9 replies to this topic

[science noob]

Situation: I've autoclaved my newly prepared LB + Agar which came out still in its liquid form from the autoclave.

Question: How long can I keep this LB Agar solution in the 60C oven (to keep it at its liquid state) before pouring onto culture plates? I intend to pour it the following day.

Would it be better to let the agar set at RT and then to microwave it when I need it?

[phage434]

I would keep it at 55 (53 if you are brave and pour fast). Higher temperatures make more condensation on your plates and it's hard to pick up the bottle. Normally I would not do either, and plan ahead to pour plates. Both approaches have issues. I suspect that there is some degradation of the agar over time -- probably not an problem with plain LB, but definitely an issue if you have added heat sensitive components such as antibiotics. If you do reheat agar in a microwave, then it is best to use a larger bottle less than half full, since the agar will splatter. It's actually remarkably difficult to redissolve agar.

[leelee]

I always make up and sterilise more than I need (plain LB) and store it at room temp for sometimes up to months at a time.

I usually make between 200 and 300ml in a 500ml schott bottle, then microwave it to melt it before pouring. I'll often then not use the entire 300ml and so that will reset and I will remelt it again later when I need it. In this case, I have on occasion got a contaminant on the top of the set agar but this is rare (like maybe twice in 6 years?).

I've never had difficultly redissolving the agar, you just have to make sure that you heat it up to near boiling and mix by swirling really well. I hold it up to the light to make sure the texture is consistent, you can easily see when it is ready. It would take me maybe 5 minutes to do.

I prefer this to making plates and storing them in the fridge long term. Especially as I don't use many plates at a time and use a range of different antibiotics depending on what I'm doing.

That, and the condensation completely grosses me out!! I have issues with that kind of thing, gives me the heebie jeebies!

[Leishman001]

Hi:

There have been several very good answers to your questions. Here's my two cents.

I prepare LB-agar in 500 ml batches in Kimax bottles and autoclave to sterilize. I put stir bars in the bottles before autoclaving (make sure the bottle bottom is flat or the bar will not stir properly). 500 ml of LB-agar is enough to be poured into a standard sleeve of 25 petri plates and takes about 1 hour to cool down to where you can add antibiotics and pour. I stir the agar on a stir plate during the cooling process to avoid localized cooling in the bottle, avoiding "chunks" of agar when pouring.

To test if the agar is cool enough to add antibiotics without cooking them, I CAREFULLY touch the bottle to the inner skin of my forearm (wait at LEAST 45 minutes before attempting this to avoid burns). If I can hold the bottle there for 5-10 seconds, the agar is probably cool enough to pour. I add the antibiotics, give the agar a quick final stir and pour. To prevent the stir bar in the bottle from flopping around, I use a second magnet on the outside of the bottle to hold the first in place.

If I want to make batches to cool to store, I make and sterilize the LB-agar the same way and put it in a quiet place with no traffic to cool. Air currents generated by moving people increase the chance of a contaminant getting under the cap and ruining your agar. Keep the cap loose during the cooling process to avoid a vacuum but cap it once the agar is mostly cool. I usually store the agar at room temp for about a month.

To re-liquify the agar, we have access to a steam chamber (not an autoclave, although that can also be used). This melts the agar in about 1-1.5 hours and avoids the spurting caused by microwaving the solid agar.

To answer your first question, you can put the molten agar at 55-60 C to store it for a few hours. If you incubate at this temperature for longer, the sugars in the agar will start to carmelize and the agar will turn dark and eventually appear almost black. Don't use it once it starts to change colour.

[Leishman001]

Oh yeah, condensation on the lids can be controlled a bit by stacking the plates in one "tower" after pouring and leaving them at room temperature over night. Repackage them in the morning and store at 4 C.

[science noob]

Thanks everyone!

To deter abit from the topic, what would happen if I took too long to pour a freshly autoclaved LB agar? The liquid consistency seemed to have thickened but not set and I managed to pour the agar as a thick liquid solution into my plates. All looks ok albeit small air bubbles (which did not affect streaking)

Would this cause uneven distribution of my antibiotic? I've plated some cells and there seems to be healthy colonies thriving on them.

[Trof]

We make 5x concentrated LB and autoclave in 0.5L bottles. Then use this stock to make agar plates (5x LB, water and agar -> autoclave) or liquid medium (just dilute with dH2O under hood).
The LB agar from autoclave is checked regulary and mixed each time, when you can touch it by hand and the agar is stil pretty liquid, we add the antibiotics in the hood, mix and pour.

Once, my agar got too cold in the lower part, it couldn't be thoroughly mixed and on half of the plates were chunks. I think there could be uneven distribution of antibiotics, but I used these plates anyway for something unimportant, just not the colonies growing on chunks.
If it is important I would do the agar again, and pour immediatelly.

[badguy]

My lab's practice is do not keep the autoclave agar in the oven for not more than 24 hours, and mix well before keep inside the oven as agar will tend to set at the bottom.

[science noob]

We make 5x concentrated LB and autoclave in 0.5L bottles. Then use this stock to make agar plates (5x LB, water and agar -> autoclave) or liquid medium (just dilute with dH2O under hood).
The LB agar from autoclave is checked regulary and mixed each time, when you can touch it by hand and the agar is stil pretty liquid, we add the antibiotics in the hood, mix and pour.

Once, my agar got too cold in the lower part, it couldn't be thoroughly mixed and on half of the plates were chunks. I think there could be uneven distribution of antibiotics, but I used these plates anyway for something unimportant, just not the colonies growing on chunks.
If it is important I would do the agar again, and pour immediatelly.

Explain "colonies growing on chunks"?
Mine was somewhat viscous but still mixed well enough to allow me to pour the plates. Situation is that it became somewhat bubbly and thick straight after pouring it. I still got distinct colonies on the 16-streak method.

[Trof]

If you imagine a plate created by pretty liquid agar but containing some chunks of one that was obviously too cooled, the chunks kind of tower above the surface. So you know in which part the agar was too solid to have mixed properly. So I assumed the chunks contained less antibiotics than the evenly mixed liquid part, so those colonies growing on the chunk parts I counted as suspicious and don't use.
But that doesn't matter, is by a no way a good laboratory practice, I just had an agar ready so I though I would use it, If I found that the colonies I picked don't contain plasmid (i.e. were not selected right) I would just throw it all away after first try, but it was fine and I learned how to make a better agar next time.

Anyway if you had just a more viscous agar I think it will be OK.