Plate Pouring Protocol

[@addgene]

Addgene does not regularly monitor comments posted here, so we may not see your question immediately. We’d be happy to answer any questions sent to [e-mail] as soon as possible. Please include the name of the video along with any questions so our support team can help. Thanks!

[@flyingmorningdew]

this is an amazing video perfectly made, major kudos to Addgene for sharing the science!

[@andrerodon3921]

Nice video, but the high pressure in the autoclave is not to keep the solution from boiling over - it raises the temperature at which water boils, thereby allowing for sterilization of the solution/substrate by killing microbes that can survive the normal boiling point of water.

[@vikramjitsingh4538]

very nicely explained and the comic illustration lightens the mood as compared to some dead serious science type labs out there.......awesome

[@tartanhandbag]

if your agar goes solid, you should preferably NOT autoclave it again as this will degrade the nutrients eg. caramalising sugars, which can have metabolic consequences. this of course is dependant on what you're doing with the bacteria and so is permissable in certain scenarios.

[@addgene]

Thanks for your comment! We agree that autoclaving the media again should be considered only on a case-by-case basis depending what is in the medium. For example, it may be okay to autoclave or microwave plain LB agar but like you said, once sugars (or antibiotics) have been added, it is best not to do this.

[@thinginground5179]

As a follow up, it’s fine to just microwave your agar all the way back up to its liquid point if it’s solidifies. Simple.

[@audreyoswita984]

Lol this is so funny! Good job guys!

[@joekub7]

great job telling inexperienced you tubers to light their burner before spraying alcohol.

[@opokuissac2523]

Nice presentation

[@petergiangrasso5519]

More importantly, don't stack the plates too high!

[@brandonfriesen9820]

Hey; Spraying alcohol before you light your burner is a great way to sterilize everything all at once!

[@joekub7]

Brandon Friesen my point was: alcohol is flammable; you should at least warn ppl to ventilate the area before igniting the burner

[@sheri-.-]

Thanks for adding some humor ❤️👌

[@HuyenPhan-ir2wo]

This was in my homework assignment as an OS.

[@geinstein8407]

I do think it would be better practice to pour plates in front of your flow hood instead

[@christinerodriguez-rutkows7150]

Is there a way to cool plates faster, such as in a 4'C fridge? Is there a downside for cooling them too quickly?

[@addgene]

Yes, that would cause the agar to solidify more quickly. Provided that your antibiotic was well mixed and distributed throughout the liquified agar prior to cooling, this approach should be OK. However, be aware condensation may form more rapidly when quickly cooling the plates, and it may drip down on the agar, so be sure to check your plates frequently.

[@sansgaming226]

Thank you, very cool.

[@naveen08563]

How do you dry them overnight at room temp. after adding antibiotic? antibiotic half life is 12-14 hrs, right? I think this procedure is for plates without antibiotic. Or else, we can immediately store the plates after solidification at 4C if the antibiotic is added. In this procedure, the colonies are supposed to be grown in -ve plate too because the antibiotic has already been degraded.

[@addgene]

Dear Naveen, Thanks for your comment! This procedure is for plates with antibiotic. The plates are fine after cooling overnight at room temperature. While some of the antibiotic may have degraded, the remaining active dosage is typically sufficient for selection. You can also put the plates at 4 degrees C sooner, though typically this results in a higher degree of condensation on the lids. We hope this helps! The Addgene Team

[@VanessaOliveira-um7cb]

I appreciate the funny moments!

[@skaweber]

What is the exact type of pipet are you using to pipet the liquid agar onto the petri dishes?

[@addgene]

Dear Scott - we were using a 10 mL serological sterile pipette. Cheers!

[@christinerodriguez-rutkows7150]

What do you use for absorbent material?

[@udayawimalasiri5957]

Informative and interesting. Thanks for sharing.

[@Bing4000]

thank you so much for this wonderful video ill make sure to subscribe to your channel

[@3dp_edc]

What kind of bottles are those that you use for pours, looking for something easier to pour out of than the mason jars i am currently using, one screw up and it pour down the side rendering the rest of the batch useless since it has then came in contant with the outer jar meaning its no longer sterile.

[@addgene]

Hi Dylan, you can try using a bottle that has a narrower mouth to minimize spills as shown in the video - for example, from Corning, Fisher, etc. Try searching for "laboratory glassware" and these types of bottles will come up. You can also try autoclaving your media in a flask (cover with foil for autoclaving) for easier pouring.

[@3dp_edc]

@@addgene Thanks for the response i found something that looks really promising that i ordered should be here soon. Media Storage Bottle basically the same thing you have.

