natgillin/translations-raw huggingface dataset:
Cover the test windows with the dilution of FITC conjugate used to determine the titre.
Testna okenca prekrijemo z razredčino konjugata FITC, uporabljenega za določitev titra.
The volume of conjugate applied on the windows must be identical to the volume of antibody applied.
Količina konjugata, nanesenega na okenca, mora biti enaka količini nanesenih protiteles.
Shake the droplets of conjugate off the slide.
S stekelca otresemo kapljice konjugata.
Rinse and wash as before (5.3.3).
Splaknemo in operemo kot prej (5.3.3).
Carefully remove excess moisture.
Previdno odstranimo odvečno vlago.
Pipette 5 - 10 µl of 0,1M phosphate-buffered glycerol (Appendix 4) or a commercially antifading mountant on each window and apply a coverslip.
Na vsako okence odpipetiramo 5–10 µl 0,1 M glicerola s fosfatnim pufrom (Dodatek 4) ali podobnim prekrivnim pufrom proti bledenju in pokrijemo s krovnim stekelcem.
Reading the IF test:
Branje IF-testa:
Examine test slides on an epifluorescence microscope with filters suitable for excitation of FITC, under oil or water immersion at a magnification of 500-1000.
Stekelca pregledamo pod epifluorescenčnim mikroskopom s filtri, primernimi za vzbujanje FITC, v imerzijskem olju ali vodi pri povečavi 500–1000.
Scan windows across two diameters at right angles and around the perimeter.
Okenca pregledamo vzdolž dveh pravokotnih premerov in po obodu.
For samples showing no or low number of cells observe at least 40 microscope fields.
Pri vzorcih, ki ne kažejo nobenih celic ali le malo celic, opazujemo vsaj 40 mikroskopskih polj.
Check the positive control slide first.
Najprej pregledamo stekelce pozitivne kontrole.
Cells must be bright fluorescent and completely stained at the determined antibody titre or working dilution.
Celice morajo biti svetlo fluorescenčne in povsem obarvane pri določenem titru protiteles oziroma delovne razredčine.
The IF test (Section VI.A.5.) must be repeated if the staining is aberrant.
Če pri obarvanju pride do odstopanj, je treba IF test (oddelek VI.A.5.) ponoviti.
Observe for bright fluorescing cells with characteristic morphology of R. solanacearum in the test windows of the test slides (see website 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 to the positive control strain at the same antibody dilution.
Intenziteta fluorescence mora biti enaka kot pri sevu pozitivne kontrole pri enaki razredčini protiteles.
Cells with incomplete staining or with weak fluorescence must be disregarded.
Celice z nepopolnim obarvanjem ali šibko fluorescenco se ne upoštevajo.
If any contamination is suspected the test must be repeated.
Če sumimo kontaminacijo, moramo test ponoviti.
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 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 immunofluorescence test.
S specifičnostjo testa imunofluorescence 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 are likely to occur in potato heel end core and stem segment pellets.
V krompirjevih peletah stebelnih delov in stolonov se bodo verjetno pojavile 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 at the titre or working dilution of the antibodies as in 5.3.
Upoštevamo samo fluorescentne celice tipične velikosti in morfologije pri titru oziroma delovni razredčini protiteles, kot je opisano v 5.3.
Interpretation of the IF reading:
Interpretacija rezultata IF testa:
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 5).
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 5).
The IF reading is positive for samples with at least 5 × 103 typical cells per ml of resuspended pellet.
Rezultat testa IF je pozitiven pri vzorcih, ki imajo vsaj 5 × 103 značilnih celic na ml resuspendirane pelete.
The sample is considered potentially contaminated and further testing is required.
Vzorec velja za potencialno kontaminiranega in potrebni so nadaljnji testi.
The IF reading is negative for samples with less than 5 × 103 cells per ml of resuspended pellet and the sample is considered negative.
Rezultat IF testa je negativen pri vzorcih z manj kot 5 × 103 celic na ml resuspendirane pelete, in vzorec velja za negativnega.
Further testing is not required.
Nadaljnji testi niso potrebni.
PCR tests
Testi PCR
When the PCR test is used as the principal screening test and found to be positive, isolation or IF must be performed as a second compulsory screening test.
