We thank Dr. Bing-Yan Wang, Dr. Gena Tribble (The University of Texas, School of Dentistry), Dr. Richard Lamont (University of Louisville, School of Dentistry), and Dr. Samuel Shelburne (MD Anderson Cancer Center) for providing laboratory and clinical strains of the mitis group streptococci. We also thank Dr. Keith Blackwell (Department of Genetics, Harvard Medical School) for the C. elegans strains. Finally, we thank Dr. Danielle Garsin and her lab (The University of Texas, McGovern Medical School) for providing reagents and worm strains to conduct the study. Some worm strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

1. Preparation of THY (Todd-Hewitt Yeast Extract) Agar Plates
<ol>
	<li>For 1 L of media, add 30 g of Todd-Hewitt powder, 2 g of yeast extract and 20 g of agar to a 2 L Erlenmeyer flask. Add 970 mL of deionized water to the contents of the flask and include a stir bar. Autoclave the media at a temperature of 121 &#176;C and pressure of 15 lb/inch2 for 30 min. Thereafter, set the media on a stir plate and allow for cooling with gentle stirring.</li>
	<li>Pour the media into appropriately sized sterile Petri d...

<ol>
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The authors declare that they have no competing financial interests.

The methods described can be used for other pathogenic bacteria such as Enterococcus faecium, which also produces H2O2 grown under anaerobic or microaerophilic conditions26. Typically, for most pathogenic organisms, it takes several days to weeks to complete the survival assays. However, due to the robust production of H2O2 by members of the mitis group, these assays could be completed within 5-6 h under the conditions described. This ensures th...

Mitis group streptococci are human commensals of the oropharyngeal cavity1. However, these organisms can escape this niche and cause a variety of invasive diseases2. The infections caused by these microorganisms include bacteremia, endocarditis, and orbital cellulitis2,3,4,5,6. Furthermore, they are emerging as causative agents of bloodstream infections in immunocompromised, neutropenic, and cancer patients that have undergone chemotherapy5,7,8,9.
The mechanisms underlying mitis group pathogenesis is obscure, because few virulence factors have been identified. The mitis group is known to produce H2O2, which has shown to play an important role in oral microbial communities10. More recently, several studies have highlighted a role for H2O2 as a cytotoxin that induces epithelial cell death11,12. S. pneumonia, which belongs to this group, has been shown to produce high levels of H2O2 that induces DNA damage and apoptosis in alveolar cells13. Using an acute pneumonia animal model, the same researchers demonstrated that production of H2O2 by the bacteria confers a virulence advantage. Studies on pneumococcal meningitis have also shown that pathogen-derived H2O2 acts synergistically with pneumolysin to trigger neuronal cell death14. These observations clearly establish that H2O2 produced by this group of bacteria is important for their pathogenicity.
Interestingly, it has also been shown that members of the mitis group S. mitis and S. oralis cause death of the nematode C. elegans via the production of H2O215,16. This free-living nematode has been used as a simple, genetically tractable model to study many biological processes. More recently, the worm has emerged as a model to study host-pathogen interactions17,18. In addition, several studies have highlighted the importance of studying oxidative stress using this organism19,20,21. Its short life cycle, ability to knockdown genes of interest by RNAi, and use of green fluorescent protein (GFP)-fused reporters to monitor gene expression are some of the attributes that make it an attractive model system. More importantly, the pathways that regulate oxidative stress and innate immunity in the worm are highly conserved with mammals20,22.
In this protocol, it is demonstrated how to use C. elegans to elucidate the pathogenicity caused by streptococcal-derived H2O2. A modified survival assay is shown, and members of the mitis group are able to kill the worms rapidly via the production of H2O2. Using members of the mitis group, a sustained biological source of reactive oxygen species (ROS) is provided, as opposed to chemical sources that induce oxidative stress in the worms. Furthermore, the bacteria are able to colonize the worms rapidly, which allows for H2O2 to be directly targeted to the intestinal cells (compared to other sources that have to cross several barriers). The assay is validated either 1) by determining the survival of the skn-1 mutant strain or 2) by knocking down skn-1 using RNAi in worms relative to the N2 wild-type and vector control treated worms. SKN-1 is an important transcription factor that regulates the oxidative stress response in C. elegans23,24,25. In addition to survival assays, a worm strain expressing a SKN-1B/C::GFP transgenic reporter is used to monitor activation of the oxidative stress response via the production of H2O2 by the mitis group.

