The overall goal of this procedure is to observe fluorescently labeled bacterial symbiance within their nematode host. This is accomplished by first labeling the bacteria with a fluorescent protein through conjugation. The second step is to isolate AIC nematodes by harvesting eggs.
Next, the AIC nematodes are grown in combination with their fluorescently labeled bacterial symbiant to allow natural association ultimately the localization of the bacterial symbiant within the nematode host. And the frequency of this association within the nematode population can be determined by fluorescence microscopy. This method can help answer key questions in the symbiosis field, such as where do bacteria localize within their host, and what is the distribution of bacterial carriage across a host population?
After overnight growth of the bacterial strains, subculture, the donor recipient and helper strains into nutrient rich growth media lacking antibiotics in a one to 100 ratio of culture to medium, then grow the cultures at the temperature appropriate for each strain until they reach mid log stage growth. Next, combine the strains in a single micro centrifuge tube and centrifuge the mixed cultures for two minutes At 17, 900 Gs, decant the supernatant and spen the pellet in 30 microliters of fresh media. Then spot the suspension onto a nutrient rich media plate without any antibiotics, and allow the spot to dry when the suspension has dried.
Incubate the plate inverted overnight at a temperature optimal for the recipient bacterium and permissible for the donor and helper if applicable. Now, scrape up the spot and streak for single colonies on a selective antibiotic plate to obtain a pure culture of bacteria streak, a single colony for isolation. Finally, ensure that the resulting colonies are the recipient symbiant and not the donor strain.
By screening for the presence of recipient specific phenotypes. For example, cino abdi bacteria can be distinguished from e coli by performing a catalyst test screened for the presence of the plasmid by confirming fluorescence under the wavelength corresponding to the fluorescent plasmid protein. After growing the natural bacterial symbiant overnight spread 600 microliters of the bacterial culture onto eight to 10, 10 millimeter lipid aerates, and then incubate the plates in the dark without moisture at 25 degrees Celsius.
After two days, add 5, 000 infective juvenile nematodes in 500 microliters of media to the bacterial lawns. After incubating the plates at 25 degrees Celsius for three more days, place 20 microliters of water onto a microscope slide. Then use a sterile stick to scrape up a small amount of nematodes from the bacteria lawn.
Place the stick into the water to allow the nematodes to swim off. Look at this slide under low magnification. If eggs and females are visible, continue when the females contain eggs, place a few milliliters of water on the surface of the plates, gently swirl the plates and then pour the water into a 50 milliliter conical tube.
The nematodes should come off the plates and no longer be visible on the plate surface. Allow the nematodes to settle to the bottom of the tube, then pipe at the excess water from the top of the tube and refill the tube with clean water. After allowing the adult nematodes to settle and pipetting off the excess water once more, fill the conical tubes with egg solution.
Then mix the tubes by gentle inversion for exactly 10 minutes at room temperature. After shaking immediately centrifuge the conical tubes for exactly 10 minutes at 1, 250 Gs and room temperature with the break on. Then quickly decant the supernatant, resuspend the pellet with egg solution by pipetting and fill the conical tube with fresh egg solution.
Mix the egg suspension well by inverting the tube three to five times and then after immediately pelleting the eggs and decanting the supernatant as just shown. Resuspend the pellet in misogyny, broth or LB by pipetting, and then transfer the egg suspension to a 15 milliliter conical tube. Now fill the 15 milliliter tube with lb, wash the eggs three times in the broth using the same rapid step technique as just demonstrated, and then dilute the resuspended P nematode eggs to at least 10 eggs per microliter.
Transfer the eggs to a six centimeter Petri dish containing five milliliters of LB and antibiotics against colonizing bacteria. The plate may be stored wrapped in perfil for up to four days after overnight growth and selection of the fluorescent bacteria. Use a sterile stick to spread 600 microliters of the bacterial culture onto 10 millimeter lipid aerates, and to incubate the culture at 25 degrees Celsius.
After two days, dispense 500 to 5, 000 a nematode eggs onto each lipid plate. If the plate has been stored, wash the eggs in 15 milliliters of LB as just demonstrated at least once prior to inoculating the eggs onto the previously prepared bacterial lawns. Then incubate the nematode bacteria co cultures in the dark without moisture at 25 degrees Celsius until infective juveniles appear as a fuzzy white ring on the edge of the plate.
When the infective juveniles have been observed. Remove the lid of the lipid agar plate and place the bottom of the plate into the bottom of an empty 100 millimeter by 20 millimeter Petri dish. Then fill the Petri dish with enough water to come up to about half the height of the smaller plate and incubate the water trap until the progeny infective juveniles have emerged into the water.
After collecting the nematodes from the water or at the desired life stage from the appropriate bacterial lawn, dissolve a few grains of ole in 30 microliters of water, and at one to two microliters of this paralyzing agent for each 50 microliters of nematode sample. Then transfer about 20 to 30 microliters of the paralyzed nematode sample to a microscope slide and place a cover slip on top of the sample. View the nematodes using light microscopy to ensure nematodes are in the field of view.
To identify bacterial localization. Photograph the nematodes under fluorescent microscopy in addition to the light microscopy setting, and then superpose the images. It may be necessary to try several magnifications and views of the nematode to find the bacteria.
A population of the nematodes from two media were counted and scored for colonization by the bacterial symbiant. For robust statistics, it is best to count at least 100 nematodes per sample with at least 30 falling into each category as seen in the table. These nematodes are colonized at a level of approximately 14.6%when grown on lipid agar and 68.6%when grown on liver, kidney agar.
Other nematode and bacterial species have been shown to have different levels of colonization. This schematic illustrates the general appearance of Steiner NEMA females. The inset shows the differential interference contrast image of an S fot graphic female at 20 x magnification.
The black arrow in the inset indicates the vulva, whereas the white arrows indicate visible eggs. This image shows a developed but unhatched volt nematode egg at 40 x magnification, and these eggs isolated from s Volta nematodes were imaged under 10 x magnification. Here, representative microscope images of Steiner Nima nematodes associated with Xeno aptus bacteria are shown in this first image.
Alvin's nematodes were associated with their bacterial symbiant expo expressing GFP to create this composite image. A phase contrast image was overlaid with a fluorescent image. The arrow indicates the bacteria present within the infective juvenile nematode.
This image shows an S carpa capsi juvenile nematode with GFP expressing X nla. The image was constructed in a similar manner as the previous image and depicts the juvenile nematode with green fluorescent protein labeled bacteria visualized as the green rods localized throughout the nematode intestinal lumen. In addition to the described procedure, other methods like genetic manipulation of the bacterial symbiant prior to association with the nematode host can be incorporated in order to answer additional questions such as what are the molecular mechanisms necessary for the host microbe association.
