This protocol describes how to screen for bacteria with impact on C.elegans oxidative thermal stress resistance in two C.elegans strains on 48 bacterial isolates in parallel. This approach is versatile and scalable. It affords rapid screening of multiple conditions with impact on C.elegans against health, which is applicable to the study of mechanisms of health and diseases.
Using stress resistance as a proxy for health, this pipeline is readily applicable to the preclinical screening of drugs, antiemetics, and probiotics on nematode mutant, knockdown, transgenic, and disease models. The capacity to test many conditions in parallel enables the study of complex interactions involved and serve physiological processes. These include host microbe interactions, metabolic disorders, stress handling, and aging.
It is critical to avoid contamination throughout. Do not allow worms to run out of food, and perform key steps as soon as the worms reach the required stage. Start with the preparation of a concentrated E.coli OP50 bacteria culture by inoculating four one liter bottles of lysogeny broth with two milliliters of OP50 starter culture each.
Then place the bottles in a shaking incubator for six hours at 37 degrees Celsius and 160 G, followed by pelleting the bacteria at 3057 G and 20 degrees Celsius for 15 minutes. Afterwards, discard the supernatant and re-suspend the pellets with six milliliters of OP50 medium. Collect the concentrated culture in a sterile 50 milliliter conical tube.
Inoculate eight six centimeter NGM plates per strain with 100 microliters of saturated OP50 culture grown overnight at 37 degrees Celsius per plate. Then keep plates at 20 degrees Celsius for two days before use. Then cut a square agar chunk of 0.5 centimeters with worms from a recently starved NGM plate using a scalpel, and transfer onto each of the eight inoculated six centimeter NGM plates.
Incubate these plates at 20 degrees Celsius for three days. Next, re-suspend the worms by adding up to three milliliters of sterile M nine buffer to the six centimeter NGM plates using a P1000 pipette, and collect the worm solution from all eight plates per strain in a single 15 milliliter conical tube. Then centrifuge at 142 times G for two minutes at four degrees Celsius, and carefully remove the supernatant using a P5000 pipette or a water pump equipped with a sterile Pasteur pipette or tip.
Afterward, add 10 milliliters of sterile M nine buffer to wash the worm pellet. Next, remove the supernatant and transfer the worms onto a 15 centimeter OP50 inoculated NGM plate supplemented with concentrated OP50 using a pipette. Grow each worm strain on a 15 centimeter diameter NGM plate for three to four days at 15 degrees Celsius by re-feeding the worms with 0.5 milliliters of concentrated OP50 daily.
After collecting and washing an M nine buffer, transfer each worm strain culture to more NGM plates, and propagate worms at 20 degrees Celsius until 95%of the population are gravid adults. Then bleach the gravid adult worms by following the standard egg preparation method, and transfer the eggs onto two unseeded 15 centimeter NGM plates for 24 hours at 15 degrees Celsius to allow for all the L one larvae to hatch and grow synchronously in the subsequent steps. First, collect all the bacterial mass previously grown on a six centimeter LB agar plate, and transfer it to a labeled 1.5 milliliter microcentrifuge tube containing one milliliter of M nine buffer.
Then vortex the microcentrifuge tube until the bacterial pellets are fully re-suspended. Next, spin down at 9, 300 times G for five minutes at room temperature, and remove 700 microliters of supernatant. Again re-suspend the bacterial pellet by vortexing.
Following, transfer 200 microliters of each bacterial suspension into a single well of an empty sterile 96 well plate. From this plate, inoculate eight 96 well NGM agarose plates with 10 microliters of bacterial solution using a multi-channel pipette, and incubate with the lid on at 25 degrees Celsius for 24 hours. Afterward, seal the 96 well suspension plate with clean adhesive ceiling film, and store it at 15 degrees Celsius for up to five days.