Če test PCR uporabljamo kot glavni presejalni test in je odčitek pozitiven, je treba kot drugi obvezni presejalni test opraviti izolacijski test ali IF test.
When PCR is used as the second screening test and found to be positive, further testing according to the flow scheme is required to complete the diagnosis.
Če test PCR uporabljamo kot drugi presejalni test in je odčitek pozitiven, je za zaključek diagnoze potrebno nadaljnje testiranje glede na diagram poteka.
Full exploitation of this method as principal screening test is only recommended when specialised expertise has been acquired.
Popolno izkoriščanje te metode kot osnovnega presejalnega testa se priporoča samo, če smo pridobili strokovno usposobljenost.
Note: Preliminary testing with this method should permit reproducible detection of 103 to 104 cells of R. solanacearum per ml added to sample extracts which previously tested negative.
Opomba: Predhodno testiranje s to metodo mora omogočati ponovljivo detekcijo 103 do 104 celic bakterije R. solanacearum na ml, dodanih ekstraktom vzorcev, ki so bili prej testirani kot negativni.
Optimisation experiments may be required to achieve maximum levels of sensitivity and specificity in all laboratories.
Če želimo doseči največjo stopnjo občutljivosti in specifičnosti v vseh laboratorijih, bodo morda potrebni eksperimenti za optimizacijo.
Use validated PCR reagents and protocols (see Appendix 6).
Uporabimo potrjene reagente za PCR in protokole (glej Dodatek 6).
Preferably select a method with an internal control.
Po možnosti izberemo metodo z notranjo kontrolo.
Use appropriate precautions to avoid contamination of sample with target DNA.
Z ustreznimi previdnostnimi ukrepi se izogibamo kontaminaciji vzorca s ciljno DNK.
The PCR test should be performed by experienced technicians, in dedicated molecular biology laboratories, in order to minimise the possibility of contamination with target DNA.
Test PCR smejo izvajati le izkušeni strokovnjaki v temu namenjenih laboratorijih za molekularno biologijo, da bi zmanjšali možnost kontaminacije s ciljno DNK.
Negative controls (for DNA extraction and PCR procedures) should always be handled as final samples in the procedure, to make evident whether any carry over of DNA has occurred.
Negativne kontrole (za ekstrakcijo DNK in postopke PCR) je treba vedno obravnavati kot končne vzorce v postopku, ki pokaže, ali je prišlo do prenosa DNK.
The following negative controls should be included in the PCR test:
V test PCR vključimo naslednje negativne kontrole:
Sample extract that previously tested negative for R. solanacearum,
ekstrakt vzorca, ki je bil predhodno testiran negativno za R. solanacearum,
Buffer controls used for extracting the bacterium and the DNA from the sample,
pufrske kontrole za ekstrakcijo bakterije in DNK iz vzorca,
PCR-reaction mix.
reakcijsko zmes PCR.
The following positive controls should be included:
Vključimo naslednje pozitivne kontrole:
Aliquots of resuspended pellets to which R. solanacearum has been added (preparation see Appendix 3 B).
alikvote resuspendiranih pelet, ki jim je bila dodana bakterija R. solanacearum (za pripravo glej Dodatek 3B),
A suspension of 106 cells per ml of R. solanacearum in water from a virulent isolate (e.g. NCPPB 4156 = PD 2762 = CFBP 3857; see Appendix 3 B).
suspenzijo 106 celic na ml bakterije R. solanacearum v vodi iz virulentnega izolata (npr. NCPPB 4156 = PD 2762 = CFBP 3857; glej Dodatek 3B),
If possible use also DNA extracted from positive control samples in the PCR test.
če je mogoče, pri PCR testu uporabimo tudi DNK, ekstrahirano iz pozitivnih kontrolnih vzorcev.
To avoid potential contamination prepare positive controls in a separate environment from samples to be tested.
Da ne bi prišlo do kontaminacije, pripravimo pozitivno kontrolo v okolju, ločenem od vzorcev, ki jih nameravamo testirati.
Sample extracts should be as free as possible from soil.
