The video demonstrates a laboratory protocol for preparing and processing sputum samples using functionalized magnetic nanoparticles to aid in the detection of Mycobacterium tuberculosis (TBC).
đ What the procedure involves
1. Starting materials
Tubes prefilled with 250 µL of functionalized magnetic nanoparticles.
1 mL of sputum sample is added to each tube.
2. Handling viscous sputum If the sputum is too thick to mix:
Prepare a solution of 0.4% sodium hydroxide + 0.05% NâacetylâLâcysteine (NALC) in water.
Add it at a 1:1 ratio with the sputum to liquefy it.
3. Mixing steps
Mix the sample by inverting the tube 20 times.
Let it sit for 5 minutes.
Mix again 10 times.
4. Magnetic separation
Place the tubes on a magnetic rack.
Nanoparticles migrate to the side; the center of the liquid becomes clear.
Highly viscous samples may take up to 15 minutes to fully separate.
5. Removing the supernatant
Carefully open the tube without disturbing the nanoparticles.
Use a pipette to remove the sputum by inserting the tip straight down the center to avoid pulling nanoparticles.
Repeat for both negative and positive samples.
6. Important caution If the sputum is brown or if too many nanoparticles are accidentally removed, microscopy and detection will not work properly.
York tubes will come to you prefilled with 250 microliters of functionalized magnetic nanoparticle which you can see here what we need to do is add your
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sample to the nanoparticle so what you'll do here is you'll take one milliliter of your sample
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and then go ahead and add it directly to your tube now if your sputum sample is extremely
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viscous and does not want to mix what you can do is make a solution of 0.4
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percent sodium hydroxide 0.05 percent and acetate cysteine in water and add
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that in a ratio of 1 to 1/2 years few them in order to liquefy it so here I'm going to prepare my negative again 1
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milliliter of sputum and then you can discard your pipe back now we mix the
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samples and we mix by an inversion 20 times if your sample will not mix again
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add the 0.4 percent sodium hydroxide 0.05 percent and asked he'll cysteine in
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water to your solution once they are mixed 20 times you let them sit for 5 minutes once they have sat for 5 minutes
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you pick them back up and mix again 10 times and then they go on to the magnetic tube breath if your sample is
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very viscous it may take up to 15 minutes for the nanoparticle to separate and come to the side of the magnet as
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you can see here our sample is not extremely viscous so it is separating quite easily you'll see that the center
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of the liquid becomes clear again as the nanoparticle moves to the side once it's separated fully you are free to remove
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the supernatant or the sputum so you gently open this and you're careful not to disturb the nanoparticles in here then you're gonna
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take one of your pipettes and you're going to gently remove the sputum so I'm going to go straight down the center of the tube so that I don't grab any
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nanoparticle and I'm gonna remove all the sputum that I can find so that's for the negative now we're gonna do the same
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thing for the positive again gently opening leaving it in the tube wrap and then going straight down the center of
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the tube to remove the sputum if you remove too much nanoparticle in this process or if your sputum is very
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Brown and that means you've sucked up your nanoparticle your detection and microscopy will not run very well for you
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