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3.7: Tryptone Broth - Biologie

3.7: Tryptone Broth - Biologie


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Bakteriële triptofanase dek die aminosuur triptofaan in piruvaat, ammoniak en indool. Die medium wat gebruik word om vir hierdie ensiem te toets, is 1% triptoon in water.

Prosedure

  1. Kry 'n buisie triptoon sous.
  2. Gebruik 'n inentingslus en spoel 'n bietjie van jou toegewysde organisme in die sous.
  3. Inkubeer die buis vir ten minste 48 uur.
  4. Na die inkubasieperiode, voeg verskeie druppels Kovac se reagens by die buis.

Interpretasie

Die chemikalieë in Kovac se reagens reageer met die indool om 'n rooi kleur te produseer. In 'n positiewe toets sal 'n rooi laag op die oppervlak van die sous dryf.

Indool Positief


Lysogeniese sous

Lysogeniese sous (LB) is 'n voedingsryke medium wat hoofsaaklik vir die groei van bakterieë gebruik word. Die skepper daarvan, Giuseppe Bertani, het bedoel LB om voor te staan lysogeniese sous, [1] maar LB het ook gekom om in omgangstaal te beteken Luria sous, Lennox sous, lewens sous of Luria-Bertani medium. Die formule van die LB medium is in 1951 gepubliseer in die eerste koerant van Bertani oor lysogenie. In hierdie artikel het hy die gemodifiseerde enkelbars eksperiment en die isolasie van die fage P1, P2 en P3 beskryf. Hy het die ontwikkel LB medium te optimaliseer Shigella groei en plaakvorming. [1] [2]

LB media formulerings is 'n industrie standaard vir die verbouing van Escherichia coli so ver terug as die 1950's. [3] [4] [5] [6] [7] Hierdie media is wyd gebruik in molekulêre mikrobiologie toepassings vir die voorbereiding van plasmied DNA en rekombinante proteïene. Dit is steeds een van die mees algemene media wat gebruik word vir die instandhouding en kweek van laboratorium rekombinante stamme van Escherichia coli. [8] Vir fisiologiese studies moet die gebruik van LB-medium egter ontmoedig word. [9]

Daar is verskeie algemene formulerings van LB. Alhoewel hulle verskillend is, deel hulle oor die algemeen 'n ietwat soortgelyke samestelling van bestanddele wat gebruik word om groei te bevorder, insluitend die volgende:

Natriumione vir vervoer en osmotiese balans word deur natriumchloried verskaf. Tryptoon word gebruik om essensiële aminosure soos peptiede en peptone aan die groeiende bakterieë te verskaf, terwyl die gisekstrak gebruik word om 'n oorvloed van organiese verbindings te verskaf wat nuttig is vir bakteriese groei. Hierdie verbindings sluit vitamiene en sekere spoorelemente in.

In sy oorspronklike 1951-vraestel het Bertani 10 gram NaCl en 1 gram glukose per 1 L oplossing gebruik Luria in sy "L-bouillon" van 1957, het Bertani se oorspronklike resep presies gekopieer. [6] Resepte wat later gepubliseer is, het tipies die glukose uitgelaat.


3.7: Tryptone Broth - Biologie

Jy ent 'n onbekende organisme in triptoon sous. Na 24 uur inkubasie by 37C voeg jy 10 druppels Kovac's by. ’n Rooi ring ontwikkel. Wat weet jy van jou organisme? Kies alles wat van toepassing is.[/caption] A.) Die organisme produseer desulfhidrase. B.) Die organisme produseer triptofonase. C.) Die organisme het suur geproduseer. D.) Die organisme het indool geproduseer. E.) Die organisme kan triptofaan benut. F.) Die organisme kan stysel benut.

Jy ent 'n onbekende organisme in triptoon sous. Na 24 uur inkubasie by 37C voeg jy 10 druppels Kovac's by. ’n Rooi ring ontwikkel. Wat weet jy van jou organisme? Kies alles wat van toepassing is.[/caption]
A.) Die organisme produseer desulfhidrase.
B.) Die organisme produseer triptofonase.
C.) Die organisme het suur geproduseer.
D.) Die organisme het indool geproduseer.
E.) Die organisme kan triptofaan benut.
F.) Die organisme kan stysel benut.

B.) Die organisme produseer triptofonase.
D.) Die organisme het indool geproduseer.
E.) Die organisme kan triptofaan benut.

Gebruik jou beursie-saldo of laai jou beursie onmiddellik met PayPal of kredietkaart om te koop. $5 per volledige oplossing.


VEREISTES VIR DIE VOORBEREIDING VAN TRYPTICASE SOJABOUMEDIUM (TSB)

  • Steriele kegelfles / Erlenmeyerfles
  • Spatel
  • Pankreas vertering van kaseïen
  • Papaïese vertering van sojaboonmeel
  • Dekstrose (glukose)
  • Dikaliumwaterstoffosfaat
  • Natriumchloried
  • Meetsilinder
  • 1N HCl
  • 1N NaOH
  • pH-strook
  • Weegskaal
  • Gedistilleerde water
  • Botter Papier

Elektroniese aanvullende materiaal is aanlyn beskikbaar by https://doi.org/10.6084/m9.figshare.c.4971935.

