7 replies to this topic

[ionchannelbk]

Hello folks,

 

Currently I am working on a phage display project. What I do is I feed caterpillar larva with bait containing phages, and then collecte the larva haemolymph to identify phages which translocate from guts to "blood". Now my problem is during the feeding the caterpillar larva get phage contamination on its cuticle surface. This surface contamination is very hard to get rid of and it contaminates the collected haemolymph sample. Does anyone have some good ideas to get rid of the surface phage contamination after larva feeding on bait without killing the insect?

 

Thanks!

[hobglobin]

Perhaps waiting for moulting so that the insect has a "new" cuticle? You might start the feeding with the bait quite short before the moulting happens (usually you can do this with easily with rearing at constant temperatures from the egg, as then the duration of every larval stage is quite constant).

[ionchannelbk]

Thanks, hobglobin. That is good idea theoritically. But practically there still be contamination concern, because the old cuticle may contaminate the container and the post-moulting larva will crawl around, etc. But thank you for your idea.

[hobglobin]

But can't you keep the larvae individually when feeding them with bait and clean up thoroughly in this phase (removing old cuticles etc) and sterilise the cages or boxes frequently (or using UV light or a DNA killing spray)? And I wonder how much bait they have to feed on that it's spread everywhere?

Edited by hobglobin, 13 September 2013 - 09:17 AM.

[Phil Geis]

What about using a FISH probe for the bacteriophage on a frozen or fixed section of infected caterpillar.  You could validate the approach if signal was seen only on exterior at time zero.

[ionchannelbk]

Thanks, Phil Geis. FISH probe is an interesting idea. Can a FISH probe detect phage in a living larva?

[Phil Geis]

Not sure but should have a chance - need to have an accessible a viral genome that could hybridize. Would take some method development. I'm sur there are better references but here's something.
http://www.ntu.org/g...iabilities.html

[AquaPlasmid]

Interesting project! But some phages might be carried into blood via macrophages that eat up bacteria infected with your phages in the gut.

 

Washing/cleaning the larva off phages is nearly impossible. There are millions of phages on the surface.

 

Fish on slides can show the distribution of phages but you couldn't recover the phages that have entered the blood stream for sequencing.

 

You couldn't use antibody binding to select those entering the blood as all of them have your inserted coat.

 

Just curious. Are you trying to identify blood-entering phages for treating bacterial infection?! We have used directed evolution to generate phages that can overcome phage-resistant bacteria by random mutagenesis by treating the resistant phages with AquaMutant solution. Maybe you could also create mutant phage library using AquaMutant to increase your odds of finding phages that can enter the blood.

 

Hope you find a way to separate phages in the blood.

 

AP