bacterial inoculation and paraformaldehyde inactivation for caenorhabditis elegans research

Bakteriel metabolisk inaktivering i Caenorhabditis elegans ved hjælp af paraformaldehyd

Caenorhabditis elegans was originally proposed as a model organism in 19651 and has since been widely adopted in studies of genetics, development, behavior, aging, and metabolism2. Due to their large brood size and transparent cuticle, C. elegans is particularly well-suited for high-throughput screening with fluorescent reporters3. Their short life cycle, hermaphroditic reproduction, and genetic homology with humans also make C. elegans a valuable model system for studies on development4 and aging biology5. Moreover, C. elegans are relatively easy to maintain. Worms can be grown in liquid culture or on solid agar plates and consume a diet of live Escherichia coli OP50 bacteria4.
However, the live food source of C. elegans can confound studies of metabolism, drug supplementation, and behavior. Because live bacteria have their own metabolism, experimental conditions that affect the bacteria also alter the nutrients and metabolites available to the worms. For example, differences in bacterial iron, amino acid, and folate concentrations have diverse effects on C. elegans&#39; development, physiology, and lifespan6. Many common lab practices can elicit such changes in the nutrient composition and metabolites produced by OP50. Specifically, exposure to 5-fluoro-2&#39;-deoxyuridine (FUdR), a compound commonly used to prevent reproduction in C. elegans, elicits broad changes in OP50 gene expression, including amino acid biosynthesis pathways7. Live bacteria can also confound studies in which C. elegans are supplemented with small molecules because bacteria can partially or completely metabolize the active compounds. Moreover, the effects of these small molecules on the bacteria can, in turn, alter C. elegans physiology, as was reported with the lifespan-extending drug metformin8. Finally, live bacteria can change the worm&#39;s environment in ways that alter behavior, such as secreting attractive odorants9, producing exogenous neuromodulators10, and creating oxygen gradients in a dense bacteria lawn11.
To mitigate the confounding effects of bacterial metabolism on C. elegans research, multiple methods for killing bacteria have been developed (Table 1). Three common strategies for killing OP50 are UV-irradiation, heat-killing, and antibiotic treatment. While straightforward and relatively low-cost, each of these methods can have undesirable effects on both bacteria and C. elegans. UV-killing via a UV crosslinker12 is low-throughput and the rate is limited by the number of plates that can fit in the UV crosslinker. Moreover, the efficacy of UV-killing can vary from plate to plate within a batch, and testing for growth on all plates can become difficult in large experiments. Heat-killing OP50 by exposing culture to temperatures of &#62;60 &#176;C comes with a separate set of challenges. High heat can damage nutrients essential for the worm and destroy the cellular structure of bacteria, creating a softer texture that decreases the amount of time worms spend on the food13. This method also cannot be used throughout the life cycle of C. elegans because worms fed heat-killed bacteria can arrest early in development13. Antibiotic treatment is a third common method for suppressing bacterial metabolism14, but antibiotics can also alter worm growth and metabolism15.