<table border="0" cellpadding="0" cellspacing="0" style="width:845px;" width="845">
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Media and chemicals</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Agarose&#160;</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:119px;">A9539-50G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Bacto peptone&#160;</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">DF0118-17-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">BD Bacto Todd Hewitt Broth</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">DF0492-17-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">BD BBL Sheep Blood, Defibrinated&#160;&#160;</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">B11947</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">BD Difco Agar&#160;</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">DF0145-17-0</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">BD Difco LB Broth</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">DF0446-17-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Blood agar (TSA with Sheep Blood)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">R01200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Calcium Chloride</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP510-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Carbenicillin</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP26481</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Catalase&#160;</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td>C1345-1G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Cholesterol</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">ICN10138201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">IPTG</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">MP21021012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Magnesium sulfate</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP213-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Nystatin</td>
			<td style="width:179px;">Acros organics</td>
			<td style="width:119px;">AC455500050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Potassium Phosphate Dibasic</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP363-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Potassium phosphate monobasic</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP362-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Sodium Azide</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:119px;">S2002-25G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Sodium chloride&#160;</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP358-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Sodium Hydroxide</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">SS266-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">8.25% Sodium Hypochlorite</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Sodium Phosphate Dibasic&#160;</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP332-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Streptomycin Sulfate&#160;</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP910-50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Tetracyclin</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:119px;">87128-25G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">(&#8722;)-Tetramisole hydrochloride</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:119px;">L9756</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Yeast extract</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP1422-500&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Consumables&#160;</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">15mL Conical Sterile Polypropylene Centrifuge Tubes&#160;</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">12-565-269</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Disposable Polystyrene Serological Pipettes 10mL</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-574</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Disposable Polystyrene Serological Pipettes 25mL</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-575</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Falcon Bacteriological Petri Dishes with Lid (35 x 10 mm)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">08-757-100A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">No. 1.5&#160; 18 mm X 18 mm Cover Slips</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">12-541A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Petri Dish with Clear Lid (60 x 15 mm)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875713A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Petri Dishes with Clear Lid (100X15mm)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875712</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plain Glass Microscope Slides (75 x 25 mm)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">12-544-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software&#160;</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Prism</td>
			<td style="width:179px;">Graphpad</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Bacterial Strains</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">S. oralis ATCC 35037</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">S. mitis ATCC 49456</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">S. gordonii DL1 Challis&#160;&#160;</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">E. coli OP50</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">E. coli HT115</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Worm Strains</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Strain</td>
			<td style="width:179px;">Genotype</td>
			<td style="width:119px;">Transgene</td>
			<td>Source</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">N2</td>
			<td style="width:179px;">C. elegans wild isolate</td>
			<td style="width:119px;"></td>
			<td>CGC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">EU1</td>
			<td style="width:179px;">skn-1(zu67) IV/nT1 [unc-?(n754) let-?] (IV;V)</td>
			<td style="width:119px;"></td>
			<td>CGC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">LD002</td>
			<td style="width:179px;">IdIs1</td>
			<td style="width:119px;">SKN-1B/C::GFP + rol-6(su1006)</td>
			<td>Keith Blackwell</td>
		</tr>
	</tbody>
</table>

studying oxidative stress caused by the mitis group streptococci in caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Members of the mitis group S. mitis, S. oralis, and S. gordonii rapidly killed the worms, as opposed to S. mutans, S. salivarius, and non-pathogenic E. coli OP50 (Figure 3A). The median survival for S. mitis, S. oralis, and S. gordonii was 300 min, 300 min, and 345 min, respectively. To determine if the killing was mediated by H2O2, catalase...

Watch this Scientific Journal Video about Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans at JoVE.com