Gepubliseer deur die Royal Society onder die bepalings van die Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, wat onbeperkte gebruik toelaat, mits die oorspronklike outeur en bron gekrediteer word.

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CRISPR-Cas Ensieme

Robert D. Fagerlund,. Peter C. Fineran, in Methods in Enzymology, 2019

2.1.2 Buffers en reagense

5 mL en 0,5 L Lysogeny Broth (LB) media (10 g/L bakto-tryptoon, 5 g/L gisekstrak, 5 g/L NaCl)

Strep lise buffer: 50 mM HEPES-NaOH, pH 7,5, 0,5 M KCl, 10% v/v gliserol

Strep-elueringsbuffer: 50 mM HEPES-NaOH, pH 7,5, 0,5 M KCl, 10% v/v gliserol, 3 mM d -desthiobiotien

Strep SEC buffer: 10 mM HEPES-NaOH, pH 7,5, 0,5 M KCl, 10% gliserol

cOmplete™ EDTA-vrye protease-inhibeerder (Roche)

100 mM fenielmetaansulfonielfluoried (PMSF)

Strep-taktienhars (IBA Wetenskappe)

Vloeibare stikstof of etanol-droë ysbad


Tryptic Soy Broth (TSB) Gedehidreerde en voorbereide kultuurmedia, BD Diagnostics

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Die gebruik van terminale oordragase-gemedieerde dUTP Nick-eindetikettering (TUNEL) en Caspase 3/7-toetse om epidermale seldood by paddas met chytridiomycosis te meet

Ons kwantifiseer epidermale seldood by paddas met chytridiomycosis met behulp van twee metodes. Eerstens gebruik ons ​​terminale transferase-gemedieerde dUTP nick-eind-etikettering (TUNEL) in situ histologie om verskille tussen klinies geïnfekteerde en onbesmette diere te bepaal. Tweedens doen ons 'n tydreeksanalise van apoptose oor infeksie deur 'n kaspase 3/7-proteïenanalise te gebruik.

Abstrak

Amfibieë ervaar wêreldwyd 'n groot verlies aan biodiversiteit en een van die hoofoorsake is die aansteeklike siekte chytridiomycosis. Hierdie siekte word veroorsaak deur die swampatogeen Batrachochytrium dendrobatidis (Bd), wat padda-epidermis infekteer en ontwrig, patologiese veranderinge is egter nie eksplisiet gekenmerk nie. Apoptose (geprogrammeerde seldood) kan deur patogene gebruik word om gasheerweefsel te beskadig, maar kan ook 'n gasheermeganisme van siekteweerstand vir patogeenverwydering wees. In hierdie studie kwantifiseer ons epidermale seldood van besmette en onbesmette diere deur twee verskillende toetse te gebruik: terminale transferase-gemedieerde dUTP nick-eind-etikettering (TUNEL), en kaspase 3/7. Met behulp van ventrale, dorsale en dy velweefsel in die TUNEL-toets, neem ons seldood in die epidermale selle waar in situ van klinies besmette diere en vergelyk seldood met onbesmette diere deur gebruik te maak van fluoresserende mikroskopie. Om te bepaal hoe apoptosevlakke in die epidermis verander oor die verloop van infeksie, verwyder ons toonpuntmonsters tweeweekliks oor 'n tydperk van 8 weke, en gebruik 'n kaspase 3/7-toets met onttrekte proteïene om aktiwiteit binne die monsters te kwantifiseer. Ons korreleer dan kaspase 3/7-aktiwiteit met infeksielading. Die TUNEL-toets is nuttig vir lokalisering van seldood in situ, maar is duur en tyd intensief per monster. Die kaspase 3/7-toets is doeltreffend vir groot monstergroottes en tydsverloop-eksperimente. Omdat paddapunt-biopsies egter klein is, is daar beperkte uittreksel beskikbaar vir monsterstandaardisering via proteïenkwantifiseringsmetodes, soos die Bradford-toets. Daarom stel ons voor om die veloppervlakte te skat deur fotografiese ontleding van toonbiopsies om die verbruik van ekstrakte tydens monsterstandaardisering te vermy.

Inleiding

Amfibieë ervaar tans een van die grootste verliese aan globale biodiversiteit van enige gewerwelde taksa 1 . 'n Groot oorsaak van hierdie afnames is die dodelike velsiekte chytridiomycosis, veroorsaak deur die swampatogeen Batrachochytrium dendrobatidis, Bd 2 . Die patogeen infekteer oppervlakkig die epidermis, wat kan lei tot die ontwrigting van velfunksie wat lei tot ernstige elektrolietverlies, hartstilstand en dood 3 . Verskeie potensiële gasheer immuunmeganismes teen Bd word tans bestudeer, soos antimikrobiese peptiede 4 , 5 , kutane bakteriese flora 6 , immuunselreseptore 7 , 8 en limfosietaktiwiteit 9 , 10 . Min studies ondersoek egter of epidermale apoptose en seldood 'n immuunmeganisme teen hierdie dodelike patogeen is.