One solution to eliminate the metabolic effects of live bacteria while preserving bacterial structure and essential nutrients is to kill OP50 with paraformaldehyde (PFA)16. PFA is a polymer of formaldehyde that can crosslink proteins within cells17 to prevent bacterial replication without destroying internal cell structures like the inner plasma membrane18. Due to this preservation of internal cellular structure, PFA-treated bacteria exhibit no growth or metabolic activity but remain an edible and nutrient-rich food source for C. elegans16. Here, a detailed protocol is provided which shows how to metabolically inactivate bacteria using paraformaldehyde.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Method</td>
			<td>Required Materials</td>
			<td colspan="2">Scalable?</td>
			<td>Nutritional?</td>
			<td colspan="4">Effects on Worm?</td>
		</tr>
		<tr>
			<td rowspan="4">UV</td>
			<td rowspan="4">UV-crosslinker</td>
			<td colspan="2">Limited by:</td>
			<td rowspan="4">Yes</td>
			<td colspan="4">Variable effects on lifespan on NGM12, 23, 24</td>
		</tr>
		<tr>
			<td colspan="2">Number of plates that fit in UV-crosslinker</td>
			<td colspan="4">Variable effects on lifespan on FUdR24, 26, 27</td>
		</tr>
		<tr>
			<td colspan="2">Irradiation time per plate</td>
			<td colspan="4" rowspan="2">Decreased food preference16</td>
		</tr>
		<tr>
			<td colspan="2">Ability to check every plate for growth8</td>
		</tr>
		<tr>
			<td rowspan="3">Heat</td>
			<td rowspan="3">&#62;60 &#176;C incubator</td>
			<td colspan="2" rowspan="3">Yes</td>
			<td rowspan="3">No: destroys cell wall, decreased nutritional value</td>
			<td colspan="4">Developmental arrest 13</td>
		</tr>
		<tr>
			<td colspan="4">Decreased food preference13</td>
		</tr>
		<tr>
			<td colspan="4">Extends lifespan on NGM31</td>
		</tr>
		<tr>
			<td rowspan="3">Antibiotics</td>
			<td rowspan="3">Antibiotics (kanamycin, carbenicillin, etc.)</td>
			<td colspan="2" rowspan="3">Yes</td>
			<td rowspan="3">Yes</td>
			<td colspan="4">Delays growth and development15</td>
		</tr>
		<tr>
			<td colspan="4">Extends lifespan in liquid media19</td>
		</tr>
		<tr>
			<td colspan="4">Extends lifespan on NGM15</td>
		</tr>
		<tr>
			<td rowspan="3">PFA</td>
			<td rowspan="3">0.5% Paraformaldehyde</td>
			<td colspan="2" rowspan="3">Yes</td>
			<td rowspan="3">Yes</td>
			<td colspan="4">Small brood size decrease16</td>
		</tr>
		<tr>
			<td colspan="4">Small development time increase16</td>
		</tr>
		<tr>
			<td colspan="4">Decreased food preference16</td>
		</tr>
	</tbody>
</table>
Table 1. Comparisons of methods to kill OP50. UV-killing, heat-killing, antibiotic-treatment, and PFA-treatment have varied effects on the nutritional status of the bacteria and the health of worms fed treated bacteria. These methods for replicatively inactivating E. coli also differ in their required materials and scalability.

