/sci/ - Science & Math

Here's an idea: don't put all 9 L of your media in all at once.

Or it's possible that there's a failed heating element or sensor in there. Place a thermometer in your media and watch the temperature of it. Then you'll know what's causing it.

Biologists can't into troubleshooting.

>>3534510
pretty much. some kind of heater pack/coil, take that and the thermostat out along with your mulitmeter, and check for continuity. also find any pcbs and check for blown fuses.

>>3534510
That would be the obvious solution, but we're already using up almost every drop of media we use each day, so to run a cycle at less than max capacity would be a waste of time and preparation. Where the problem arises from is that last time we had it calibrated/validated and had a temperature mapping done, it was done with the chamber completely empty, so of course it's going to get up to the proper temperature. What needs to happen, is it needs to be calibrated with a full load of 9 liters of SDA, so that we know it will get our most viscous media up to the proper temperature.

I like to think I have at least adequate troubleshooting skills, but the real problem is the pharmaceutical industry. To have a piece of equipment validated requires so much bullshit paperwork that by the time I got everything in order it would probably be more cost effective to order premade media.

I work with fucking idiots too. Two Ph.D. microbiologists can't fucking understand that heating water is easier than heating something as thick as fucking honey....

>>3534510
That's a problem. If he's doing serious work, he needs to validate every batch of plates he makes from the media. He needs to test some, assign a batch #, etc. Doing it in smaller batches will generate a ton of paperwork and creates a lot of room for error.

My advice is to take 2 hotplate stirrers, 2 2L beakers, and 2 temp probes. Ensure that all equipment is identical. Fill one with water (1L) and one with media. Heat up both starting at the same time and note temperature over time. This should adequately demonstrate your problems.

I had a similar problem when I worked as a microbiologist, as our autoclaves didn't reach adequate pressure for liquid sterilization. We had a lot of boil-overs and it sucked to clean an autoclave in a cleanroom setting. You might want to make a custom setting (if it's a digitally controlled autoclave) for it to do your media. If the interface on the machine won't let you, look up the equipment number and the software/manual should be on file at your company/university.

Just curious, are you doing work with fungi?

Also, if micro gets boring, come join me in law school. It's sweet and the job prospects for corporate patent attorneys are actually good.

>>3534527
OP here, Autoclave runs fine. We just had a temperature mapping done two weeks ago. The thing is, the dumbfuck did it while it was FUCKING EMPTY. So now our autoclave tries to heat up 9 liters of liquid as thick as honey just like it tries to heat up compressed air.

I know what the solution is to the problem, I just need the tools to show my boss that we HAVE a problem.

Also, thanks for the help everybody. /sci/ never disappoints me :D

>>3534545
ok, then do you know what teh time involved to boil water is when the machine is running properly? if so, put water in there with a meat thermometer, and test it against the known time for water. that will tell you if the problem is the media (what is the media, btw?) or the machine.

use water as the control group

>>3534478
LOL when a microbiologist doesn't know about tyndallization. Over-reliance on gadgetry leaves gaps in your practical knowledge. Tyndalization is excellent for sterilizing both liquid and solid medias.

Using that you should be able to compare your contamination rates and determine if the clave is malcalibrated.

Also when tyndalizing, it is not a problem to heat for a longer period of time to make sure the media is thoroughly heated. An hour to 40 minutes every time for three days, and you should know where the fault is.

>>3534556
THIS. Every batch of media I make I have to growth promote and check for proper pH. So tiny-ass batches are a complete waste of time and resources.

I don't really do much work with fungi in particular. Right now I do quality control work for a pharma company. Just found a huge fucking Aspergillus niger colony in a supposed negative control plate from last wednesday. I think I would like it more if the whole industry wasn't so regulated. I'm trying to get into remediation work, but that's such a small field it's hard to find work. Even if I found a job, my partner has an even sweeter gig with NASA that he doesn't want to give up, so I'm kinda stuck here :/

Autoclaves need sterilizatin maintenance now and then.

Fuck, I do piercings and tattoos and know that basic thing - hello, extremophiles.

Sounds like your lab supervisor needs to go back to school.

>>3534599

Oh, and he's a microbiologist as well?