Ekstrakti vzorcev morajo biti čim bolj očiščeni zemlje.
It could therefore, in certain cases, be advisible to prepare extracts from washed potatoes if PCR protocols are to be used.
Zato bi bilo v nekaterih primerih morda priporočljivo, da pripravimo ekstrakte iz opranih krompirjev, če nameravamo uporabiti protokole PCR.
Standardized positive and negative control material available for use with this test are listed in Appendix 3).
Standardizirani pozitivni in negativni kontrolni materiali, ki so na voljo za uporabo s tem testom, so navedeni v Dodatku 3.
Use positive and negative control samples as described above (see Appendix 3).
Uporabimo vzorce pozitivne in negativne kontrole, kot je opisano zgoraj (glej Dodatek 3).
A variety of methods are available for purification of target DNA from complex sample substrates, thus removing inhibitors of PCR and other enzymatic reactions and concentrating target DNA in the sample extract.
Za prečiščevanje ciljne DNK iz kompleksnih vzorčnih medijev je na voljo vrsta metod, s katerimi odstranimo inhibitorje za PCR in druge encimske reakcije in koncentriramo ciljno DNK v ekstraktu vzorca.
The following method has been optimised for use with the validated PCR methods shown in Appendix 6.
Naslednja metoda je bila optimirana za uporabo z potrjenimi metodami PCR, prikazanimi v Dodatku 6.
Pipette 220 µl of lysis buffer (100 mM NaCl, 10 mM Tris-HCl [pH 8,0], 1 mM EDTA [pH 8,0]) into a 1,5 ml Eppendorf tube.
Odpipetiramo 220 µl lizirnega pufra (100 mM NaCl, 10 mM Tris-HCl [pH 8,0], 1 mM EDTA [pH 8,0]) v 1,5 ml Eppendorfovo epruveto.
Add 100 µl sample extract and place in a heating block or water bath at 95 °C for 10 min.
Dodamo 100 µl ekstrakta vzorca in za 10 min postavimo v grelno enoto ali vodno kopel pri 95 °C.
Put tube on ice for 5 min.
Epruveto za 5 min damo na led.
Add 80 µl Lysozyme stock solution (50 mg Lysozyme per ml in 10 mM Tris HCl, pH 8,0) and incubate at 37 °C for 30 min.
Dodamo 80 µl osnovne raztopine lizocima (50 mg lizocima na ml v 10 mM Tris HCl, pH 8,0) in 30 min inkubiramo pri 37 °C.
Add 220 µl of Easy DNA® solution A (Invitrogen), mix well by vortexing and incubate at 65 °C for 30 min.
Dodamo 220 µl Easy DNA® raztopine A (Invitrogen), dobro premešamo z vrtinčenjem in inkubiramo 30 min pri 65 °C.
Add 100 µl of Easy DNA® solution B (Invitrogen), vortex vigorously until the precipitate runs freely in the tube and the sample is uniformly viscous.
Dodamo 100 µl Easy DNA® raztopine B (Invitrogen) in močno vrtinčimo, da oborina v epruveti prosto teče ter da vzorec postane enakomerno viskozen.
Add 500 µl of chloroform and vortex until the viscosity decreases and the mixture is homogeneous.
Dodamo 500 µl kloroforma in vrtinčimo, dokler se viskoznost ne zmanjša in mešanica postane homogena.
Centrifuge at 15000 g for 20 min at 4 °C to separate phases and form the interphase.
Centrifugiramo pri 15000 g 20 min pri 4 °C, da ločimo faze in dosežemo interfazo.
Add 1 ml of 100 % ethanol (–20 °C) vortex briefly and incubate on ice for 10 min.
Dodamo 1 ml 100 % etanola (–20 °C), na kratko vrtičimo in za 10 min. inkubiramo na ledu.
Centrifuge at 15000 g for 20 min at 4 °C and remove ethanol from pellet.
Centrifugiramo pri 15000 g 20 min pri 4° C in iz pelete odstranimo etanol.
Add 500 µl 80 % ethanol (–20 °C) and mix by inverting the tube.
Dodamo 500 µl 80 % etanola (–20 °C) in premešamo, tako da obrnemo epruveto.
