natgillin/translations-raw huggingface dataset:
Centrifuge for 7 minutes at 7000 g, remove the supernatant and resuspend the pellet in 75 µl 0,01M PB (see Appendix 7).
Centrifugiramo 7 min pri 7000 g, odstranimo supernatant in resuspendiramo peleto v 75 µl 0,01 M PB (glej Dodatek 7).
The remaining sample solution (49 µl) can be stored at –20 °C after addition of one volume of 96 % ethanol.
Preostalo raztopino vzorca (49 µl) lahko shranimo pri –20 °C, potem ko smo dodali 1 količino 96 % etanola.
In case the FISH assay requires repeating, remove the ethanol by centrifugation and add an equal volume of 0,01 PB (mix by vortexing).
Če je treba test FISH ponoviti, etanol odstranimo s centrifugiranjem in dodamo enako količino 0,01 PB (premešamo z vrtinčenjem).
Air-dry the slides (or on slide dryer at 37 °C) and fix them by flaming.
Stekelca posušimo na zraku (ali v sušilniku za stekelca pri 37 °C) in jih fiksiramo s plamenom.
At this stage the procedure may be interrupted and the hybridisation continued the following day.
Na tej točki je postopek mogoče prekiniti in nadaljevati s hibridizacijo naslednji dan.
Slides should be stored dust-free and dry at room temperature.
Stekelca je treba shraniti v suhem okolju brez prahu pri sobni temperaturi.
Hybridisation
Hibridizacija
Dehydrate the cells in a graded ethanol series of 50 %, 80 % and 96 % for one minute each.
Celice dehidriramo v stopenjski seriji etanola 50 %, 80 % in 96 %, v vsakem po eno minuto.
Air dry the slides in a slide-holder.
Stekelca pustimo, da se posušijo v držalu.
Prepare a moist incubation chamber by covering the bottom of an air-tight box with tissue or filter paper soaked in 1x hybmix (Appendix 7).
Pripravimo vlažno inkubacijsko komoro, tako da pokrijemo dno hermetične škatle z vpojnim ali filtrirnim papirjem, namočenim v 1x hybmix (Dodatek 7).
Pre-incubate the box in the hybridisation oven at 45 °C for at least 10 minutes.
Škatlo vsaj 10 minut predinkubiramo v hibridizacijski pečici pri 45 °C.
Apply 10 μl of hybridisation solution (Appendix 7) to eight windows (windows 1, 2, 4, 5, 6, 7, 9 and 10; see Fig 7.1) of each slide leaving the two centre windows (3 and 8) empty.
Nanesemo 10 μl hibridizacijske raztopine (Dodatek 7) na 8 okenc (okenca 1, 2, 4, 5, 6, 7, 9 in 10; glej sliko 7.1) vsakega stekelca, tako da sredinski okenci (3 in 8) ostaneta prazni.
Apply coverslips (24 × 24 mm) to the first and last four windows without trapping air.
Prva 4 in zadnja 4 okenca pokrijemo s krovnimi stekelci (24 × 24 mm), ne da bi ujeli zrak.
Place the slides in the pre-warmed moist chamber and hybridise for five hours in the oven at 45 °C in the dark.
Stekelca postavimo v vnaprej ogreto vlažno komoro in hibridiziramo 5 ur v pečici pri 45 °C v temi.
Prepare three beakers containing 1 l of Milli Q (molecular grade) water, 1 l of 1x hybmix (334 ml 3x hybmix and 666 ml Milli Q water) and 1 l of 1/8x hybmix (42 ml 3x hybmix and 958 ml Milli Q water).
Pripravimo 3 čaše, ki vsebujejo 1 l Milli Q (molekulske stopnje) vode, 1 l 1x hybmixa (334 ml 3x hybmixa in 666 ml Milli Q vode) in 1 l 1/8x hybmixa (42 ml 3x hybmixa in 958 ml Milli Q vode).
Pre-incubate each in a waterbath at 45 °C.
Vsako predinkubiramo v vodni kopeli pri 45 °C.
Remove the coverslips from the slides and place the slides in a slide holder.
S stekelc odstranimo krovna stekelca in stekelca damo v držalo.
Wash away excess probe by incubation for 15 minutes in the beaker with 1x hybmix at 45 °C.
Speremo odvečno sondo, tako da 15 minut inkubiramo v čaši z 1 × hybmix pri 45 °C.