Seldood, hetsy deur apoptose (geprogrammeerde seldood) of nekrose (ongeprogrammeerde dood), in die epidermis kan 'n patologie van Bd infeksie. Vorige navorsing dui daarop dat Bd infeksie kan apoptose veroorsaak omdat ontwrigting van intrasellulêre aansluitings waargeneem word wanneer veleksplantings aan soöspoor-supernatante blootgestel word in vitro 11 . Daarbenewens, degeneratiewe epidermale veranderinge in Bd-besmette paddas word waargeneem met behulp van elektronmikroskopie 12 , 13 . Transkriptomiese ontledings dui daarop dat apoptose-bane opgereguleer word in besmette vel 14, en amfibiese splenosiete ondergaan apoptose wanneer hulle blootgestel word aan Bd supernatante in vitro 15 . Ten spyte van die groeiende hoeveelheid bewyse wat daarop dui Bd kan apoptose en gasheersel dood veroorsaak in vitro, in vivo studies wat apoptosemeganismes ondersoek of kwantifiseer deur die vordering van infeksie, ontbreek. Verder is dit onbekend of die gasheer apoptose gebruik as 'n defensiewe immuunstrategie om te bestry Bd infeksie, of as apoptose 'n patologie van siekte is.

In hierdie studie het ons daarop gemik om epidermale seldood en apoptose by besmette diere op te spoor in vivo deur gebruik te maak van twee metodes: kaspase 3/7 proteïentoets, en terminale transferase-gemedieerde dUTP nick-eind-etikettering (TUNEL) in situ toets. Aangesien elke toets verskillende aspekte van seldood 16 opspoor, bied hierdie metodes saam 'n volledige begrip van die meganismes betrokke by seldood, en verseker 'n akkurate maatstaf van die effek. Die kaspase 3/7-toets kwantifiseer die aktiwiteit van effektorkaspases 3 en 7, wat kwantifisering van beide die intrinsieke en ekstrinsieke apoptose-bane moontlik maak. Daarteenoor bespeur die TUNEL-toets DNA-fragmentasie, wat veroorsaak word deur seldoodmeganismes insluitend apoptose, nekrose en piroptose 17 . Ons gebruik die TUNEL-toets om die ligging van seldood binne die epidermis van beide klinies besmette en onbesmette diere te ondersoek deur drie verskillende velafdelings te gebruik: die rug, die vent en die bobeen van Pseudofryne corroboree. Hierdie metode identifiseer die anatomiese plek van seldood, sowel as om die ligging daarvan binne spesifieke epidermale lae te onderskei. Ons gebruik dan die kaspase 3/7-toets om 'n tydreeks-kwantifisering van apoptose deur 'n 8-week infeksie in Litoria verreauxii alpina. Ons neem toonpuntmonsters tweeweekliks van dieselfde diere en is in staat om patogeeninfeksielading met kaspase 3/7-aktiwiteit te korreleer.

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Protokol

James Cook Universiteit goedgekeur diere-etiek in aansoeke A1875 vir P. corroboree en A1897 en A2171 vir L. v. alpina.

1. Veeteelt en Monitering

  1. Huisdiere individueel, in 'n omgewing wat geskik is vir die spesie, met 'n toepaslike water-, voer- en skoonmaakskedule. Kontroleer diere daagliks.
    1. Gebruik volwasse individue van die kritiek bedreigde Pseudofryne corroboree (vir die TUNEL Assay, Afdeling 4) en die bedreigde Litoria verreauxii alpina (vir die Caspase 3/7-toets, Afdeling 5), geskenk vanaf gevangenskaplike teelfasiliteite. Hou die diere by 15 - 18 °C, op 'n mos en gruis substraat, mis hulle daagliks met tru-osmose water, en voer 3 keer weekliks met ingewande gelaaide krieke.
    1. Euthaniseer met 'n oordosis tricaine metaansulfonaat (MS222) (0.1% w/v MS222 tot pH 6 -ه met natriumbikarbonaat in verouderde kraanwater). Hou diere in MS222 vir 10 minute nadat alle beweging opgehou het en hulle geen reaksie op stimuli toon nie.

