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The overall goal of this procedure is to use the larvae of the wax moth Galleria melanoma to analyze the pathogenesis of the bacterial pathogen legionella pneumophila. To begin. This is accomplished by infecting larvae with l pneumophila strains by injection during the next 72 hours.

Induction of larval mortality is indicated by a loss of mobility and the appearance of black pigmentation larvae are sacrificed for their hemolymph at various time points. The hemolymph is then plated to enumerate the bacteria and visualize the insect cells. Ultimately, the analysis of bacterial growth and the use of immunofluorescence microscopy provide insight into the intracellular niche legionella established during an in vivo infection and differences in the virulence between strains.

Main advantage of this technique over the legionella mouse and cell culture models is that it is ethically more acceptable and it allows rapid and cost-effective analysis of a large number of Legionella strains in a multicellular organism demonstrating the procedure will be clear. Harding, a PhD student from my lab Always work with L Pneumophila at biosafety Containment level two in microbial safety cabinets according to local rules, first incubate the bacterial plates for four days. Then one day before infecting the larvae Resus suspend one loop full of bacteria containing several colonies in one milliliter of prewarm, A YE broth and measure the suspensions absorbance at 600 nanometers.

Next inoculate a fresh three milliliter a YE culture at an OD 600 of 0.1. Incubate the cultures at 37 degrees Celsius on a shaker at 200 RPM for 21 hours to the post exponential phase so they can be used for infection. Measure the suspensions absorbance at 600 nanometers.

Then dilute the culture to give a billion CFU per milliliter. In DPBS, prepare a container for the G melanoma larvae by placing a circular 10 centimeter filter paper at the bottom of a 10 centimeter Petri dish using blunt tip tweezers. Place 10 healthy larvae of about the same size onto the filter paper, discard unhealthy brown colored or blotchy looking insects.

Healthy larvae are uniformly creamy colored with no areas of dark discoloration and are able to write themselves quickly. If turned over, prepare the injection platform by taping filter paper to a safety cabinet. Then tape a P 1000 tip onto the filter paper.

Now sterilize a 20 microliter microtiter syringe by aspirating 70%ethanol and incubating the syringe in 70%ethanol for at least 10 minutes. Remove any residual ethanol from the syringe by aspirating and expelling sterile water several times. Then aspirate 10 microliters of the 1 billion CFU per milliliter L pneumophila suspension.

Take one larva and gently but firmly. Turn it on. Its back bent over the P 1000 tip.

Place the tip of the syringe over the front right pro leg of the larvae. Then gently insert the tip of the needle into the pro leg. Make certain that the needle is inside the larva and smoothly.

Inject all of the 10 microliter bolus. If the syringe is inserted correctly, it should be possible to pick up the larvae using only the syringe and transfer it into the chamber. Inject a total of 10 larvae per condition and inject another 10 larvae with just DPBS as controls.

The injection process is the most technically challenging of the procedure and will require practice. Finally, tape the dishes closed and place them in secondary containment. Store them at 37 degrees Celsius for the duration of the experiment.

Mortality should be analyzed at several points post infection. To examine the mortality of the infected larvae, use blunt tip tweezers to turn over the larvae and look for movement of the legs. Healthy larvae should write themselves quickly.

Pigmentation indicates a strong immune reaction to the infection. If there is any movement at all, count the larvae as alive at various times. Randomly select three larvae and place them into a 14 milliliter tube.

Put the tube on ice for five to 10 minutes until no leg movement is observed. Then transfer the larvae to a petri dish. Now use a scalpel to make an incision between two segments near the tail to collect each larvas hemolymph.

Keep the incisions away from the gut to prevent contamination by gut bacteria. Squeeze the hemolymph out of the larvae into a sterile 1.5 milliliter centrifuge tube. A single larvae gives between 15 and 50 microliters of he hemolymph.

Now the hemolymph must be used within 10 minutes or it will coagulate to determine viability of insect. He cytes mix 20 microliters of the hemolymph with 20 microliters of trian blue solution in one well of a 96 well plate load three wells this way. Incubate the reactions for five minutes at room temperature.

Then for each reaction, use 10 microliters to count the viable non blue cells with a hemo cytometer. To process the hemo cytes for microscopy. Pipette the hemolymph collection onto a 10 to 15 millimeter glass cover slip in a 24 well plate.

Then add 0.5 milliliters of DPBS and mix the hemolymph well with gentle ation. Using an aerosol tight centrifuge plate holder centrifuge the plate for 10 minutes at 500 G at room temperature. Then examine each well using an inverted microscope to check that the he cytes have adhered.

Next, remove the supernatant and carefully wash the cells three times by adding 0.5 milliliters of DPBS to the wall of the well. Rock the plate a few times and aspirate off the DPBS. Details on fixation and staining are provided in the text protocol.

To quantify the bacterial CFUs, make serial tenfold dilutions of the hemolymph in sterile a YE media. Next, divide the base of a CYE plate containing SPECT mycin into six equal sectors. Plate three drops of 25 microliters of each dilution starting with the most dilute dilution in each section of the plate.

Incubate these plates and later count the colonies. G melan larvae were infected with wild type and ICM deficient. L pneumophila strains Infection with 10 million CFU of strain one 30 B resulted in 100%mortality within 24 hours.

However, the delta A strain which does not have a functional ICM T four SS secretion system was a virulent to determine if uptake of the bacteria is crucial in its pathogenesis. In this model, larvae were pretreated with cyto D and then infected treatment with the inhibitor alone did not affect survival at 24 hours, however, pretreated infected larvae displayed significantly greater survival compared to controls. In order to validate the expression and determine the subcellular localization of an effector protein in g Melan Legionella expressing a fragment of the well-defined LCV localized effector.

SID C fused to four and terminal HA tags were used to infect the larvae and analyzed by microscopy to follow the bacterial replication Kinetics larvae were sacrificed at several time points post-infection and the CFU per gram of extracted hemolymph was determined after an initial dip at five hours, the CFUs of wild type bacteria increases. However, the delta a strain undergoes no replication and is cleared by 18 hours. He cytes were extracted from the infected insects and viable cells were counted Using the trian blue exclusion method at five hours post-infection, there was no difference.

Incyte counts between the strains. However, by 18 hours there was a significant drop in hemo concentration in wild type, but not delta A infected larvae. This in vivo result suggests that L pneumophila replicates within he cytes then lys them.

After watching this video, you should have a good understanding of how to infect larvae, extract, hemo, lymph, and process. He cytes to analyze various aspects of legionella pathogenesis.