Centrifuge at 15000 g for 10 min at 4 °C, save the pellet and remove ethanol.
Centrifugiramo pri 15000 g 10 min pri 4 °C, shranimo peleto in odstranimo etanol.
Allow the pellet to dry in air or in a DNA speed vac.
Peleto pustimo, da se posuši na zraku ali v vakuumski centrifugi.
Resuspend the pellet in 100 µl sterile UPW and leave at room temperature for at least 20 minutes.
Peleto resuspendiramo v 100 µl sterilne ultra čiste vode in vsaj 20 minut pustimo pri sobni temperaturi.
Store at –20 °C until required for PCR.
Hranimo pri –20 °C, dokler je potrebno za PCR.
Spin down any white precipitate by centrifugation and use 5 µl of the supernatant containing DNA for the PCR.
Morebitno belo oborino odvrtimo s centrifugiranjem in uporabimo 5 µl supernatanta, ki vsebuje DNK za PCR.
Other DNA extraction methods, e.g. Qiagen DNeasy Plant Kit, could be applied providing that they are proven to be equally as effective in purifying DNA from control samples containing 103 to 104 pathogen cells per ml.
Druge metode za ekstrakcijo DNK, npr. Qiagen DNeasy Plant Kit, lahko uporabimo, če so dokazano enako učinkovite za prečiščevanje DNK iz kontrolnih vzorcev, ki vsebujejo 103 do 104 patogenih celic na ml.
Prepare test and control templates for PCR according to the validated protocols (Section VI.A.6.).
Po potrjenih protokolih pripravimo testne in kontrolne matrice za PCR (oddelek VI.A.6).
Prepare one decimal dilution of sample DNA extract (1:10 in UPW).
Pripravimo eno desetkratno razredčino vzorčnega ekstrakta DNK (1:10 v ultra čisti vodi).
Prepare the appropriate PCR reaction mix in a contamination-free environment according to the published protocols (Appendix 6).
Ustrezno reakcijsko zmes PCR pripravimo v nekontaminiranem okolju po objavljenih protokolih (Dodatek 6).
Where possible, it is recommended to use a multiplex PCR protocol that also incorporates an internal PCR control.
Kjer je to mogoče, je priporočljivo uporabiti multipli protokol PCR, ki vključuje tudi notranjo kontrolo PCR.
Add 2-5 µl of DNA extract per 25 µl PCR reaction in sterile PCR tubes according to the PCR protocols, (see Appendix 6).
Dodamo 2-5 µl ekstrakta DNK na 25 µl reakcijske zmesi PCR v sterilne epruvete za PCR po protokolih za PCR (glej Dodatek 6).
Incorporate a negative control sample containing only PCR reaction mix and add the same source of UPW as used in the PCR mix in place of sample.
Vključimo tudi vzorec za negativno kontrolo, ki vsebuje samo reakcijsko zmes PCR, in dodamo isti vir ultra čiste vode, kot smo ga uporabili za zmes PCR pri vzorcu.
Place tubes in the same thermal cycler which was used in preliminary testing and run the appropriately optimised PCR programme (Appendix 6).
Epruvete damo v isti ciklični termostat, ki smo ga uporabili pri predhodnem testiranju, in zaženemo ustrezno optimirani program PCR (Dodatek 6).
Resolve PCR amplicons by agarose gel electrophoresis.
Pomnožke PCR odkrijemo z elektroforezo v agaroznem gelu.
Run at least 12 µl of amplified DNA reaction mixture from each sample mixed with 3 µl loading buffer (Appendix 6) in 2,0 % (w/v) agarose gels in tris-acetate-EDTA (TAE) buffer (Appendix 6) at 5 to 8 V per cm.
Vsaj 12 µl pomnožene reakcijske zmesi DNK iz vsakega vzorca, zmešane s 3 µl nanašalnega pufra (Dodatek 6), vlijemo v 2,0 % (w/v) agarozne gele v tris-acetatnem-EDTA (TAE) pufru (Dodatek 6), 5-8 V na cm.
Reveal DNA bands by staining in ethidium bromide (0,5 mg per L) for 30 to 60 minutes taking appropriate precautions for handling this mutagen.