    2. Toets vir Bd Infeksie

    1. Depper vir Bd
      1. Depper elke dier met 'n nuwe steriele rayon depper (een depper per dier). Gebruik die volgende deppermetode: vyf keer op die vent, vyf keer op elke bobeen, sy en ledemaat. Dit is 'n totaal van 45 houe.
      2. Draai die depper liggies tydens en tussen hale om effektiewe vaslegging van DNA te verseker. Breek die depperpunt af in 1,5 mL mikrosentrifugeerbuise en stoor by -20 °C tot ekstraksie.
      1. Onttrek genomiese DNA uit die deppers deur 50 µL van 'n kommersieel beskikbare DNA ekstraksie reagens met 30 - 40 mg 0.5 mm silika krale by te voeg. Bead klits die monsters in 'n kraal klitser sel ontwrigter teen maksimum spoed vir 2 min. Na die kraalklitserstap, sentrifugeer die monsters vir 1 min by 2 000 x g.
      2. Inkubeer die monsters by 100 °C vir 10 min, en laat afkoel. Sentrifugeer die monsters by 5 000 x g vir 3 min, en dra dan die supernatant oor na individuele 1,5 ml mikrosentrifugeerbuise en stoor by -20 °C tot qPCR.
      3. Gebruik kwantitatiewe PCR (qPCR) na aanleiding van Boyle et al. 2004 18 om die infeksielading te ontleed. Gebruik die volgende wysigings:
        1. Verdun DNA-ekstraksiemonsters 6:100 met dubbel gedeïoniseerde water. Voeg 0,7 µL beeserumalbumien (BSA) by elke put om te help om PCR-inhibisie te voorkom. Begin elke monster in enkelvoud. Gebruik 'n negatiewe (geen sjabloon) kontrole en 'n positiewe kontrole met 'n reeks verdunningstandaarde op elke plaat om soöspoor (infeksie) lading te skat.
        1. Kultuur Bd
          1. Berei kultuurbouillon voor deur 16 g triptoon, 2 g gelatienhidrolisaat en 4 g laktose (TGHL) by 1 L gedeïoniseerde water te voeg. Outoklaveer (121 °C vir 40 min) en laat die sous afkoel.
          2. Ent in Bd isoleer (in hierdie geval, geïsoleer van Nieu-Suid-Wallis isolaat, AbercrombieR-L.booroologensis-2009-LB1, Passasie nommer 11) in 'n TGHL-bouillon en groei vir 7 dae by 23 °C, dra dan die souskultuur oor na TGHL-agar plate.
          3. Om plate te maak, voeg 16 g triptoon, 2 g gelatienhidrolisaat, 4 g laktose (TGHL) en 10 g bakteriologiese agar by 1 L gedeïoniseerde water en outoklaveer (121 °C vir 40 min). Sodra die mengsel koel genoeg is om aan te raak, maar voordat dit stol, gooi die TGHL-agar in 92 mm deursnee kultuurplate sodat hulle 1/4 tot 1/3 vol is, in 'n Klas II biologiese veiligheidskas.
          4. Wanneer die agar gestol en heeltemal afgekoel het, ent elke plaat met 0,5 mL van Bd uit die vloeibare sous, en versprei eweredig. Laat toe Bd sousmengsel om vir ongeveer 1 uur op plaat droog te word en verseël dan plate met plastiek paraffienfilm. Inkubeer plate met die agarkant na onder by 23 °C vir 5 - 7 d.
          1. Kontroleer soöspore-motiliteit onder 'n omgekeerde ligmikroskoop om lewensvatbaarheid daagliks voor inenting te verseker. Wanneer soöspore vrystelling hoog is, met baie dierespore wat buite die sporangia swem, is die kultuur gereed vir inenting.
          2. Oorstroom elke bord met 3 mL verouderde kraanwater of kunsmatige damwater, deur die water in die bord te gooi en teen kamertemperatuur vir 10 minute te broei om die soöspore in die water vry te laat. Na die inkubasietydperk, gooi die soöspoorsuspensie in 'n nuwe steriele houer.
          3. Skat soöspoorkonsentrasie volgens vervaardiger se instruksies deur 'n hemositometer te gebruik. Sodra die konsentrasie van die soöspoorsuspensie bekend is, verdun die suspensie tot 'n konsentrasie van 1 x 10 6 soöspore per 3 ml met verouderde kraanwater.
          1. Ent elke dier in deur 3 ml inokulummengsel oor sy vent 19 in individuele 50 ml houers te gooi. Laat oortollige inokulum in die basis van die inokulasiehouer versamel. Laat elke dier vir 24 uur in individuele inentingshouer om infeksie te verseker, en na die 24 uur inenting, keer elke individu terug na 'n ontsmette terrarium.
            1. Ontsmet terraria met 'n 13% volume/volume (v/v) kommersiële bleikmiddeloplossing 20 , spoel ten minste twee keer met water uit en laat dan vir nie minder nie as 24 uur droog word.
            1. Maak diere dood wat kliniese tekens van chytridiomycosis toon, soos beskryf in stap 1.3.1, en 'n gelyke aantal Bd-negatiewe beheer diere.
            2. Dissekteer vel (dorsale, ventrale en dy) monsters van elke dier. Bevestig die velmonsters vir 2 uur in 4% v/v fosfaatgebufferde formaldehied. Die kort en konsekwente bindingstyd laat die weefsels volledig vas, maar maak ook voorsiening vir effektiewe en akkurate immunohistochemie-kleuring. Dra dan oor na 80% etanol tot inbedding vir snit.
            3. Bed vel in paraffienwas vir histologiese voorbereiding volgens standaardmetodes 21 . Kortliks is die protokol soos volg:
              1. Dehidreer weefsels in 'n gegradeerde reeks etanol, en maak die etanol skoon met xileen. Bed die weefsel in paraffienwas, en plaas al drie velmonsters vir elke individu in een paraffienblok.
              1. Kleur die eerste skyfie met hematoksilien gevolg deur eosien-teenkleuring (H&E). Hierdie skyfie is om die ligging van te visualiseer Bd sporangia in die vel.
              2. Kleur die tweede skyfie na 'n kommersieel beskikbare TUNEL-toets vir histologiese voorbereiding en volg vervaardiger se instruksies, wat hieronder beskryf word.
                1. Eerstens, deparaffieniseer die weefselafdelings in 'n coplin-fles deur die skyfies met drie veranderinge xileen vir 5 min per was te was, en volg met twee veranderinge van 100% etanol vir 5 min elke was. Volg met een was van 95% etanol vir 3 min en dan 70% etanol vir 3 min. Voltooi met een was PBS vir 5 min.
