dear Sophia,

Before plating the autoclave agar-media, either to the plates or to the prepared media add the desired concentration of HgCl2 from the prepared stock solution and mix it thoroughly. Allow the plates to solidify followed by addition of sample - spread plate technique or any other technique.

All the best

I would be careful with mercury of any type, and strongly advise against autoclaving it - even as as HgCl2

Here is what PubChem has to say about it:

"Mercury chloride (HgCl2). A highly toxic compound that volatizes slightly at ordinary temperature and appreciably at 100 degrees C. It is corrosive to mucous membranes and used as a topical antiseptic and disinfectant."

https://pubchem.ncbi.nlm.nih.gov/compound/Mercuric-chloride

Make your plates as usual and smear HgCl2 solution on it. If you worry about contamination filter sterilize the solution.

Thank you Bhavya G. and Yoram Gerchman for your informative answers.

If you are unable to autoclave HgCl2, are there other methods for sterilizing and disposing of the agar plates after use?

Also, is it safe to smear the HgCl2 solution in a biosafety cabinet or would you require a special fume hood?

as Yoram Gerchman already mentioned you can opt for filter sterilize the mercuric chloride solution.

regrading disposing of plates its better to follow the guidelines of your lab. As it also contains microbes once your utilized for the growth.

Yes, its safe to do in bio-safety cabinet.

We have done this in our lab and what we found is the best way is to wait for the agar to cool to a temperature you can handle safely with gloves on, quickly spike in the HgCl2 and move fast for pouring your plates.

Doing this in a regular fume hood is advisable if you can manage to have a bunsen burner in there to keep the flame, although a biosafety cabinet is also okay if it maintains your sterile field. One thing to keep in mind is you should not UV the cabinet with plates inside of it as Hg is sensitive to UV light and will volatilize.

As for disposal, it depends on what you're plating. In Canada, we work in a level 1 which means no pathogens, as such we prioritize disposing of the Hg in a solid waste container. If the microbes you are working with are pathogenic, it is more likely they will prioritize you get rid of the biohazardous material first by bleaching, then disposing into Hg solid waste.

As for sterilizing the HgCl2 I would not recommend filter sterilizing with a syringe or vacuum setup because Hg has a high affinity for many filtration membranes (particularly those made with PES).

What we have done is autoclave and filter sterilize ultra filtered water (0.2 µm pore size) and then created a stock from HgCl2 acidified with HNO3. Something to keep in mind is it is highly unlikely something grows in this type of stock because 1) the Hg concentration is high 2) the HNO3 drops the pH to ~2 or 3 and 3) there are no nutrients to support rapid growth. If you store this stock in the fridge in the dark there is even less of a reason to suspect you will contaminate. I would argue you're more likely to contaminate while pouring plates than anything else.

You want to make sure your stock is at the concentration is should no matter how you handle it as the microbes will not be exposed to the full concentration of Hg amended to plates given that most of it will be below the surface of the plate. If they are aerobes, they will not dig or grow into the agar anyway. So double check your total Hg concentration before moving on. Best practice for Hg dictates that you have a principal stock conserved with HNO3 from which you prepare a fresh working solution daily.

For some more information on it please see my papers where I discuss my Hg stock handling methods:

Article Heliobacteria Reveal Fermentation As a Key Pathway for Mercu...

Article A physiological role for HgII during phototrophic growth

I hope this helps, good luck.

Daniel Gregoire