Hi Noor,

I faced a similar problem earlier. But, you can fix it. You have to prepare a higher volume of sample and then concentrate it in lower volumes after each centrifugation process. You can centrifuge at 30,000 g for 30 to 45 mins. Discard the supernatant from the tube and add a certain volume of the sample. You may not see the particles in the first trial, but if you repeat it again and again there will be enough particles. That way, the AgNP concentration will increase.

To measure the concentration, weigh an Eppendorf tube before adding your sample. Then weigh after drying the tube with the concentrated sample (after the repeated centrifugation process).

Based on this result, you will have an idea of the stock concentration.

To check the MIC, you can use the Micro-broth dilution method. Use 96 well plates, add an incremental concentration of AgNP (serial dilution) in MHB media. Grow bacteria and maintain it at 0.5 McFarland standard (1.5 x 10^8 cells). Add 10 ul of it into each well. Make sure to keep positive, negative and media control.

To check bacterial viability, you can use Resazurin assay or plating methods (Drop-culture method).

If you want, you can refer to:

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I have centrifuge in lab with speed limit upto 14000 rpm. I centrifuged the nanoparticles for 20 mins. I obtained minute pellet, I washed it with distilled water and also pellet color turned black after centrifugation while the color of solution was brownish yellow. not enough to carry out MIC experiment. Satwik Majumder Satwik MajumderSatwik MajumderSatwik MajumderSatwik Majumder

Hi Noor Ul Ain ,

If you are using the Eppendorf tubes (of 1.5 mL) for centrifugation and washing:

Add 1 mL of your samples in 10 tubes (considering you prepare 10 mL of sample in total). Centrifuge them. After your first centrifugation process, you will receive a pellet which will be pretty low in all the 10 tubes.

Now add 1 mL of DI water to the 1st tube, disperse/mix (vortex) the pellet. Take 1mL from that 1st tube and resuspend it into the 2nd tube. Disperse the pellet. Again take that 1mL from the 2nd tube and add it to the 3rd tube. Do this until the 10th Tube. Now centrifuge the 10th Tube. You should have enough pellets (particles) by then. Wash the sample in the 10th Tube.

If you feel that the concentration is still not enough then prepare 50 mL of sample. By this, what I mean is that you should increase the volume during sample preparation.

Although, turning brownish-black is normal after washing. I recommend you do a wavescan and confirm whether the wavelength is still between 400 to 470 nm or not. If not, then your particles are not stable. You should recheck the protocol you are following.

I hope this helps.

Satwik Majumder Thank you so much for your detailed response. Yes the pellet was quite low. I will try the process that you have recommended.

Dear Researcher,

Use high concentrations of primary materials to have finally enough concentrations of Ag NPs and on the other hand, apply ultra-centrifuge for precipitation of Ag NPs.

Hi Noor Ul Ain I hope you had a detailed answer from Satwik Majumder and should work after multiple centrifugations.

The chemical reduction method yields a very low amount of the AgNPs so here are few suggestions.

You may increase the molarity of AgNO3.

Scale up and use a 15 ml centrifugation tube for more pallet.

I hope it will help.

goodluck.

Hammad Arshad thanks for your help. I have read that increasing the molar concentration of AgNO3 leads to agglomeration and sedimentation of the nanoparticles.

But I am considering to increase it from 1 mM to 2 mM. So would it increase the yield of silver nanoparticles?

Hi Noor Ul Ain , it will increase the concentration slightly. But follow what I said earlier. That will work.

Noor Ul Ain the yield would be increased a bit.

Satwik Majumder hello! I tried the strategy you told me but still the yield was low. Would adding capping agent or molar concentration of silver nitrate help in increasing the silver nanoparticles?

Noor Ul Ain Hi... yes you may try increasing the molar concentration. Also check the reducing agent you are using, You may have to change its concentration as well if you increase the molar concentration of silver nitrate. Good luck.

Satwik Majumder also upon centrifugation the color of nanoparticles turned black. does it indicate any oxidation or aggregation of the nanoparticles?

Noor Ul Ain Did you check the wavelength after centrifugation? Is it still between 400 to 470 nm?

Satwik Majumder it is at 400 nm