                2. Behandel die weefsel vooraf met vars verdunde proteïenverterende ensiem (proteïnase K teen 'n konsentrasie van 20 μg/ml verdun in PBS), en voeg direk by die skyfie. Laat inkubeer vir 15 min. Was met twee veranderinge van PBS in 'n coplin-fles vir 2 min elk.
                3. Tik die oortollige vloeistof af en blus in 3.0% waterstofperoksied in PBS in 'n coplin-fles vir 2 min by kamertemp. Spoel twee keer met PBS, vir vyf minute elke was.
                4. Tik die oortollige vloeistof af, dien dan 75 µL/5 cm 2 van die ewewigsbuffer direk op die weefsel op die skyfie toe. Inkubeer vir 10 s. Tik die oortollige vloeistof rondom die gedeelte af en dien 55 µL/5 cm 2   van werkende terminale deoksinukleotidieltransferase (TdT) ensiem toe. Inkubeer in 'n bevochtigde kamer vir 1 uur by 37 °C.
                5. Na die TdT-inkubasie, plaas die skyfie in 'n coplin-fles met werksterkte stop/was buffer, roer vir 15 s en inkubeer vir 10 min by kamertemperatuur. Was die skyfie met drie veranderinge van PBS vir 1 min per was. Tik die oortollige vloeistof af.
                6. Dien anti-digoksigenien-konjugaat (rhodamien) wat tot kamertemperatuur verhit is op die weefsel toe, 65 µL/5 cm 2 . Inkubeer in 'n bevochtigde kamer vir 30 min by kamertemperatuur en vermy blootstelling aan lig. Was met vier veranderinge van PBS vir 2 min per was. Tik die oortollige vloeistof af.
                7. Finish by adding 15 µL of 0.5 - 1 µg/mL DAPI (4',6-diamidino-2-phenylindole) in slide mounting medium to the slide, which acts as a counter stain. Then cover with a cover slip and seal with nail polish or rubber cement. Allow slides to dry in the dark as the assay is light sensitive.
                1. For the positive controls, instead of step 4.5.2.2, pre-treat the tissue with DN buffer (30mM trizma base, pH 7.2, 4mM MgCl2, 0.1mM DDT) and let incubate at room temperature for 5 minutes. Then dissolve Dnase I in DN buffer for a final concentration of 0.1ug/mL, and apply it directly to the slide. Incubate for 15 minutes at room temperature.  Then, wash the slide with 5 wash of dH2O for 3 minutes each wash. Blot off the excess liquid. Then resume TUNEL assay as directed in section 4.5.2.3.
                2. For the negative controls, do not add the terminal deoxynucleotidyl tranferase (TdT) enzyme to those samples (as described in section 4.5.2.4).
                1. Take photos at random intervals along each skin section at 200X using a fluorescent microscope, using filters for both the rhodamine stain, which reveals the apoptotic cells and appears red, and DAPI which reveals all nuclei and appears blue, so that an overlay of cells can be generated. Ensure at least 100 cells are photographed per skin section per sample. Store the slides in a dark microscope box at -20 °C.
                2. Count cells (blue DAPI stained) per image. Ensure at least 100 cells are counted per skin section. To reach 100 cells, count all cells within the image. If less then 100 cells are present in one image, count another image until at least 100 cells are reached per skin section per animal.
                3. Next, count the number of TUNEL positive cells in the same images (red rhodamine stained). TUNEL positive cells, which indicate apoptosis and not necrosis or background, are single cells with clear cell edges (membranes are intact with no lysis). Sometimes the cells shrink to form apoptotic bodies.
                4. Locate the areas counted in the TUNEL assay and analyze the corresponding regions on the H&E stained histosections. Confirm that the H&E stained sections correspond to sites of Bd infection, which ensures Bd-infected sites are used when counting apoptotic cells in the TUNEL assay.
                1. Collect toe tips from each animal (both Bd- exposed and Bd- negative) once weekly through week 3 post inoculation, and fortnightly through the end of the experiment, up to 8 toes per individual.
                  1. Cut the toe tip at the second phalange with flame-sterilized scissors, and place in a 1.5 mL microtube and freeze immediately at -80 °C.
                  1. Place samples in 100 µL of sample buffer (25 mM HEPES pH 7, 5 mM MgCl2) with two stainless steel beads (3.2 mm) in a 1.5 mL screw cap microtube. Lyse samples by 4 cycles of 1 min bead beating at maximum speed (in the same bead beater as used in step 1.2.1) followed by 3 min on ice. After lysis, centrifuge samples at 12,000 x g and 4 °C for 5 min. Collect supernatant to use in the assay.
                  1. In a 384 well plate, mix 10 µL of protein extract with 10 µL of Bradford reagent, and incubate for 2 min at room temperature. Perform each sample in duplicate, along with a series of 5 fold BSA dilution standards. Read the absorbance at 595 nm on an absorbance plate reader.
                  1. Before extracting the proteins from the sample for the caspase assay, photograph each toe at 40X magnification using an inverted light microscope.
                  2. Analyze the toe sample using a computer imaging software, by estimating area of the toe. Do this by drawing a line around the edge of the toe clip, and have the imaging software estimate area within the shape. Then multiply that area by pi (3.14), which will approximate the surface area of the 3-dimentional skin sample (as length x cross-sectional area (pi x diameter) gives the outer surface area of a tube).
                  1. In a 384 well luminescence plate, add 10 µL of commercially available caspase 3/7 reagent and 10 µL of protein extract. Run each sample in triplicate.
                  2. Mix the reagents by shaking the plate slowly for 15 s. Incubate the plate away from light for 30 min at room temperature. Measure luminescence using a luminescent plate reader.