Transfer the slide holder to 1/8 hybmix washing solution and incubate for a further 15 minutes.
Držalo prestavimo v pralno raztopino 1/8 hybmix in inkubiramo nadaljnjih 15 minut.
Dip the slides briefly in Milli Q water and place them on filter paper.
Stekelca na hitro potopimo v vodo Milli Q in jih damo na filtrirni papir.
Remove excess moisture by covering the surface gently with filter paper.
Odvečno vlago odstranimo, tako da površino narahlo prekrijemo s filtrirnim papirjem.
Pipette 5 to 10 μl of anti-fading mountant solution (e.g. Vectashield, Vecta Laboratories, CA, USA or equivalent) on each window and apply a large coverslip (24 × 60 mm) over the whole slide.
Na vsako okence odpipetiramo 5-10 μl raztopine prekrivnega pufra proti bledenju (npr. Vectashield, Vecta Laboratories, CA, USA ali ekvivalentni) in pokrijemo celo stekelce z velikim krovnim stekelcem (24 × 60 mm).
Reading the FISH test
Branje testa FISH:
Observe the slides immediately with a microscope fitted for epifluorescence microscopy at 630 or 1000 × magnification under immersion oil.
Stekelca takoj opazujemo z mikroskopom, opremljenim za epifluorescenčno mikroskopiranje, pri 630 ali 1000-kratni povečavi v imerzijskem olju.
With a filter suitable for fluorescein isothiocyanate (FITC) eubacterial cells (including most gram negative cells) in the sample are stained fluorescent green.
S filtrom, primernim za fluorescein izotiocianat (FITC), so evbakterijske celice (kar vključuje večino gram negativnih celic) v vzorcu obarvane fluorescentno zeleno.
Using a filter for tetramethylrhodamine-5-isothiocyanate, Cy3-stained cells of R. solanacearum appear fluorescent red.
S filtrom za tetrametilrodamin 5-izotiocianat so celice bakterije R. solanacearum, obarvane s Cy3, videti fluorescentno rdeče. Primerjamo morfolocijo celic z morfologijo pozitivnih kontrol.
Compare cell morphology with that of the positive controls.
Celice morajo biti svetlo fluorescentne in povsem obarvane.
Cells must be bright fluorescent and completely stained The FISH test (Section VI.A.7.) must be repeated if the staining is aberrant.
Če pri obarvanju pride do odstopanj, je treba test FISH ponoviti (oddelek VI.A.7.).
Observe for bright fluorescing cells with characteristic morphology of R. solanacearum in the test windows of the test slides (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main).
V testnih okencih testnih stekelc iščemo svetle fluorescenčne celice z značilno morfologijo bakterije R. solanacearum (glej spletno mesto http://forum.europa.eu.int/Public/irc/sanco/Home/main).
The fluorescence intensity must be equivalent or better than that of the positive control strain.
Intenziteta fluorescence mora biti enaka ali večja kot pri sevu pozitivne kontrole.
This may be the case when all slides in a batch show positive cells due to the contamination of buffer or if positive cells are found (outside of the slide windows) on the the slide coating.
Do tega lahko pride, če vsa stekelca v seriji kažejo pozitivne celice zaradi kontaminacije pufra ali če najdemo pozitivne celice (zunaj okenc stekelc) na zgornji plasti stekelca.
There are several problems inherent to the specificity of the FISH test.
S specifičnostjo testa FISH je povezanih več problemov.
Background populations of fluorescing cells with atypical morphology and cross reacting saprophytic bacteria with size and morphology similar to R. solanacearum may occur, although much less frequent as in the IF test, in potato heel end core and stem segment pellets.
V krompirjevih peletah stebelnih delov in stolonov se lahko – čeprav veliko redkeje kot pri testu IF – pojavijo populacije fluorescentnih celic ozadja z atipično morfologijo in navzkrižno reagirajoče saprofitske bakterije, ki so po velikosti in morfologiji podobne bakteriji R. solanacearum.
Consider only fluorescing cells with typical size and morphology.
Upoštevamo samo fluorescenčne celice značilne velikosti in morfologije.
Interpretation of the FISH test result:
Interpretacija rezultata testa FISH:
Valid FISH test results are obtained if bright green fluorescent cells of size and morphology typical of R. solanacearum are observed using the FITC filter and bright red fluorescent cells using the rhodamine filter in all positive controls and not in any of the negative controls.
