevialsinacontrolledratefreezingunit.Fromroomtemperaturecoolat1°C/minto -40°C.If thefreezingunitcancompensatefortheheatoffusion,maintainrateat 1°C/minthroughtheheatoffusion.At- 40°Cplungeintoliquidnitrogen.Alternatively,placethevialsinNalgene1°Cfreezing apparatus.Placetheapparatusat- 80°Cfor1.5to2hoursandthenplungeampulesintoliquid nitrogen.(Thecoolingrateinthisapparatusisapproximately1°C/min.) Toestablishaculturefromthefrozenstateplaceanampuleinawaterbathsetat+35°C.Immersethe ampuletoaleveljustabovethesurfaceofthefrozenmaterial.Donotagitatetheampule. Immediatelyafterthawing,donotleaveinthewaterbath,asepticallyremovethecontentsofthe ampuleandinoculateaT25tissuecultureflaskcontaining10mLofBodomediumbacterized withKlebsiellapneumoniaesubsp.pneumoniae(ATCC®700831). Incubatehorizontallywiththecapscrewedontightlyat22°C