Wax worms inoculated with GFP tagged Listeria--Hemolymph extraction

Yesterday (day 1) I helped with a project to view the activity of  Listeria monocytogenes in the hemolymph of Galleria mellonella. The stain of Listeria we are using has a GPF tag. The protocols I used yesterday and today (day2) are described below.

Day 1-Inocculation of worms
1. From agar plate of GFP tagged Listeria, suspend a loopful of bacteria into 500μL of sterile PBS.
2. Dilute the suspesion 1:10 with PBS for two cell suspensions to be used for injections
3. For both the diluted and undiluted solutions, inject a group of 15 wax worms (Galleria mellonella) with 20μL.
     a. Use 70% alcohol to clean worms before injection
     b. Using a microstep-repeat injector with 31G needle and 1mL syringe, inject worms with appropriate suspension in the 2nd to last hind segment.
     c. Incubate worms at 37C groups of 8 per petri plates for 24 hours.

Day 2-Extraction and preparation of hemolymph
1. Prepare the following solutions
     a. 0.05% N-Phenylthiourea in PBS for use as anticoagulant buffer (AC buffer)
     b. 2% Formaldehyde in AC buffer
     c. 0.2% TritonX detergent in PBS
     d. 0.1% DAPI stain in PBS
2. Bleed worms into 1.5mL epp tubes containing10μL AC buffer.
     a. Using forceps to hold worms over tubes, puncture the worm with sterile 16G needle and allow hemolymph to drip into tube (250μLs were collected into each tube, with 3 tubes total)
3. Pellet the cells at 12,000 rcf for 1 minute, wash with PBS and re-pellet.
 4.Discard supernatant and resuspend in 50μL of 2% Formaldehyde.
5. Incubate at room temp for 30 minutes.
6. Pellet the cells at 12,000 rcf for 1 minute, wash with PBS, and re-pellet.
7. Discard supernatant and resuspend in 50μL of 0.2% TritonX detergent
8. Incubate at room temp for 15 minutes.
9. Pellet the cells at 12,000 rcf for 1 minute, wash with PBS and re-pellet.
10.Discard supernatant and resuspend in 50μL of 0.1% DAPI
11.Incubate at room temp for 15 minutes.
12.Pellet the cells at 12,000 rcf for 1 minute, wash in PBS and re-pellet.
13.Discard supernatant and resuspend in 50μL PBS.
14.Pellet again and remove 30μL of supernant.
15. Resuspend the remaining culture and place 10μL onto a slide.
16. Observe under microscope to view Listeria and hemocytes.

 //BEE