Veljavne rezultate testa FISH dobimo, če opazimo celice, ki so po velikosti in morfologiji značilne za bakterijo R. solanacearum, in so s filtrom FITC videti fluorescentno svetlo zelene, s filtrom rodaminom pa fluorescentno svetlo rdeče. Opaziti jih moramo v vseh pozitivnih kontrolah in v nobeni od negativnih kontrol.
If bright fluorescing cells with characteristic morphology are found, estimate the average number of typical cells per microscope field and calculate the number of typical cells per ml of resuspended pellet (Appendix 4).
Če najdemo svetle fluorescentne celice z značilno morfologijo, določimo povprečno število značilnih celic na mikroskopsko polje in izračunamo število značilnih celic na ml resuspendirane pelete (Dodatek 4).
Samples with at least 5 × 103 typical cells per ml of resuspended pellet are considered potentially contaminated.
Vzorci z najmanj 5 × 103 značilnih celic na ml resuspendirane pelete veljajo za potencialno kontaminirane.
Further testing is required.
Potrebni so nadaljnji testi.
Samples with less than 5 × 103 typical cells per ml of resuspended pellet are considered negative.
Vzorci z manj kot 5 × 103 značilnih celic na ml resuspendirane pelete veljajo za negativne.
The FISH test is negative if bright red fluorescent cells with size and morphology typical of R. solanacearum are not observed using the rhodamine filter, provided that typical bright red fluorescent cells are observed in the positive control preparations when using the rhodamine filter.
Test FISH je negativen, če s filtrom rodaminom ne opazimo fluorescentnih živo rdečih celic z značilno velikostjo in morfologijo bakterije R. solanacearum, pod pogojem, da značilne živo rdeče fluoresecentne celice opazimo v preparatih pozitivne kontrole pri uporabi filtra rodamina.
ELISA tests
Testi ELISA
ELISA can only be used as an optional test in addition to IF, PCR or FISH due to a relatively low sensitivity of this test.
Test ELISA lahko uporabimo le kot dodatni test poleg testov IF, PCR ali FISH, saj ima ta test dokaj nizko občutljivost.
When DAS ELISA is used enrichment and the use of monoclonal antibodies are compulsory (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main).
Če uporabimo DAS ELISA, je obvezna obogatitev in uporaba monoklonalnih protiteles (glej spletno mesto http://forum.europa.eu.int/Public/irc/sanco/Home/main).
Enrichment of the samples before using ELISA may be useful in order to increase the sensitivity of the test, but it can fail due to competition by other organisms in the sample.
Obogatitev vzorca pred uporabo testa ELISA je lahko koristna, saj poveča občutljivost testa, vendar lahko spodleti zaradi tekmovanja z drugimi organizmi v vzorcu.
Note: Use an validated source of antibodies to R. solanacearum (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main) It is recommended that the titre is determined for each new batch of antibodies.
Opomba: Uporabimo potrjen vir protiteles za R. solanacearum (glej spletno mesto http://forum.europa.eu.int/Public/irc/sanco/Home/main). Priporočljivo je, da se titer določi za vsako novo serijo protiteles.
The titre is defined as the highest dilution at which optimum reaction occurs when testing a suspension containing 105 to 106 cells per ml of the homologous strain of R. solanacearum and using appropriate secondary antibody conjugates according to the manufacturer’s recommendations.
Titer je definiran kot najvišja razredčina, pri kateri pride do optimalne reakcije, če testiramo suspenzijo, ki vsebuje 105 do 106 celic na ml homolognega seva bakterije R. solanacearum, z ustreznimi konjugati sekundarnih protiteles v skladu z navodili proizvajalca.
During testing, the antibodies should be used at a working dilution close to or at the titre of the commercial formulation.
Med testiranjem protitelesa uporabljamo pri delovni razredčini blizu titra ali v vrednosti titra komercialne formulacije.
Determine the titre of the antibodies on a suspension of 105 to 106 cells per ml of the homologous strain of R. solanacearum.
Titer protiteles določimo na suspenziji 105 do 106 celic na ml homolognega seva bakterije R. solanacearum.
Include a sample extract that previously tested negative for R. solanacearum and a suspension of a non-cross reacting bacterium in phosphate buffered saline (PBS) as negative controls.
Kot negativno kontrolo vključimo ekstrakt vzorca, ki je bil že testiran kot negativen za R. solanacearum, in suspenzijo ne-navzkrižno reagirajoče bakterije v fosfatnem pufru s soljo (PBS).