PLEASE tell that fucker to go back to school.

>>3534545
yep. It's a calibration error, then. Leave it in longer. Still, use a thermometer with the media to determine its temperature.

http://en.wikipedia.org/wiki/Heat_equation

Just scroll down a bit for a simplified equation. I'm assuming water and your media has different specific heat capacities, of course.

>>3534575
OP here, did you fucking read what I posted? Tyndallization is heating at 121 for 15 minutes in a pressure cooker. Do you have any earthly idea what an autoclave is supposed to do? Where the problem lies is in the programming of the autoclave. It thinks it's heating to 121 for 15, but it's not actually reaching that temperature. I'm unable to simply change the settings in the autoclave to compensate, though. Every preprogrammed cycle has to be validated and approved using USP specs (shitload of paperwork). Unfortunately, what I can't seem to get across to my idiot supervisors is that that preprogrammed cycle is rigged to heat an empty chamber (only compressed air) to 121 degrees for 15 minutes, and not a full load of media.

>>3534584
I feel your pain, I did the same shit for a contract pharma company. QA did all manner of paperwork magic to cover up problems in my reports. My boss/the owner talked to me about it, tried to get me to fudge things... I quit before shit got serious and I still have my paperwork with me if the FDA come a'calling. Fortunately, I had finished LSATS and my lease was about to expire, so I got out of dodge and ran off to law school.

The regulation is there for a reason, but the paperwork is a fucking bitch and a half. I spent more time doing paperwork than actually doing shit in my lab.

A. niger is serious business. Try the heating test to show your supervisors and work on an autoclave program that gets the job done. Once you have the program, talk to QA about getting a proper validation study done. It'll be a bitch, but likely worth it.

>>3534613

Get some remote temp data collectors that can withstand high temperatures and humidities, and toss them int the autoclave. Run a few cycles, then toss that data into a computer and graph that shit out for your idiot lab super.

>>3534608
I need to just pony up and order a set of thermocouples to test the damn thing with. It's so frustrating working with people who have no problem-solving skills.

Ahh, the joys of being an engineer.

>>3534620
That's the problem. We just had that exact process done less than three weeks ago. Everything was perfectly fine......for an empty chamber. Nobody bothered to run it with a full load of media or even freaking water! AAAARGHJlHAFDH!!!!

>>3534633

Complain higher up the chain for the morons not testing at full load like ANY real test should do.

I'll try to take a crack at it. The trouble I can see right away is you have a limited time (15mins) to reach a certain temp (121 C), your media is denser and more viscous (meaning higher heat cap. and takes longer for circulation).

Solutions: some one proposed it already, less media. This, you can't really do it without knowing the heat cap. of the media. Or you can heat the thing longer (20-25 mins), providing that the autoclave has a temp setting. And yeah, it'd be easier if you could put some sort of paddle or stirrer inside the media to improve circulation.

Well, the simple formula for heating is

Q = (Tf-Ti)*Cw*m

Q: heat, Ti, Tf: initial and final temperature
Cw: heat capacity of water, m: mass of water.
there is element of pressure as well (since you increase pressure of the autoclave to heat water pass it normal boiling point) but I assume the media you try to heat up doesn't just boil at 121 degree C.

or you can divide the two side with time (15 mins) to find the wattage (power I guess) of the whole thing since Q is measured as J.

>>3534615
Same guy here, fun story.

My boss hired a consultant to design our cleanroom. Degowning was to happen in a room with a double-ended autoclave that also served as the entry point for fresh vials. She argued that custom metal boxes should adequately insulate the vials against contamination. There was no pressure differential between the two rooms.

I stayed really late one day, knowing my boss/the owner would be the only other person there. I got a bottle of scotch, 2 glasses, and a power point set up in the conference room. 60+ slides, about 1/3 of the bottle, and a long heart-to-heart later, he caved and we fired the consultant.

It's amazing that well-educated, ostensibly successful people, can be so ignorant.
>>3534636
The problem is that there are sometimes ulterior motives for doing shit studies. Sometimes, a company is too cheap or just doesn't care to correct errors.

I'm almost getting nostalgic for my days as a microbiologist. ALMOST. Rocking a bespoke suit and mopping the floor with wannabe John Yoo's and prosecutors at mock trial is just way more fun.