Proge DNK razkrijemo tako, da jih 30-60 min barvamo z etidijevim bromidom (0,5 mg na L), pri tem pa upoštevamo ustrezne varnostne ukrepe za ravnanje s to mutageno snovjo.
Observe stained gel under short wave UV transillumination (λ = 302 nm) for amplified PCR products of the expected size (Appendix 6) and document.
Obarvani gel opazujemo pod kratkovalovno UV presvetlitvijo (λ = 302 nm) in iščemo pomnožene produkte PCR pričakovane velikosti (Dodatek 6) ter dokumentiramo.
For all new findings/cases verify authenticity of the PCR amplicon by performing restriction enzyme analysis on a sample of the remaining amplified DNA by incubating at the optimum temperature and time with an appropriate enzyme and buffer (see Appendix 6).
Za vsa nova odkritja/primere preverimo avtentičnost pomnožka PCR, tako da opravimo restrikcijsko encimsko analizo na vzorcu preostale pomnožene DNK: inkubiramo pri optimalni temperaturi in času z ustreznim encimom in pufrom (glej Dodatek 6).
Resolve the digested fragments by agarose gel electrophoresis as before and observe characteristic restriction fragment pattern under UV transillumination after ethidium bromide staining and compare with the undigested and digested positive control.
Tako kot prej, raztopljene fragmente PCR odkrijemo z elektroforezo v agaroznem gelu in po obarvanju z etidijevim bromidom opazujemo značilni restrikcijski vzorec fragmentov pod UV presvetlitvijo ter primerjamo z raztopljeno in neraztopljeno pozitivno kontrolo.
Interpretation of the PCR test result:
Interpretacija rezultata testa PCR:
The PCR test is negative if the R. solanacearum-specific PCR amplicon of expected size is not detected for the sample in question but is detected for all positive control samples (in case of multiplex PCR with plant specific internal control primers: a second PCR-product of expected size must be amplified with the sample in question).
Test PCR je negativen, če za dotični vzorec ne odkrijemo za bakterijo R. solanacearum značilnega pomnožka PCR pričakovane velikosti, vendar ga odkrijemo za vse vzorce pozitivne kontrole (pri multiplem PCR z za rastlino specifičnimi PCR primerji notranje kontrole: z dotičnim vzorcem je treba pomnožiti drugi produkt PCR pričakovane velikosti).
The PCR test is positive if the R. solanacearum-specific PCR amplicon of expected size and restriction pattern (when required) is detected, providing that it is not amplified from any of the negative control samples.
Test PCR je pozitiven, če odkrijemo za bakterijo R. solanacearum značilen pomnožek PCR pričakovane velikosti in restrikcijskega vzorca (če je potrebno), pod pogojem, da ni bil pomnožen iz nobenega od vzorcev negativne kontrole.
Reliable confirmation of a positive result can also be obtained by repeating the test with a second set of PCR primers (Appendix 6).
Zanesljivo potrditev pozitivnega rezultata dobimo tudi, če ponovimo test z drugo množico PCR primerjev (Dodatek 6).
Note: Inhibition of the PCR may be suspected if the expected amplicon is obtained from the positive control sample containing R. solanacearum in water but negative results are obtained from positive controls with R. solanacearum in potato extract.
Opomba: Inhibicijo PCR lahko sumimo, če pričakovani pomnožek dobimo iz vzorca pozitivne kontrole, ki vsebuje bakterijo R. solanacearum v vodi, medtem ko iz pozitivnih kontrol z bakterijo R. solanacearum v ekstraktu krompirja dobimo negativne rezultate.
In multiplex PCR protocols with internal PCR controls, inhibition of the reaction is indicated when neither of the two amplicons are obtained.
Pri multiplih protokolih PCR z notranjo kontrolo PCR je indicirana inhibicija reakcije, če ne dobimo nobenega od obeh pomnožkov.
Contamination may be suspected if the expected amplicon is obtained from one or more of the negative controls.
Kontaminacijo lahko sumimo, če pričakovani pomnožek dobimo iz ene ali več negativnih kontrol.
When the FISH test is used as the first screening test and found to be positive, Isolation or the IF test must be performed as a second compulsory screening test.