As positive control use aliquots of sample extract, that previously tested negative, mixed with 103 to 104 cells per ml of R. solanacearum biovar 2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857, see Appendix 2 A and B).
Kot pozitivno kontrolo uporabimo alikvote ekstrakta vzorca, ki so bili že testirani kot negativni, pomešane z 103 do 104 celicami na ml bakterije R. solanacearum biovar 2 (npr. sev NCPPB 4156 = PD 2762 = CFBP 3857, glej Dodatek 2 A in B).
For comparison of results on each plate use a standard suspension of 105 to 106 cells per ml in PBS of R. solanacearum.
Za primerjavo rezultatov na obeh ploščah uporabimo standardno suspenzijo 105 do 106 celic na ml v PBS bakterije R. solanacearum.
Ensure positive controls are well separated on the microtitre plate from the sample(s) under test.
Poskrbimo, da so pozitivne kontrole na mikrotitrski ploščici zagotovo ločene od testiranih vzorcev.
Standardised positive and negative control materials available for use with this test are listed in Appendix 3 A.
Standardizirani pozitivni in negativni kontrolni materiali, ki so na voljo za uporabo s tem testom, so navedeni v Dodatku 3, točka A.
Two ELISA protocols have been validated.
Potrjena sta dva protokola za test ELISA:
Indirect ELISA (Robinson Smith et al., 1995)
Posredni ELISA (Robinson Smith in sod., 1995)
Use 100 to 200 µl aliquots of sample extract.
Uporabimo 100-200 µl alikvota ekstrakta vzorca.
(Heating at 100 °C for four minutes in a waterbath or heating block may reduce non-specific results in some cases).
(Segrevanje 4 minute pri 100 °C v vodni kopeli ali grelni enoti lahko v nekaterih primerih zmanjša število nespecifičnih rezultatov).
Add an equal volume of double strength coating buffer (Appendix 4) and vortex.
Dodamo enako količino karbonatnega premaznega pufra z dvojno koncentracijo (Dodatek 4) in močno premešamo.
Apply 100 µl aliquots to each of at least two wells of a microtitre plate (e.g. Nunc-Polysorp or equivalent) and incubate for one hour at 37 °C or overnight at 4 °C.
V vsako od vsaj dveh vdolbinic na mikrotitrski ploščici odmerimo 100 µl alikvote (npr. Nunc-Polysorp ali ekvivalentni). Inkubiramo eno uro pri 37 °C ali čez noč pri 4 °C.
Flick out the extracts from the wells.
Otresemo ekstrakte iz vdolbinic.
Wash the wells three times with PBS-Tween (Appendix 4), leaving the last washing solution in the wells for at least five minutes.
Vdolbinice trikrat speremo z raztopino PBS-Tween (Dodatek 4), pri čemer zadnjo izpiralno raztopino pustimo v vdolbinicah vsaj pet minut.
Prepare the appropriate dilution of antibodies against-R. solanacearum in blocking buffer (Appendix 4).
Pripravimo ustrezno razredčino protiteles za bakterijo R. solanacearum v blokirnem pufru (Dodatek 4).
For validated commercial antibodies, use the recommended dilutions (usually twice as concentrated as the titre).
Za veljavna komercialna protitelesa uporabimo priporočene razredčine (običajno dvakrat bolj koncentrirane kot titer).
Add 100 µl to each well and incubate for one hour at 37 °C.
V vdolbinice nanesemo po 100 µl razredčine in inkubiramo eno uro pri 37 °C.
Flick out the antibody solution from the wells and wash as before (4).
Otresemo raztopino iz vdolbinic. Vdolbinice speremo kakor prej (4).
Prepare the appropriate dilution of secondary antibody-alkaline phosphatase conjugate in blocking buffer.
Pripravimo ustrezno razredčino konjugata alkalne fosfataze sekundarnih protiteles v blokirnem pufru.
Flick out conjugated antibody from wells and wash as before (4).
Otresemo konjugat protiteles iz vdolbinic. Vdolbinice speremo kakor prej (4).
Add 100 µl alkaline phosphatase substrate solution (Appendix 4) to each well.
V vsako vdolbinico dodamo 100 µl raztopine substrata alkalne fosfataze (Dodatek 4).
Incubate in the dark at ambient temperature and read absorbance at 405 nm at regular intervals within 90 minutes.
Inkubiramo v temnem prostoru pri sobni temperaturi in odčitavamo absorbanco pri 405 nm v rednih presledkih v 90 minutah.