To start, assess the developmental stage of a synchronized worm population by looking at the plates. Once more than 90%of worms have reached L four stage, collect the worms in up to 10 milliliters of sterile M nine solution in 15 milliliter conical tubes. Next, wash the worms extensively for four times by spinning down at 142 times G for two minutes at four degrees Celsius, while adding 10 milliliters of fresh sterile M nine between each wash to get rid of OP50 bacteria.
Then remove the supernatant, and re-suspend the worm pellet in 10 milliliters of M nine. Transfer 50 microliters of worm solution into a 1.5 or two milliliter low surface binding tube containing 950 microliters of M nine. After gently mixing the tube contents to avoid worm sedimentation, quickly use a wetted low bind pipette tip to transfer three to four separate 10 microliter drops onto a glass slide or an NGM plate.
Under a stereo microscope at 16X magnification, count number of worms and average the counts from three to four drops to determine the number of worms per microliter in the worm solution. Adjust worm concentration to 15 worms per microliter in the 15 milliliter conical tube. Then transfer eight microliters of worm solution into each of the wells of the eight 96 well NGM agarose plates using a multi-channel pipette or repeat pipette.
After 36 hours, dispense 30 microliters of M nine into each well of the 96 well plate. Next, transfer worms to the 384 well plate according to set layouts using low retention tips, ensuring that the plate readers are set up properly. Afterward, top up the 384 well plates with more M nine, aiming for a final volume of 60 microliters per well by adding 40 microliters of M nine for thermal stress assay and 34 microliters of M nine in six microliters of TBHP for TBHP-induced oxidative stress.
Then start the assay within two minutes of adding TBHP. Next, close the plates with their transparent lid, and seal the edges of the 384 well plates with masking tape, ensuring that the tape does not go over the lid or under the plate. Then insert the plate into the plate reader.
Determine a fluorescence value below which a peak would not be significantly different from noise, and note the earliest time point when fluorescence fluctuations dampen prior to rising. Then note the time points between which minimal and maximal fluorescence values are expected to fall, as they will be used for curve fitting. Afterward, check if the amplitudes of the fluorescence peaks vary significantly between the wells.
Normalize the data prior to further analysis using the formula as described in the manuscript. First, download LFASS from GitHub, and launch MATLAB, and navigate to the LFASS folder. Then type and run fit folder in the command window, following the onscreen instructions.
After typing in fit folder, type in the name of your data folder in which the Excel file is located as My Data. Then enter two for the time interval between successive measurements in the current protocol, and type the noise threshold value. Next, assign the upper tolerance threshold by typing 0.94, and the lower tolerance threshold by typing 0.06 to constrain the sigmoid fit.
Set time intervals to find maximum and minimum values. To visually inspect the converging fits, type Y for yes or N for no for not displaying fitted and smooth curves when prompted. Next, provide new, lower, and upper time boundaries to attempt refitting.
If you wish to attempt the refit, type two. But if you wish to accept the refit and move on to the next curve, type zero. In order to choose whether to analyze curves identified as noise or refit poorly fitted curves, either type Y to approve and N to reject.
After refitting has been attempted or declined for all remaining curves, the program ends. Retrieve result files from the results folder, and save as dot XLSX for downstream analysis. Heat and oxidative stress resistance assays were performed on Bristol N two wild type worms, which were fed on either MYB11 or MYB115 bacterial isolates or E coli OP50 control bacteria.
After 36 hours, the adult wild type worm populations were exposed to 42 degrees Celsius thermal stress and 7%TBHP-induced oxidative stress. The median time of death varied between 40 to 130 minutes for the thermal stress assay, and between 90 to 240 minutes for the oxidative stress assay. To make sure the worms are grown in step 2.5, and step 2.1 may take up to two hours, draw a plate layout to prepare worms from 96 well to 284 well plates in step 4.6.
Steps can be added or substituted. For instance, host genome wide RNA screens or bacterial genetic screens can be performed by substituting gut bacterium isolates for RNI clones or bacterial mutants respectively. So we're currently using this approach to identify new probiotics and to uncover gene regulatory networks involved in host micro interactions in the context of infections and aging.
