21st Mar 2023

Does heating antibiotics destroy them? No

Whether antibiotics are destroyed by heating is probably inconsequential to most people. However, I see scientists and biohackers talk about it way more than you would expect.


In genetic engineering when scientists modify bacteria or yeast they use antibiotic selection. This means that they give the genetically modified bacteria and yeast antibiotic resistance because it makes it easier to tell which were engineered. Organisms that survive the antibiotics were most likely engineered. This is not always the case, contamination, escapers and natural mutations can give false positives. People doing genetic engineering for their first time experience these issues much more than a seasoned experimenter and so it is important to know what to blame so you can get the experiment correct.

Media is the term used to describe food organisms eat to survive. It basically contains sugars, nitrogen and other macro and micro nutrients. Generally, media is heated to sterilize it, you don't want random bacteria to grow in your media and ruin your experiment.

In a professional lab environment many scientists will use an autoclave which heats to 121C and 15 PSI. While people doing experiments in a more modest setting will use a microwave or an oven which can only go to ~100C before the liquid boils over. In most cases 100C is sufficient to sterilize media. In fact, in many cases not heating and just adding antibiotics is more than enough to sterilize media over the course of a 2-3 day experiment.

When making media scientists wait until after the media cools to add antibiotics. This is good practice. If you can wait 30 minutes no harm is done by adding the antibiotics at a later time. However, this action has led many people to believe that heating antibiotics in any way will destroy them. In fact, it is what I was taught. Only add antibiotics when the media cools to below 50C.

Because I am lazy and always try and do things different than the establishment I started adding antibiotics to my media before it cooled a long time ago and have rarely or never had problems.

I never did a head to head experiment though. I never compared some heated media to non-heated media to media without antibiotics. So I decided to give it a go.

I heated up LB Agar in a microwave and added antibiotics at ~95C.
I used standard working concentrations for bacteria
Kanamycin - 50ug/mL
Ampicillin - 100ug/mL
Chloramphenicol - 35ug/mL
Streptomycin - 100ug/mL
G418 - 200ug/mL

The length of time each antibiotic was at >90C was 5 minutes. The media was allowed to cool at room temperature so the agar plates could solidify.

I also did
LB Agar with no antibiotics
Ampicillin 100ug/mL added at ~50C

I took a tube of DH5a E. coli bacteria and grew to OD 600nm 0.6 in SOC and then plated 10uL - 4 times on each plate. I let all the plates grow overnight for 18 hours at 37C.

As you can see from the plates there is clear growth on the LB Agar plate that had no antibiotics added and there is no growth on the other plates whether the antibiotics were added at >90C or 50C. The antibiotics were not destroyed by heat at least not enough to prevent bacterial growth.

After ~40 hours there is still no growth on any of the plates but the LB Agar without antibiotics plate has some random contaminating strain of bacteria growing

The antibiotics seem to be working fine.

If you don't believe me try the experiment yourself. It is fairly easy to perform.

I am not saying "no portion of the antibiotics in the media were destroyed". What I am saying is that it is safe to heat antibiotics and still have enough of them leftover to prevent standard lab bacteria and contaminating bacteria from growing, which is their purpose in this case.

I am not saying this method is the way everyone should make their media. What I am saying is that if you do heat your media with antibiotics in it you are ok and it won't ruin your experiment.