Prepare the appropriate dilution of anti-R. solanacearum polyclonal immunoglobulins in coating buffer pH 9.6 (Appendix 4).
Pripravimo ustrezno razredčino poliklonalnih imunoglobulinov z delovanjem proti bakteriji R. solanacearum v premaznem pufru pH 9,6 (Dodatek 4).
Add 200 µl to each well.
V vsako vdolbinico nanesemo 200 µl.
Incubate at 37 °C for four to five hours or at 4 °C for 16 hours.
Inkubiramo 4-5 ur pri 37 °C ali 16 ur pri 4 °C.
Wash the wells three times with PBS-Tween (Appendix 4).
Vdolbinice trikrat izperemo z raztopino PBS-Tween (Dodatek 4).
Add 190 µl of sample extract to at least two wells.
Vsaj v 2 vdolbinici nanesemo 190 µl ekstrakta vzorca.
Also add positive and negative controls in two wells each per plate.
Na vsaki ploščici dodamo tudi pozitivno in negativno kontrolo, vsako v po dve vdolbinici.
Incubate for 16 hr at 4 °C.
Inkubiramo 16 ur pri 4 °C.
Prepare an appropriate dilution of R. solanacearum-specific monoclonal antibodies in PBS (Appendix 4) also containing 0,5 % bovine serum albumin (BSA) and add 190 µl to each well.
Pripravimo ustrezno razredčino za bakterijo R. solanacearum specifičnih monoklonalnih protiteles v PBS (Dodatek 4), ki vsebuje tudi 0,5 % bovinega seruma albumina (BSA), in dodamo 190 µl v vsako vdolbinico.
Incubate at 37 °C for two hours.
Inkubiramo 2 uri pri 37 °C.
Prepare an appropriate dilution of anti-mouse immunoglobulins conjugated with alkaline phosphatase in PBS.
Pripravimo ustrezno razredčino antimišjih imunoglobulinov, konjugiranih z alkalno fosfatazo v PBS.
Prepare an alkaline phosphatase substrate solution containing 1 mg p-nitrophenyl phosphate per ml of substrate buffer (Appendix 4).
Pripravimo raztopino substrata alkalne fosfataze, ki vsebuje 1 mg p-nitrofenil fosfata na ml pufra substrata (Dodatek 4).
Incubate in the dark at ambient temperature and read absorbance at 40 nm at regular intervals within 90 minutes.
Inkubiramo v temnem prostoru pri sobni temperaturi in odčitavamo absorbanco pri 405 nm v rednih presledkih v 90 minutah.
Interpretation of ELISA test results:
Interpretacija rezultatov testa ELISA:
The ELISA test is negative if the average optical density (OD) reading from duplicate sample wells is < 2x OD of that in the negative sample extract control well, providing the OD for the positive controls are all above 1,0 (after 90 minutes incubation with the substrate) and are greater than twice the OD obtained for negative sample extracts.
Test ELISA je negativen, če je povprečni odčitek optične gostote (OD) iz ponovitev vzorčnih vdolbinic < 2x vrednost OD v vdolbinici ekstrakta vzorca negativne kontrole, pod pogojem, da so OD pozitivnih kontrol vsi nad 1,0 (po 90 minutah inkubacije s substratom) in so več kot dvakrat večji od OD, ki je bil ugotovljen za negativne ekstrakte vzorca.
The ELISA test is positive if the average OD readings from duplicate sample wells is > 2x OD in the negative sample extract well provided that OD readings in all negative control wells are < 2x those in the positive control wells.
Test ELISA je pozitiven, če so povprečni odčitki OD iz ponovitev vzorčnih vdolbinic > 2x vrednosti OD v vdolbinici ekstrakta vzorca negativne kontrole, pod pogojem, da so odčitki OD v vseh vdolbinicah negativne kontrole < 2x vrednosti odčitkov v vdolbinicah pozitivne kontrole.
Negative ELISA readings in positive control wells indicate that the test has not been performed correctly or that it has been inhibited.
Negativni odčitki ELISA v vdolbinicah pozitivne kontrole kažejo, da test ni bil opravljen pravilno ali da je bil inhibiran.
Positive ELISA readings in negative control wells indicate that cross-contamination or non-specific antibody binding has occurred.
Pozitivni odčitki ELISA v vdolbinicah negativne kontrole kažejo, da je prišlo do navzkrižne kontaminacije ali nespecifične vezave protiteles.