Forfatter Spotlight: Fremme af Caenorhabditis elegans-forskning ved hjælp af paraformaldehydbehandlede bakterier 

Metode til metabolisk inaktivering af bakterier til Caenorhabditis elegans Research

A challenge in sea elegance research is isolating the effects of a live bacterial diet from the effects of experimental manipulations. It can be difficult to determine whether the manipulation directly impacts the host or indirectly impacts the worms through a host microbiome interactions. Paraformaldehyde treated bacteria address the challenge of isolating the effects of bacterial metabolism from host physiology.Paraformaldehyde treatment is high throughput and consistently renders the bacteria replicate and metabolically inactive. In addition to its consistency PFA treated bacteria remain a palatable and nutritious food source.

Watch this Scientific Journal Video about Bacterial Inoculation and Paraformaldehyde Inactivation for  Caenorhabditis elegans  Research at JoVE.com

Bacterial Inoculation and Paraformaldehyde Inactivation for  Caenorhabditis elegans  Research  

Bakteriel podning og paraformaldehydinaktivering for Caenorhabditis elegans Forskning

Begynd med at inokulere en enkelt bakteriekoloni i 200 ml steriliseret Luria bouillon i en 500 ml Erlenmeyerkolbe. Dyrk bakterierne natten over i en shaker-inkubator indstillet til 37 grader. Efter 14 timers kultur skal du kontrollere bakteriens optiske tæthed ved 600 nanometer.Aliquot bakterierne i 50 ml koniske rør og opbevar dem ved fire grader Celsius til videre brug. Ved hjælp af en serologisk pipette overføres 50 ml af bakteriekulturen til en ny 250 ml Erlenmeyerkolbe Mærket rørene til levende eller mock-behandlet kontrol. Brug en serologisk pipette til at overføre 50 ml af bakterierne til et 50 ml konisk rør til den mock-behandlede kontrol før vask.I den kemiske hætte tilsættes 781 mikroliter paraformaldehyd til bakteriekulturen for at opnå en endelig koncentration på 0,5%Bortskaf den brugte spids i en kemisk farebeholder til fast affald. Derefter dækkes kolben med et folieark og anbringes i en rysteinkubator ved 37 grader Celsius i en time. Når inkubationen er afsluttet, vaskes bakterierne for at fjerne eventuel resterende paraformaldehyd.Til fjernelse af det resterende paraformaldehyd overføres de behandlede bakterier fra Erlenmeyerkolben til et 50 ml konisk rør ved hjælp af en serologisk pipette. Derefter centrifugeres de behandlede bakterier ved ca. 3000 G i 20 minutter. Supernatanten kasseres i en kemisk farebeholder til flydende affald inde i kemikaliehætten.Tilsæt nu 25 ml Luria bouillon til de behandlede og kontroller bakteriepiller og hvirvel for at resuspendere bakteriepillerne. Gentag centrifugeringen og pelletopslæmningen fire gange. Som forberedelse til forskellige analyser resuspenderes pelleten i passende mængder.Til levetidsanalyser. Frø 200 mikroliter bakterier resuspenderet i 10 ml Luria bouillon på 60 millimeter plader. Opbevar bakterierne ved fire grader Celsius.For at udføre en kvalitetskontrol af bakterievæksten skal du stribe en Luria bouillonplade med en forberedt bakterie ved hjælp af en steril pipettespids. For at kontrollere forurening, bøf Luria bouillon, der bruges til vask og resuspendering på en separat plade. Placer derefter pladerne i en 37 grader Celsius inkubator natten over.Den næste dag skal du kontrollere, om der er bakterievækst. Gem bakterierne, hvis paraformaldehydpræparatet er vellykket, og kolonierne ikke vokser på LB-pladen. For at kontrollere den bakterielle metaboliske aktivitet med et respirometer skal du først hydrere patronen ved at tilsætte 200 mikroliter kalibrant til alle brønde på en 96-brøndplade.Anbring patronen i 96-brøndpladen og inkuber natten over ved 37 grader Celsius. Indstil maskinen til at blande, vægte, måle og sløjfe. Næste dag skal du placere den hydrerede patron i maskinen.Til en ny 96-brøndsplade tilsættes 160 mikroliter M9 til testbrøndene efterfulgt af 40 mikroliter prepped bakterier. Tilsæt 200 mikroliter M9 i de fire hjørnebrønde for at fungere som emner. Dispenser 160 mikroliter M9 og 40 mikroliter Luria bouillon som negative kontroller.Til sidst pipetteres 200 mikroliter M9 i de resterende ubrugte brønde. Udskift derefter den udskubede kalibreringsplade med analysepladen, før du kører programmet. Resultaterne vises som iltforbrugshastigheden.Forskellige stammer udviste forskellige inaktiveringsresponstider for paraformaldehyd. For eksempel er eksponering af HB101-bakterier til 0,25% i en time tilstrækkelig til at gøre den metabolisk inaktiv. Enterococcus faecalis krævede en koncentration på 1,0% for at opnå ensartet effekt.Caenorhabditis elegans orme udviste lignende præferencer over for de behandlede OP 50 stammer såvel som den mock-behandlede kontrol. Imidlertid afslørede et følsomt parvis assay en stærkere præference for den mock-behandlede kontrol. Orme på behandlet OP 50 har en højere pumpehastighed end orme på den mock-behandlede kontrol.Forbrug af behandlede bakterier resulterede i forsinket udviklingstid hos vildtypeorme samt nedsat æglægningskapacitet. Ormens levetid var ikke signifikant forskellig fra hinanden, uanset fødekilden. Imidlertid øgede de paraformaldehydbehandlede bakterier ormens levetid i nærvær af 5-fluor-2'deoxyuridin Forbrug af de paraformaldehydbehandlede bakterier af en langlivet ormstamme ændrede ikke dens levetid.

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Bacterial Inoculation and Paraformaldehyde Inactivation for Caenorhabditis elegans Research

Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a ...