Kamal Hamdy , I want to inject 1ul of the virus.

I alread read this paper and saw the video. But they didn't explane how they calibrate the pompe injector.

I had injected 2 uL. For this i had used Hamilton syringe, piece of thin polyethilen tubing and a glass pipette, like for the patch-clamp recordings, but with long tip. One end of tubing was mounted to the syringe's heedle, and another - to the pipette. Syringe and tubing were filled by distilled water with phenol red (you can use another dye) for sutability of control of water level. Pipette was with air and fixed in the manipulater for patch-clamp recordings. Than I dropped to piece of Parafilm 2 uL of solution using precious sampler for 1-10 uL.Than I sucked this drop to the pipette's tip by applying negative preassure by syringe. I got a system "solution-bubble-water". Than I made an injection by slowly application of positive preassure by syringe. Whan water level in pipette droped to initial level or even a bit lower i stoped applying preassure and wait for several minutes. Than removed pipette from site of injection.

I am assuming that you have the pipette attached to tubing which is attached to your syringe, is this correct?

Yes, you are right

Viacheslav...sorry I was trying to get confirmation from Khalki of the setup they are using. We use a similar method as you except we fill the line with mineral oil first. Then we place volume to inject on parafilm, draw this up into the pipette.  We then use a nanoinjector to inject the volume over several minutes.  

It is not necessary to calibrate to the pipette if you are using a pump. The pump is calibrated to the Hamilton syringe that you will be using to inject the volume.  This controls the rate and distance at which the plunger will be advanced on the microsyringe, controlling the volume of the injection.  You can vary the diameter of the tip of your pipette and still use the same pump and syringe to give the injection. Hope this helps.

Thank you Renee. But how you use a hamilton syringe and the glass pipette in the same time??!

We use a nanoinjector that has a micropipette holder attached.  If you don't want to purchase the nanoinjector, you can purchase a pipette holder. Warner Instruments carries these. Here is a link to some of the ones they have. My suggestion would be to call them and let them know exactly what equipment you are using and they can help you select the correct holder for your equipment.

https://www.warneronline.com/images/image_enlarge.cfm?image=holdergr1

Thank you Renee

I was reading with interest the discussion.

One essential factor to be considered is : There will be intracranial pressure to consider while injecting anything into the living brain. What I would advocate is to use an infusion pump (May be a 'Worker Bee and Syringe pump' of Bioanalytical System; or any such device that has accuracy upto 0.01 0or 0.02 micro L/min infusion accuracy), attach the  needle tip of the Hamilton Syringe (fixed on the infusion pump) a fine (less than or 0.5 mm id) Teflon tubing and draw in parafiffin or aCSF into the tubing and introduce one of two tiny air bubbles (may be 1 cm apart) that could be minitored with a magnifying glass, or by naked eye. The infusion needle (that you are planning to use in animals) is attached to the tip of the teflon tube, and fixed on to the 'guide on a brain stereotaxic frame'. Using the infusion pump calibrate the displacement of the air-bubble in the Teflon tube for delivering 1 microL (in 5 min at a rate of 0.02 micro L), and use a mm sacle to measure the distance travelled by the air bubble in the tube. Calibrate the movement of the air bubble for every 1 micro L without anything attached to your infusion needle, and then do the experiments with animals. The idea is to correct the lower volume injected into the brain, caused by intracranial pressure, so that the instrumental 'reading' could be erraneous - since the instrument is calibrated with time of injection. For eg. 0.02 micro L per min indicates aftter 5 min the delivery is 1 micro L; but in reality due to intracranial pressure the liquid delivered into the brin could be much less, and it will require more time to exactly reach the volume. Therefore this external 'bubble devise' will help one to correctly monitor the exact volume delivered. And leave the infusion needle for another 5 min in place to 'allow dissemintion of the fluifd into the brain site' and it will also help you to restrict 'backtrack-flow of injected material' along the infusion needle path.

All the best

Mohan

Thanks a lot Dr Lohan for all these details. Today I did the same thing as you say but i didn't use the teflon tube, I injected directly from the glass pipette. I measured the time needed to inject 1 micro L. As you said  the time indicated in the micropum to inject 1micro L is less than the real volume injected due first to the resistance of the tip of the glasse pipette, and second to the intracranial pressure.

Next time I will use a hamilton serynge connected to the glass pipette. And test the "buble device" technique.

Thank you again 

Loubna