Note: Preliminary testing with this method should permit reproducible detection of 103 to 104 colony-forming units of R. solanacearum per ml added to sample extracts that previously tested negative (preparation see Appendix 3).
Opomba: Predhodno testiranje s to metodo mora omogočati ponovljivo detekcijo 103 do 104 enot bakterije R. solanacearum, ki tvorijo kolonije, na ml dodano ekstraktom vzorcev, ki so bili prej testirani kot negativni (priprava glej Dodatek 3).
Highest sensitivity of detection can be expected when using freshly prepared sample extract and optimal growth conditions.
Največjo občutljivost detekcije lahko pričakujemo, če uporabljamo sveže pripravljen ekstrakt vzorca in imamo optimalne pogoje za rast.
However, the method can be successfully applied to extracts that have been stored under glycerol at –68 to –86 °C.
Metodo pa lahko uspešno uporabimo tudi za ekstrakte, ki so bili shranjeni pod glicerolom pri –68 do –86 °C.
The following protocol is based upon Janse (1988):
Spodnji protokol temelji na Janse (1988):
Use 10 test plants of a susceptible tomato cultivar (e.g. Moneymaker or cultivar with equivalent susceptibility as determined by the testing laboratory) at the third true leaf stage for each sample.
Za vsak vzorec uporabimo 10 testnih rastlin - dovzetnih kultivarjev paradižnika (npr. Moneymaker ali kultivar z ekvivalentno dovzetnostjo, kot določi testni laboratorij) v fazi tretjega pravega lista.
Symptoms have been show to be less severe and to develop more slowly in eggplant.
Simptomi pri jajčevcu so se pokazali kot blažji in se razvijejo počasneje.
Where possible, it is therefore recommended to use tomato seedlings.
Kjer je mogoče, je zato priporočljiva uporaba paradižnikovih sadik.
Distribute 100 µl of sample extract between the test plants.
Med testne rastline porazdelimo 100 µl ekstrakta vzorca.
Syringe inoculation
Inokulacija z brizgalko
Inoculate the plant stems just above the cotyledons using a syringe fitted with a hypodermic needle (not less than 23G).
Inokuliramo v steblo tik nad kličnima listoma z brizgalko, na kateri je nameščena igla (ne manj kot 23G).
Distribute the sample between the test plants.
Vzorec porazdelimo med testne rastline.
Slit inoculation
Inokulacija v zarezo
Holding the plant between two fingers, pipette a drop (approximately 5 - 10 µl) of the suspended pellet on the stem between the cotyledons and the first leaf.
Rastlino držimo med dvema prstoma in odpipetiramo kapljico (približno 5-10 µl) suspendirane pelete na steblo med kličnima listoma in prvim listom.
Using a sterile scalpel, make a diagonal slit, about 1.0 cm long and approximately 2/3 of the stem thickness deep, starting the cut from the pellet drop.
S sterilnim skalpelom naredimo diagonalno zarezo, dolgo približno 1,0 cm in globoko približno 2/3 debeline stebla. Zarežemo ob kapljici pelete.
Seal the cut with sterile vaseline from a syringe.
Zarezo zalepimo s sterilnim vazelinom iz brizge.
Inoculate by the same technique, five seedlings with an aqueous suspension of 105 to 106 cells per ml prepared from a 48 hr culture of a virulent biovar 2 strain of R. solanacearum as a positive control and with pellet buffer (Appendix 4) as negative control.
Inokuliramo z isto tehniko, 5 sadik z vodno suspenzijo 105 do 106 celic na ml, pripravljeno iz 48 urne kulture virulentnega biovarja 2 seva bakterije R. solanacearum kot pozitivno kontrolo, in s peletnim pufrom (Dodatek 4) kot negativno kontrolo.
Separate positive and negative control plants from the other plants to avoid cross-contamination.
Rastline pozitivne in negativne kontrole ločimo od drugih rastlin, da ne bi prišlo do navzkrižne kontaminacije.
Grow the test plants in quarantine facilities for up to four weeks at 25 to 30 °C and high relative humidity with appropriate watering to prevent waterlogging or wilting through water deficiency.
Testne rastline gojimo v karanteni do 4 tedne pri 25-30 °C in v visoki relativni vlažnosti in primerno zalivamo, da ne bi prišlo do zasičenja z vodo oziroma do venenja zaradi pomanjkanja vode.