Oprichting en Optimalisatie van een High Throughput Setup om te studeren Staphylococcus epidermidis En Mycobacterium marinum Infectie als een model voor Drug Discovery

The overall goal of this procedure is to generate large numbers of infected zebra fish embryos for high throughput compound testing to enable drug discovery. This is accomplished by first using a large breeding vessel to obtain large numbers of synchronous zebrafish eggs in a single event. Next, the eggs are aligned into an aros grid and infected with bacteria in the yolk using an automated micro injector.

When the infection has spread through the embryos, they are presorted by large particle flow cytometry before being treated with drugs. Finally, the bacterial burden levels in infected embryos are analyzed after compound treatment. Ultimately, high resolution analysis through confocal microscopy vertebra.

Automated screening and large particle sampling are used to show with more detail the effects compounds have on the infection. The main advantage of this method over existing techniques like manual injections or PIX scan analysis, is that with it we can generate a large amount of homogeneously affected embryos, which we can analyze in high throughput manner. To prepare S epiderm inoculum isolate several individual colonies from a plate of S epiderm strain, O 47 containing A PW VW 180 9 derived M cherry expression vector, and inoculate.

25 milliliters of LB supplemented with 10 micrograms per milliliter. Chloramphenicol incubate overnight at 37 degrees Celsius to midlock stage the following day, centrifuge one milliliter of the culture at 12, 000 GS for one minute and use one milliliter of sterile PBS with 0.3%volume per volume between 80 to wash the pellet three times. Measure the optical density at OD 600 and use 2%weight per volume, polyvinyl perone 40 or PVP 40 in PBS to dilute the bacterial suspension to an OD 600 equal to 0.3 or one times 10 to the eight CFUs per milliliter.

To prepare aminum inoculum isolate several individual colonies from M Meum strain M or E 11 stably expressing the PS MT 3M cherry vector from a Middlebrook seven H 10 aerate and inoculate 10 milliliters of Middlebrook seven H nine broth containing 10%volume per volume. Middlebrook A DC enrichment supplemented with 50 micrograms per milliliter of hygromycin incubate overnight at 28 degrees Celsius the following day. Centrifuge one milliliter of the culture at 12, 000 GS for one minute and use one milliliter of sterile PBS with 0.3%volume per volume between 80 to wash the pellet three times.

Measure the OD 600 of the bacterial suspension and with 2%weight per volume. PVP 40 in PBS diluted to an OD 600 of 0.3 or 0.3 times 10 to the eight CFUs per milliliter to obtain zebra fish eggs the night before collection place a maximum of 50 female zebra fish into the bottom chamber of a large breeding vessel and 70 males into the top portion of the vessel. The next morning, remove the separator from the vessel After approximately one hour through the egg collector at the bottom of the breeding vessel, collect the eggs into a 50 milliliter tube filled with egg water.

After preparing injection plates, according to the text protocol on the automated micro injector operating software, click on calibrate stage, then click on 10 24. Well grid and place the aros plate in the micro injector, calibrate the plate by clicking on the screen at the center position of the well. Then go to needle menu and click on calibrate needle holder.

Using a micro loader tip fill a 10 micron diameter tip injection needle with either 10 microliters of PVP 40 containing 100 CFUs per nanoliter of S epiderm, 30 CFUs per nanoliter of erum or PVP 40 alone for mock injections, place the needle in the automated micro injector and calibrate the XY position by lowering or moving the needle up and clicking on the screen at the position of the needle. Then calibrate the Z position of the needle by clicking on the screen at the position of the tip of the needle. Next, use a plastic transfer pipette to distribute the eggs over the aros grid and remove any excess egg water.

Then place the agros grid filled with eggs in the automated micro injector in the injection menu, adjust the injection pressure setting to 200 hectare pascals the injection time to 0.2 seconds, and the compensation pressure to 15 Hector Pascals, which correlates with one nanoliter in the Femto jet settings menu. Click on inject all and inject the entire plate of embryos after injection. Collect the eggs by washing them in a Petri dish and incubate them at 28 degrees Celsius.

Once the large particle flow cytometer is set up, enter the PMT menu and set the red channel to 650 volts and the green and yellow channels to zero volts. Then within the thresholds menu, set the optical density threshold signal to 50, corresponding with 975 millivolts and the time of flight minimum to 800, corresponding with 320 microseconds. In order to reduce the influence of debris, place the embryos in the sample cup and in the sort menu to find the maximum of 70 embryos per plate to be sorted by entering 70.

Place an empty Petri dish under the sorter and click on manual sort. When the Petri dish is filled, save the data by clicking on store. If high resolution analysis or imaging is required, embryos can be sorted individually into wells of a 96 well plate.

Place the embryos in the sample cup and define the maximum of one embryo per well to be sorted. Then position an empty 96 well plate into the left plate holder and click on fill plate. When the plate is filled, save the data by clicking on store to analyze drug treated embryos at three days post injection with erum.

Treat one group of embryos with a compound of interest in solvent and a second group with solvent alone. Place the treated embryos at four and five days post fertilization in the sample cup on the flow cytometer and set the sorting to 70 embryos per plate after anesthetizing embryos in trica, and setting up the vertebrate automated screening system and large particle sampler. According to the text protocol from the imaging object detection setup menu, select the reference images corresponding to the age of the embryos in the imaging auto store images menu, select the number of pictures to be created and orientation to be used.

Place a 96 well plate filled with embryos into the left plate holder of the large particle sampler and click on run plate. When an embryo is correctly positioned, use a 10 x plain dry objective and image the head and tail separately. Then use image processing software to stitch the images together to assess which developmental stage is best for yolk infection.

Injections with dermitis and a marum were performed at all the different stages between the one and 512 cell stage. As seen here injections with 100 CFU of dermitis performed between the 16 and 128 cell stage provided the best infection pattern. The bacteria proliferated inside the yolk for three days and spread into the body from three days post-infection onwards.

High throughput quantification of bacterial burden was performed by fluorescence intensity analysis using the large particle flow cytometer. As shown here, the amount of live bacteria present inside the embryo corresponds very well with the fluorescent signal measured. This figure demonstrates that the optimal developmental stage for injection of 30 CFUs of aminum is between the 16 and 128 cell stage for the E 11 strain and between the 16 and 64 cell stage.

With the more virulent EM strain embryos injected at these stages showed bacterial growth inside the oak and spreading of the bacteria through the embryo. After presort treatment of emerin infected embryos with different concentrations of rifampicin showed a reduction of the mycobacterial infection in a dose dependent manner. Using the drug at a dose of 200 micromolar demonstrated that it effectively halted bacterial progression of M mein E 11, 24 hours and onwards after treatment After its development.

These techniques paved the way for researchers in the field of compound testing and drug development, looking for new treatments for infection diseases such as tuberculosis or biomaterial associated infections.