Če test FISH uporabljamo kot glavni presejalni test in je odčitek pozitiven, je treba kot drugi obvezni presejalni test opraviti izolacijski test ali IF test.
When the FISH test is used as the second screening test and found to be positive, further testing according to the flow scheme is required to complete the diagnosis.
Če test FISH uporabljamo kot drugi presejalni test in je odčitek pozitiven, je za zaključek diagnoze potrebno nadaljnje testiranje glede na diagram poteka.
Note: Use validated R. solanacearum-specific oligo-probes (see Appendix 7).
Opomba: Uporabimo potrjene, za bakterijo R. solanacearum specifične oligo-sonde (glej Dodatek 7).
Preliminary testing with this method should permit reproducible detection of at least 103 to 104 cells of R. solanacearum per ml added to sample extracts which previously tested negative.
Predhodno testiranje s to metodo mora omogočati ponovljivo detekcijo vsaj 103 do –104 celic bakterije R. solanacearum na ml dodano ekstraktom vzorcev, ki so bili prej testirani kot negativni.
The following procedure should preferably be performed on freshly prepared sample extract but can also be successfully performed on sample extract that has been stored under glycerol at –16 to –24 or –68 to –86 °C.
Spodnji postopek je najbolje opraviti na sveže pripravljenem ekstraktu vzorca, uspešno pa ga lahko opravimo tudi na ekstraktu vzorca, ki je bil shranjen pod glicerolom pri –16 do –24 °C ali –68 do –86 °C.
As negative controls, use aliquots of sample extract that previously tested negative for R. solanacearum.
Kot negativne kontrole uporabimo alikvote ekstrakta vzorca, ki so bili prej testirani kot negativni za R. solanacearum.
As positive controls prepare suspensions containing 105 to 106 cells per ml of R. solanacearum biovar 2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857, see Appendix 3) in 0,01M phosphate buffer (PB) from a 3 to 5 day culture).
Kot pozitivne kontrole pripravimo suspenzije, ki vsebujejo 105 do 106 celic na ml bakterije R. solanacearum biovar 2 (npr. sev NCPPB 4156 = PD 2762 = CFBP 3857, glej Dodatek 3) v 0,01 M fosfatnega pufra (PB) iz 3-5 dnevne kulture). Pripravimo ločena stekelca za pozitivno kontrolo s homolognim sevom ali katerimkoli drugim referenčnim sevom bakterije R. solanacearum, suspendiranim v ekstraktu krompirja, kot je navedeno v Dodatku 3B. Uporaba eubakterijske oligo-sonde FITC omogoča kontrolo procesa hibridizacije, saj bodo obarvane vse eubakterije, ki so navzoče v vzorcu.
Prepare separate positive control slides of the homologous strain or any other reference strain of R. solanacearum, suspended in potato extract, as specified in Appendix 3 B. The use of the FITC-labelled eubacterial oligo-probe offers a control for the hybridisation process, since it will stain all eubacteria that are present in the sample.
Standardizirani pozitivni in negativni kontrolni materiali, ki so na voljo za uporabo s tem testom, so navedeni v Dodatku 3, točka A.
Standardized positive and negative control material available for use with this test are listed in Appendix 3A). Test control material in an identical manner as the sample(s).
Kontrolni material testiramo povsem enako kot vzorce.
Potato extract fixation
Fiksiranje ekstrakta krompirja
The following protocol is based upon Wullings et al.
Spodnji protokol temelji na Wullings in sod.
Pipette 100 µl of each sample extract into an Eppendorf tube and centrifuge for 7 minutes at 7000 g.
Odpipetiramo 100 µl vsakega ekstrakta vzorca v Eppendorfovo epruveto in centrifugiramo 7 min pri 7000 g.
Remove the supernatant and dissolve the pellet in 200 µl of fixative prepared < 24 hours previously.
Odstranimo supernatant in raztopimo peleto v 200 µl preparata za fiksiranje, pripravljenega manj kot 24 ur pred tem.
Vortex and incubate for one hour in the refrigerator.
Vrtinčimo in inkubiramo 1 uro v hladilniku.