>>3534613
>>3534613
What you don't get is that tyndalization actually refers to a process of repeatedly heating your media over a period of three days to make it sterile. It is from before the days of high pressure sterilization.

Just pack the media in a suitable container. Stick it in a normal cooking pot and boil it for forty minutes to an hour (put something under it or heat will transfer directly from the bottom).

Then leave the whole contraption in room temperature for 23hours, add water to outer pot and repeat the boiling.

Wait another day, and repeat the boiling.

You now have sterile media to pour. Use a batch of that and compare your contamination rates with media from the autoclave. If the clave is the problem, then you will have less contam with the tyndalized media than with the autoclaved media.

Years ago when I used to play with microbiology I would have less than 0,5% contamination rates with tyndalized media (and I had no friggin clean-rooms, I did it all in my kitchen with a homebuilt ghetto-glovebox).

>>3534620

not good, unless, you can submerge that thing into the media/water, it will do exactly like measure temp of an empty autoclave. Ahh like what the OP says here >>3534633

oh, I just read new info from OP, apparently you can't change the time, so I guess you'll have to do a little extra work to find the relative heat capacity of that media compared to water. Then try to work backward to find the required time.

>>3534661
Dude, I used 11 liters of TSA, 4 liters of SDA, and 6 liters of MacConkey broth today. We use waaaaaaaay too much media to be sterilizing it for days. Plus, to get something like you're suggesting validated would be a bitch and a half to do. Believe me, I wish I could just heat the media for about 4 hours to make sure it was clean, but I have time constraints and USP regs to work within.

>>3534644
DUUUUUUUUUUDE. EXACTLY WHAT I WAS LOOKING FOR. A MILLION THANKS TO YOU GOOD SIR.

>>3534644

Beat me to it.

That's the equation you use. A certain amount of material will take a certain amount of energy to heat up. The energy is dependent on:

1) mass of material
2) heat capacity (this is key)
3) the temperature difference you wish to achieve.

The temperature difference is given, and the heat capacity and mass is dependent on what you're heating. If the material you're heating has a higher heat capacity, then applying the SAME amount of energy will result in lower temperature or longer time.

>>3534698
Alternatively (and more practically), heating a material with HIGHER mass or HIGHER heat capacity over the same temperature range requires MORE energy (i.e. heat it longer with same input or apply more energy over same time).

>>3534644
>>3534698

Thank you both. I knew what I was trying to get across, I just didn't have a good way of illustrating it.

Commence beer drinking

>>3534712
I hope you can convince the other motherfucker that he's basically doing it wrong.

I'm just guessing, since I'm not a biologist, that your medium is mostly agar and water?

In which case if you want to be precise, you can also account for the heat of fusion of agar if it melts below 121C, which I would assume a sugar based polymer does.

Just because the medium is denser doesn't mean that it has a higher heat capacity. Ffs, lead is about 11 times denser than water yet has a specific heat of about 1/32 that of water.

You can also try to find a calorimeter and measure the actual heat capacity of the medium to see if it actually is greater than pure water, but I suspect the different will be negligible.

The heat capacity isn't really what matters anyway. It's the rate of thermal energy transfer inside the autoclave that's important. As long as the medium is able to absorb heat at roughly the same rate as water, which is should if it's water-based, then the settings should be fine. Maybe you've invented some amazing thermal insulator medium, but I doubt it.

Of course, if your autoclave just starts counting the 15 minutes from the minute it starts up before it's even hot then of course there's a problem (hint: you've been ripped off by the manufacturer or the programmer).

>>3534644
OP here

Quick question. Is the variable Q the energy (in joules) required to achieve the desired temperature increase (Tf-Ti)?

>>3534749
yup, it's the heat the medium has to absorb in order to get from Ti to Tf

>>3534747
My gut feeling is that the difference in heat capacity between water and his medium isn't very large.

And heat transfer limitations could also be a factor, but this would depend on the geometry of his container.

>>3534747
I think that if he has such a problem, we can assume the heat capacity of the medium to be higher than water's.