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                  Verteenwoordigende resultate

                  There were more TUNEL positive cells in the infected animals than in the uninfected control animals. Die in situ location of TUNEL positive cells differed in infected and control animals. In control animals, there was an even distribution of TUNEL positive cells throughout the dermal and epidermal skin layers at low levels (See Figuur 1A), but in the infected animals, the TUNEL positive cells were more frequent in the epidermis (Figure 1B). The sites of infection (as observed on the H&E stained slides) were observed as clumped Bd sporangia scattered through the thigh and ventral skin sections with none in the dorsum sections. While the TUNEL positive cells were more concentrated at and directly adjacent to sites of infected cells (Figure 1C), there was also more widespread TUNEL positive cells over the epidermis and in epidermal layers that were deeper than where Bd resided. In Bd infected animals there were more TUNEL positive cells in all three skin types analyzed, but a particularly high level in the thigh and venter skin (12.01 times higher (95% CI: 4.92 - 26.30) in the thigh skin and 22.31 times higher (95% CI 5.25 - 94.82) in the venter skin) (Figuur 2).

                  Over the course of infection there was a difference in caspase activity. In Bd infected animals, infection intensity was positively correlated with caspase 3/7 activity (Figuur 3). There was also a difference between infected and uninfected animals through time, post inoculation. Caspase 3/7 activity decreased within the first few weeks after inoculation (with 48.36% less activity in infected animals), and then increased toward the end of week 7 (Figuur 4) in Bd infected animals, but remained constant in uninfected individuals.


                  Figure 1: Terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ assay of infected and uninfected animals. A) Bd- control thigh skin section of Pseudophryne corroboree, and B) Bd+ thigh skin section of P. corroboree stained by in situ TUNEL assay. The blue is DAPI staining indicating nuclei of the cells, and the red is the rhodamine stain, which indicates DNA fragmentation characteristically caused by apoptosis. The yellow arrow indicates the position of the Bd cluster seen in panel C. C) P. corroboree section of thigh skin stained with H&E. The H&E section is serial to panel B. There is a cluster of empty Bd sporangia (arrow) and a few dark immature sporangia near the skin surface. For all three panels the epidermis is at the top of the photo. Comparing panels B and C shows that the rhodamine stained epidermal cells are concentrated around and below the cluster of Bd and where skin damage is visible, such as micro-vesicle formation between basal epidermal cells. 400X magnification and the scale bar indicates 0.03 mm. Aangepas van Figuur 2 in Brannelly et al. 2017 Peer J 22 . Klik asseblief hier om 'n groter weergawe van hierdie figuur te sien.


                  Figure 2: The proportion of TUNEL positive (TUNEL+) cells per skin type. The proportion of TUNEL positive apoptotic cells per skin type in P. corroboree, with infected animals indicating animals that succumbed to disease (n = 9) and uninfected control animals (n = 10). Error bars indicate 95% confidence intervals of a proportion and * indicates a significant increase in TUNEL+ cell proportions. In the dorsal skin, the infected animals had 14.38 (95% CI 3.32 - 62.24) times more TUNEL positive cells than control animals (Odds Ratio: Z = 3.57, p ɘ.01). In the thigh skin, infected animals had 12.01 (95% CI: 4.92 - 26.30 Odds Ratio: Z = 5.46, p ɘ.01) times more TUNEL positive cells than control animals (Pearson's Chi Squared: χ 2 1 = 44.30, p ɘ.01). In the venter skin, infected animals had 22.31 (95% CI 5.25 - 94.82) times more TUNEL positive cells than control animals (Odds Ratio: Z = 4.21, p ɘ.01). Aangepas van Figuur 3 in Brannelly et al. 2017 Peer J 22 . Klik asseblief hier om 'n groter weergawe van hierdie figuur te sien.