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

In this protocol we demonstrate how to use a free living method c. elegans to elucidate the pathogenicity caused by hydrogen peroxide produced via Mitis Group streptococci. The main advantage of this technique is that we can study pathogenicity and the activation of stress responses in the worm by using a sustainable biological source of reactive oxygen species as opposed to chemical sources For one liter of media add 30g of Todd Hewitt powder, 2g of yeast extract and 20g of ager to a 2 liter Erlin Myer flask.Add 970ml of deionized water to the contents of the flask and put in a stir bar. Use aluminum foil to cover the opening. Autoclave the media in the flask at a temperature of 121 degrees Celsius and pressure of 15 pounds per square inch for 30 minutes.Thereafter set the flask on a stir plate to cool down with gentle stirring. Under a laminar hood, pour the media into appropriately sized, sterile Petri dishes. Allow the media to dry for 2 hours.Thereafter store the plates at 4 degrees Celsius for up to 1 month. Before the preparation of Mitis Group streptococci, warm the THY agar plates in an incubator to 37 degrees Celsius. After that use a sterile loop to streak out desired strains of strepticocci on the places.Then put the plates in a candle jar and incubate the jar at 37 degrees Celsius overnight for around 18 hours to provide a microerophilic environment. The next day, remove the plates from the candle jar and use a sterile loop to pick isolated colonies. Inoculate 15 ml sterile conical tubes containing 2 ml of THY broth.Close the caps tight and incubate the tubes at 37 degrees Celsius under static conditions. Pre-warm THY plates in an incubator to 37 degrees Celsius add 50 microliters of solution containing 1000 units of catalase on to the THY plate. Use a sterile spreader to spread the catalase solution and allow the plates to dry on the laminar flow hood for 30 minutes.To seed the plates with the streptococcus strains, use a pipette to add 80 microliters of overnight grown cultures to the plate and use a sterile spreader to spread the bacteria completely across the agar surface. Incubate the plates at 37 degrees Celsius in a candle jar overnight. As a control, on two NGM plates, add 80 microliters of overnight grown cultures of E.coli OP-50 for seeding.Incubate the plates at 37 degrees Celsius overnight. The next day, remove the plates from the candle jar and allow the plates cool to room temperature. Using a sterile worm pick, transfer 30 L4 larvae from the NGM feeding plates to the streptococcus seeded THY plates.Incubate the plates at 25 degrees Celsius. Under a dissecting microscope, count the number of live and dead L4 larvae on each plate at several time points. Use the sterile worm pick to gently prod the worms and determine if they are dead or alive.Initially, score the worms as dead or live every 30 minutes. When worms rapidly start to die, score them at 15 minute intervals. The assay will take 5 to 6 hours to complete.After that, repeat the experiment two more times. To prepare the agarose pad, stick labtape lengthwise along two glass slides. This will determine the thickness of the agarose pads.Place a clean glass slide between the two taped slides. Dissolve agarose in deionized water to make 2 weight volume percent and heat the solution in a microwave. Use a pipette to place 100 microliters of molten agarose on the center of the clean slide.Immediately place another clean glass slide on top of the molten agarose and gently press down to make a pad. Allow the agarose to solidify and subsequently remove the top slide. The agarose pad is ready for use.Pre-warm THY plates to 37 degrees Celsius. Seed the plates with a streptococcus strains as done previously in the survival assays. Seed 3 NGM plates with E-coli OP-50 as control and incubate at 37 degrees Celsius overnight.The next day, cool the plates to room temperature. Use M9W buffer to wash L4 larvae from NGM or NGM RNAi feeding plates into 15 ml conical tubes. Spin the tubes at 450 times g, for one minute in a centrifuge.Decant the supernatant and add 10 ml of M9W to each tube. Repeat the centrifugal step three more times. Re-suspend the worms in 250 microliters of M9W and place three 5 microliter drops of the worm suspension on to a clean Petri dish lid.Under a dissecting microscope, estimate the number of worms per microliter. Using a pipette, add approximately 100 L4 larvae to each THY streptococcus seeded and NGM E.coli seeded plates. Use three plates per strain of bacteria.Incubate the plates for 2 to 3 hours at 25 degrees Celsius. Thereafter, remove the plates from the incubator. Wash them with M9W and collect the worms in 15ml conical tubes.Wash the worms three times as done previously in the centrifugal step. Remove most of the M9W by aspiration. And add 500 microliters of M9W containing 2 millimolar sodium azide or tetramisole hydrochloride to the worm pellet for anesthesia.Incubate the tube with worm pellets at room temperature for 15 minutes. Then, drop 15 microliters of the worm suspension on to a prepared agorose pad. Under a fluorescent microscope, visualize the localisation of skn-1 utilizing Fitzy and Dappy filters.Image worms at 10 times and 20 times magnifications. Score the worms based on three levels of localisation;low localisation, where no nuclear localisation is observed, medium localisation, with SKN 1 BCGFP in the anterior or posterior of the worm, and high localisation, where nuclear localisation of skn-1 BCGFP is observed in all intestinal cells. In this experiment, members of the Mitis Group rapidly killed the worms, as opposed to S.mutans, S.salivarius and non pathogenic E.coli OP-50.When catalase was supplemented to THY ager, the killing of the worms was abolished. Death of the worms was not observed on the delta spxB mutant strain compared to the wild-type and compliment strains. These data suggest that the hydrogen peroxide produced by the Mitis Group mediates killing of the worms.A significant decrease in the survival of the skn-1 knockdown worms was observed compared to the vector control treated worms. A similar killing phenotype was observed with the skn-1 mutant strain and the N2 wild-type worms. This demonstrates that skn-1 influence the survival of the worms on the Mitis Group.Localisation of skn-1 BCGFP was observed in worms exposed to the wild-type N-compliment strains and not in response to the Delta spxB mutant strain of the S cordonae. After components of the P38 map kinase pathway were knocked down, reduced localisation of skn-1 BCGFP and knockdown treated worms, relative to the vector control treated worms, was observed. By using the streptococci c.elegans system, the effects of hydrogen peroxide on endoplasmic verticular stress, mitochondrial damage, mitophogy, and tophogy can be further studied. Further more, mechanisms by which hydrogen peroxide acts as a virilescence factor to elicit immune responses by disrupting core processes of the cell can be identified.

studying-oxidative-stress-caused-mitis-group-streptococci

Research

JoVE Journal

Developmental Biology

7.0K visualizações.  University of Texas Health Science Center. Neste protocolo demonstramos como usar um método de vida livre c. elegans para elucidar a patogenicidade causada pelo peróxido de hidrogênio produzido através de estreptococos do Grupo Mitis. A principal vantagem desta técnica é que podemos estudar a patogenicidade e a ativação das respostas ao estresse no verme usando uma fonte biológica sustentável de espécies de oxigênio reativo em oposição a fontes químicas Para um litro de mídia adicionar 30g de pó todd hewitt, 2g de ext...