>>3534747
Yes, our media is water based and I'm seeing now that the heat capacity may not be the actual problem I'm dealing with now. With regards to the cycle, no it doesn't just count 15 minutes. The 121/15min cycle actually takes an hour to run. What also may affect the temperature is that we put our bottled media in big metal baskets (~4-5 kg each) in the autoclave. I'm wondering if these baskets are what is adversely affecting the temperature of the media.

>>3534758
I don't think so, metal is a pretty good heat conductor.

But if the whole thing is calibrated simply using air, and not even water, you sure do have a heat capacity problem.

>>3534766

Now that I see it. The heat capacity of air is insanely low. That would explain why it would have trouble heating up anything BUT air once it's filled to the brim. I'll bring this up in the meeting tomorrow. Also, the calibration paperwork for the autoclave is "mysteriously" missing.

>>3534766
Energy required to heat one bottle of media to 121 degrees from 89 degrees (roughly hot plate temp.) is ~67 joules. For the same mass of air (dry air, not moist air) it's ~16 joules. I'd imagine it's not a whole lot more for moist, high pressure air though.

>>3534780
Well there you go!
I hope this helps you solve everything!
You could even add some figures to look more convincing:
water is approx 1000 times denser than air;
Cv of air: 1005J/(kg*K)
Cv of water: 4186 at 25°C (and it doesnt change much anyway)

So basically, rising the temperature of a volume V of water needs almost 4000 times more heat than rising the temperature of a volume V of air.

>>3534756
He doesn't know that the autoclave is the problem. He suspects it, but the source may be something else. It could be glassware, dirty ventilation filters, etc. For all he knows, maybe the janitor comes in and sneezes on his plates just to troll him.

>>3534758
Given what I said above about heat capacity and transfer rates, I strongly doubt that it's the autoclave and I would spend some time trying to think of other possible sources. You may still be right, but if you push it and end up being wrong, you will have damaged your credibility with your supervisor. Supervisors can be quite stupid when it comes to some things, but most of the time it turns out that the supervisor isn't the idiot.

Another way that you could test this is to inoculate a small flask of medium with one of the contaminant colonies then run it through the autoclave again before plating it, preferably in a different, controlled environment. If you notice an abnormally higher rate of contamination then you can show your supervisor that the autoclave is indeed not working.

More importantly:
One medium, two media.
I mean, for fuck's sake.

>>3534850
has someone said "two mediums"?

>>3534854
I checked, and no one has made the mistake.
troll

Well, can we speculate about the possible problems, and ways to isolate/test for it. I have some experiences about these kind of situations as well. When you think something wrong, tell your supervisor, it turned out something else messed up, in turn induced the other to fail. But it almost pushed me into the black list of supervisor. I'd hate to see OP going in the same track, it seems OP's sup has the same kind of mentality as my sups.

>>3534822
I think tomorrow I'm just going to take about a liter of SDA straight out of the autoclave and let it cool enough to where it won't melt plates and then pour it up and incubate it just to see what happens. I highly doubt it's my technique considering contamination is popping up in other people's control plates also.

I'm not actually planning on calling anybody out on it, but my suggestion seems to be better than the supervisor's suggestion. They basically want to re-train me on aseptic technique and put the media in one-time-use bottles to reduce the chances of contamination.

>>3534877
Youve been bitching here for an hour.

Your machine is broken or miscalibrated and all you can say is
>USP specs (shitload of paperwork)
>validated requires so much bullshit paperwork
>Plus, to get something like you're suggesting validated would be a bitch and a half to do

fuck off you lazy piece of shit

>>3534887
I don't actually do the paperwork. It would be the supervisors who do it. To entice them to fill out the paperwork to have it re-validated I would need some serious evidence that it isn't reaching the proper temperature. That's why I'm here. The lab doesn't own their own thermocouples, so we can't record the temperature ourselves. All I can do is speculate. Even if I were to figure out that that was the problem, it isn't like I would be able to recalibrate it anyway. It needs to be done by an authorized technician so that the FDA doesn't bust us for not complying with USP regs. Just last week we had a laminar flow hood that stopped working. We all knew that the problem was something caught on the HEPA filter, but we couldn't do anything about it until an authorized technician can come fix it from San Antonio. It's seriously infuriating to see problems in front of you that you're not allowed to fix, dude so chill.