                  Figure 3: The correlation between infection intensity, Log10(zoospore equivalents), and caspase 3/7, Log10(Caspase) of inoculated Litoria verreauxii alpina over the course of the experiment. The correlation between infection intensity and caspase activity is 0.463, and the trend line has an equation of y = (0.229)x + 0.939. Aangepas van Figuur 4 in Brannelly et al. 2017 Peer J 22 . Klik asseblief hier om 'n groter weergawe van hierdie figuur te sien.


                  Figure 4: Caspase 3/7 activity through week 7 for each group of Litoria verreauxii alpina: Bd-infected animals that succumbed to disease (n = 4) and uninfected controls (n = 8). Caspase activity is defined as the luminescence reading controlled for by protein concentration per sample and then log base 10 transformed. The caspase activity (Log10 transformed) for each group per week. Foutstawe dui standaardfout aan. * indicates that the infected animals differed from the uninfected controls at that week. (Linear mixed effects model: week, F4 = 11.974, p ɘ.01 week*status, F8 = 2.139, p = 0.037 Week 3 ANOVA, F2,18 = 5.512, p = 0.014), Adapted from Figuur 5 in Brannelly et al. 2017 Peer J 22 . Klik asseblief hier om 'n groter weergawe van hierdie figuur te sien.

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                  Bespreking

                  We explored epidermal apoptosis and cell death as a potential mechanism of pathology of the deadly disease chytridiomycosis or a mechanism of disease resistance in Bd susceptible species. We used two methods of assessing cell death in the epidermis, TUNEL assay for in situ epidermal cell death analysis, and caspase 3/7 assay for monitoring epidermal cell death throughout the progress of infection. We found that cell death and apoptosis are correlated with infection load and cell death is significantly higher at the site of infection. In early infection, there is a decrease in apoptosis in the epidermis, but apoptosis increases as pathogen burden increases within the infected tissue.

                  Apoptosis in cells can be tested using numerous different approaches, each with benefits and limitations. It is important to study apoptosis and cell death using multiple assays in order to confirm findings. In this study, we explored two different assays to investigate epidermal cell death and apoptosis in frogs with chytridiomycosis. First, we trialled the TUNEL assay to assess cell death in situ. This test is time consuming, and expensive per sample, and slides must be read immediately as the fluorescent signal quickly fades. Despite these limitations, the results of this in situ experiment are important to further understanding the mechanisms of host-pathogen interactions. Information regarding localization can be determined using this method, and in our study apoptosis was present in high levels at the site of fungal infection and more diffusely at other sites. Additionally it must be noted that the TUNEL assay measures DNA damage, which can be caused by a number of different cell death mechanisms including apoptosis, necrosis and pyroptosis 17 . While there are signatures of apoptosis-associated cell death (such as single cells with membranes still intact, see protocol line 4.6.3, the classification relies on the interpretation of the researcher. Because the mechanism of cell death is not determined by the TUNEL assay, it is possible that another non-apoptosis cell death pathway may have caused the increase in TUNEL positive cells at morbidity of infected animals. The difference in what each assay measures might explain pattern differences in the two assays trialled here.

                  The caspase 3/7 assay can be used to quantify the effect of infection through time. However, as frog toe samples are small, there is limited sample for analysis and standardization. A common method for sample standardization is to determine the total protein concentration of each extract. As the Bradford protein quantification assay consumes sample, it is not an effective method for small extracts. In addition, we found that pigments within the frog skin precluded analysis by nano UV-Vis spectrometry. Small sample sizes also prevented standardization by dry weight. We suggest estimating skin surface area of the toe using photography, as the toe tip samples (these frogs were less than 3 g in body size) were too small to allow assays for both protein concentration and caspase concentration. We found that photographing the toes and using a skin surface area estimate is as effective as traditional protein concentration analyses.

                  In this study, we used two standard techniques for quantifying cell death and apoptosis, but adapted the methods for amphibians and small tissue samples. For the TUNEL assay, we euthanized the animal to ensure enough tissue was available for the assay, and allowed for the comparison of different skin locations. It is possible to adapt this method to smaller tissue samples, such as a toe webbing biopsy, which would not require euthanasia of the animal. Depending on the size of the animal, biopsies could allow for a time series test similar to our approach for the caspase 3/7 assay.

                  In future studies, we suggest the use of additional assays for exploring cell death and apoptosis over the course of chytridiomycosis infection because there is still much to learn of the causal mechanisms of chytridiomycosis pathogenesis. These results suggest that apoptosis and epidermal cell death may be important in the pathogenesis of Bd however, more research is needed in order to determine the influence of apoptosis on disease outcomes, particularly in hosts that do not succumb to infection.

                  Intekening vereis. Beveel asseblief JoVE aan by jou bibliotekaris.

                  Openbaarmakings

                  The authors declare no competing financial interests.