[@bkerbal8444]

can i make plates at home without being able to steralise things. my partner is interested in this field as a hobby (i know nothing about it) and wanted to get the stuff to make plates at home but not sure if there is a point if we dont have an autoclave. pls someone let me know asap if its possible to do at home so i know if i should order the powders and dishes

[@addgene]

While we haven't verified this method at Addgene, this paper tested home pressure cookers for laboratory sterilization! journals.plos.org/plosone/article?id=10.1371/journal.pone.0208769

[@themadhatter196]

I would edit the order of events starting at 3:45.... The order of lighting a burner and then spraying alcohol. Instead spray the alcohol first and wipe down. Then light the burner. The order in the video is a recipe for a giant fireball. Aerosolized alcohol + ignition source=🔥 Don't under estimate inexperienced people.

[@dannydelao375]

Does the flame act as a laminar flow hood as in it prevents contaminants from falling on the plates ?

[@addgene]

Hi, yes, the flame creates a slight air current that prevents any small particles in the air from falling into the plate

[@geinstein8407]

Shore but I still think using your flow hood would be much better practice

[@katrina3985]

Super helpful!! 👍😁

[@MycoDogs]

very nice

[@maryknollhilario363]

What's the use of alcohol lamp??

[@addgene]

We use the alcohol lamps for sterilization, because our lab doesn't have gas lines.

[@mr.buttercutter9294]

Sir please explain the importance of control plate.im still waiting for your reply

[@addgene]

The control plates test whether the antibiotics on the plates are added and working properly. If you plate a strain resistant to that antibiotic, it should grow. But, if you plate a strain sensitive to that antibiotic, it should not grow.

[@mr.buttercutter9294]

Thanks for your explanation

[@muslimlegends2700]

How much Agar conc should i use for one petri plate?

[@addgene]

Hi there. Please check our protocols page for more details. We used 12 g agar / L. (1.2%). www.addgene.org/protocols/pouring-lb-agar-plates/

[@dylanbaker5745]

Why would you not spread both nonresistant and resistant bacteria on the same plate? For example half and half

[@addgene]

Hey Dylan, Thanks for your question. You could potentially do this if you are testing the plate to see if the antibiotic functions. You just have to label the separate halves of the plate and be very careful not to cross contaminate. Some Addgenies used to streak out multiple E. coli strains on single plates to save time and plates.

[@anderfabi600]

Is not your samples contaminated? why not use a biological safety cabinet?

[@addgene]

Dear Fabian, Thank you for your question. Although a biological safety cabinet might be more sterile, it is not necessary for the plate pouring procedure. So long as the agar is autoclaved, you're working in a well ventilated lab space, and you do not leave the plates open, contamination should not be a big issue. Cheers!

[@anderfabi600]

Contamination. That is my headache. In the laboratory where I am doing my thesis there is no biological security booth. The culture media are served in a clean month and near a bunsen burner, but it is useless.

[@olivedollop7363]

who else is triggered by the way he says agar?

[@dallasneudorf]

Im triggered by you using the word triggered

[@olivedollop7363]

Studied in the UK my whole life so maybe it's pronounced different your country :) so you can understand my amusement

[@olivedollop7363]

@Bagsy I'd pronounce that like the way they say agar in the video actually. Ooooohhhh!

[@rishisharma1126]

pls also share Hindi language video microbiology practical topic

[@BM-qb3oo]

What kind of reputable lab pours petris in open air with just a flame??????????????

[@ShyamBhakta]

Doesn't produce contamination if your lab/building's HVAC system is properly tuned with high air exchange rate, filters cleaned, etc.

[@Dylabong420]

so i am suppose to light the flame on the bench before i spray the bench with 70% Ethanol? HAHAHA jk Dont do that kids!

[@ClashClash89]

nice pointer. i immediately felt that "staring down a barrel" sensation as well when i heard those two steps in that particular order. This part was more of a 'do as i do and not as i say'-segment, at least to those who looked at the video and didnt treat this as an audio-instruction.

[@sumonhasan6161]

Hi how Prize autoclave device?

[@youssefrochdi1994]

An autoclave like the one in the video is very expensive. It can cost up to 50.000$

[@Wernerrrrr]

If I fail I will blame you! :)

[@jk-ed3pi]

BBBBBBBRRRRRRAAAAAAATTTTTT

[@ricoraymendoza5956]

weirdoes. hhaaha

[@erkzneedtu]

cringe ha haa

[@annasofiabottecchi5632]

sei un po' noioso