                  Erkennings

                  We thank the following people who assisted with husbandry and data collection: D. Tegtmeier, C. De Jong, J. Hawkes, K. Fossen, S. Percival, M. McWilliams, L. Bertola, M. Stewart, N. Harney, and T. Knavel and M. Merces for assistance with dissections. We would also like to thank M. McFadden, P. Harlow and Taronga Zoo for raising the L. v. alpina, and G. Marantelli for raising the P. corroboree. We thank F. Pasmans, A. Martel for advice on apoptosis assays, C. Constantine, A. Kladnik and R. Webb for assistance with TUNEL assay, and T. Emeto and W. Weßels for help with protocol and kit for caspase 3/7 assay. This manuscript and protocol is adapted from Brannelly et al 2017 Peer J 22 .


                  Aansoeke

                  • LB is a rich medium, used as general-purpose bacterial culture medium especially Enterobacteriaceae members (E.coli is one among them).
                  • Lysogeny broth is also used for coliphage plaque assays.

                  Cold Spring Harbor Protocol recommends pH to be 7.0. However, depending on the application pH can be 7.0 - 8.0. Adjust the pH with 1N NaOH and 1N HCl. Although the protein/peptide component of the medium shows some degree of buffering capacity, which is not sufficient for the rapidly growing bacterial population. For general purposes, this is not an issue. However, some labs prefer to make LB in 5-10mM TRIS buffer of the desired pH.


                  7. Freezing and recovery of C. elegans aandele

                  Caenorhabditis elegans can be frozen and stored indefinitely in liquid nitrogen ( − 196 ° C) (Brenner, 1974). The keys to a successful freeze are using animals at the correct stage of development, the addition of glycerol to the freezing media, and a gradual cooling to -80 ° C. Freshly starved young larvae (L1-L2 stage) survive freezing best. Well-fed animals, adults, eggs and dauers do not survive well. It is best to use several plates of worms that have just exhausted the E coli OP50 lawn and that contain lots of L1-L2 animals. A 15% final volume of glycerol in the freezing solution is used. A 1 ° C decrease in temperature per minute is desirable during freezing. This can be achieved by placing the worms (in freezer vials) in a styrofoam container at -80 ° C. The styrofoam container can be either a commercial shipping box (with walls at least ¾ inch thick) or a small styrofoam box with slots for holding vials. After 12 or more hours at -80 ° C, the freezer vials should be transferred to their permanent freezer location for long term storage.

                  The CGC uses two solutions for freezing C. elegans : a Liquid Freezing Solution (Brenner, 1974) and Soft Agar Freezing Solution (Leon Avery, personal communication). For long term storage of stocks in liquid nitrogen, Liquid Freezing Solution is recommended. When this solution is used, the worms settle to the bottom of the freezer vial, and no viable animals can be easily retrieved without thawing the entire contents of the vial. A Soft Agar Freezing Solution is useful for freezing working stocks of C. elegans . The addition of the agar helps keep the worms suspended throughout the solution. A small scoop of the frozen contents can be taken, and the remainder can be left in the vial and returned to the freezer for later use. Vials frozen using Soft Agar Freezing Solution should be stored at -80 ° C. If kept in liquid nitrogen, the contents become too hard, and the vial will need to be warmed before it is possible to remove a scoop of the contents. The warming period reduces the number of times live worms can be recovered from the vial. When stored at -80 ° C there is no need to allow the vial to be warmed the contents are soft enough to be used right away. We recover worms 3-4 times from each vial of soft agar stock.

                  Worms can be frozen in 1.8 ml cryotubes. The CGC uses Nunc Cryotube Vials (#65234) with internal threads and freezes six vials of each strain it receives. Two vials are frozen using Soft Agar Freezing Solution and are stored at − 80 ° C for use as working stocks. Four vials are frozen using Liquid Freezing Solution one is thawed as a tester, and the other three are put in at least two different liquid nitrogen tanks. To fill strain requests, the working stocks that are kept at − 80 ° C are used. When the last vial of a stock stored at − 80 ° C is emptied, the worms are once again frozen using Soft Agar Freezing Solution in order to replace these vials. In theory, the stocks kept in liquid nitrogen will never need to be used since the soft agar stocks are continually replaced as they are depleted.

                  The recovery of C. elegans from stocks stored in liquid nitrogen is in the range of 35-45% of the total number of animals frozen. This number decreases only slightly after many years of storage in liquid nitrogen. The recovery of stocks stored at − 80 ° C for many years ( > 10) is not as high as liquid nitrogen, but worms can be safely stored this way for many years (CGC, unpublished data). Of course, a power failure can result in the loss of all stocks kept at − 80 ° C, so it is very wise to keep at least one copy of all stocks in liquid nitrogen. Some mutants strains (especially certain Dpy mutants) do not survive freezing as well as wild-type animals.

                  Protocol 7. Freezing C. elegans using Liquid Freezing Solution

                  S Buffer [129 ml 0.05 M K2HPO4, 871 ml 0.05 M KH2PO4, 5.85 g NaCl]


                  Kyk die video: Inoculating a TS broth from a bacterial culture (Oktober 2022).