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Invisiblemicro
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Re: Don't trust UV lights for lab sterilization [Re: Alan Rockefeller]
    #18702592 - 08/14/13 12:34 AM (10 years, 6 months ago)

UV lights will not kill everything.

I can guarantee that. Bacterial endospores will not be killed by UV light.

Along with many other things. And I've seen it in a laminar flow hood but in a ROOM?

No, it's a supplement to a hood.

UV light =/= sterilization.


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InvisibleOeric McKenna
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Re: Don't trust UV lights for lab sterilization [Re: micro]
    #18702637 - 08/14/13 12:47 AM (10 years, 6 months ago)

Yup. you got it Terry. stick to what works.
The only cleaner I've ever advocated for brewing and mycology is bleach.

I used to have this saying
" If it touches the beer, first it touches the bleach" 

In brewing, there are so many marketed products that people use that just don't compare to bleach.
When something works great, stick with it!  :borat:

Oh hey, if you use one of those big pumpable sprayers, you can get a superfine mist high into the air.
Dragging things down to die. The sound of it as it rains down to the floor in a holocaust style spore death......SEXY!


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OfflineRogerRabbitM
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Re: Don't trust UV lights for lab sterilization [Re: Alan Rockefeller]
    #18703248 - 08/14/13 06:48 AM (10 years, 6 months ago)

Quote:

Alan Rockefeller said:
What kind of UV lights are you using?




This was going to be my question too.  Is it UVA, UVB, or UVC?

Additionally, I don't think 4 UV lights would be sufficient for a whole lab anyway.

Adding vinegar to diluted bleach solution as we've been recommending for at least 5 years will lower the pH enough to make it more effective because organisms will allow the pH balanced liquid to penetrate the cell walls.
RR


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OfflineTerry M
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Re: Don't trust UV lights for lab sterilization [Re: RogerRabbit]
    #18703508 - 08/14/13 09:00 AM (10 years, 6 months ago)

Quote:

RogerRabbit said:
Quote:

Alan Rockefeller said:
What kind of UV lights are you using?




This was going to be my question too.  Is it UVA, UVB, or UVC?

Additionally, I don't think 4 UV lights would be sufficient for a whole lab anyway.

RR




I  used I used these germicidal lamps.

And I'm quite sure the dosage was more than enough. The amount of short wavelength UV light power needed to kill better than 99% of M. tuberculosis (one of the hardiest bacterium) is 10,000 µWSec/cm2. One G15T8 Germicidal 15 watt Fluorescent Light Bulb puts out (conservatively) 3.6 watts at 254 nm. At a distance of 4 meters, and assuming that from this distance the lamp can be considered a point source, that's 0.1767 watts per square meter at a surface on which the light shines, or 17.67 µW/cm2. The time needed to kill 99% of M. tuberculosis with this amount of incident light is therefore 565.9 seconds, or 9.43 minutes. And I was using a 120 minute exposure time!


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Offlinedrake89
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Re: Don't trust UV lights for lab sterilization [Re: Terry M]
    #18703712 - 08/14/13 10:07 AM (10 years, 6 months ago)

Quote:

Terry M said:
Quote:

RogerRabbit said:
Quote:

Alan Rockefeller said:
What kind of UV lights are you using?




This was going to be my question too.  Is it UVA, UVB, or UVC?

Additionally, I don't think 4 UV lights would be sufficient for a whole lab anyway.

RR




I  used I used these germicidal lamps.

And I'm quite sure the dosage was more than enough. The amount of short wavelength UV light power needed to kill better than 99% of M. tuberculosis (one of the hardiest bacterium) is 10,000 µWSec/cm2. One G15T8 Germicidal 15 watt Fluorescent Light Bulb puts out (conservatively) 3.6 watts at 254 nm. At a distance of 4 meters, and assuming that from this distance the lamp can be considered a point source, that's 0.1767 watts per square meter at a surface on which the light shines, or 17.67 µW/cm2. The time needed to kill 99% of M. tuberculosis with this amount of incident light is therefore 565.9 seconds, or 9.43 minutes. And I was using a 120 minute exposure time!




i know you're  a smart guy, but it seems that 4m, 12ft(ish) is way far for a little bit of wattage like that to have much effect.  Did you take the inverse square law into account?  I bet you did and all of this is heresay since I'm sure there's abundant literature that you've read.  Just doesn't feel right in my gut, as W would say.


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OfflineTerry M
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Re: Don't trust UV lights for lab sterilization [Re: drake89]
    #18704849 - 08/14/13 03:06 PM (10 years, 6 months ago)

Quote:

drake89 said:
Quote:

Terry M said:
Quote:

RogerRabbit said:
Quote:

Alan Rockefeller said:
What kind of UV lights are you using?




This was going to be my question too.  Is it UVA, UVB, or UVC?

Additionally, I don't think 4 UV lights would be sufficient for a whole lab anyway.

RR




I  used I used these germicidal lamps.

And I'm quite sure the dosage was more than enough. The amount of short wavelength UV light power needed to kill better than 99% of M. tuberculosis (one of the hardiest bacterium) is 10,000 µWSec/cm2. One G15T8 Germicidal 15 watt Fluorescent Light Bulb puts out (conservatively) 3.6 watts at 254 nm. At a distance of 4 meters, and assuming that from this distance the lamp can be considered a point source, that's 0.1767 watts per square meter at a surface on which the light shines, or 17.67 µW/cm2. The time needed to kill 99% of M. tuberculosis with this amount of incident light is therefore 565.9 seconds, or 9.43 minutes. And I was using a 120 minute exposure time!




i know you're  a smart guy, but it seems that 4m, 12ft(ish) is way far for a little bit of wattage like that to have much effect.  Did you take the inverse square law into account?  I bet you did and all of this is heresay since I'm sure there's abundant literature that you've read.  Just doesn't feel right in my gut, as W would say.




Yes, of course I used the inverse square law. Let's do the math! :smile:

The 3.6 watts of power at 254 nm is distributed evenly over a sphere 4 meters in radius. That's what the assumption of a point source of light gives. This mathemagically uses the inverse square law, because the area of the sphere surface goes as the radius squared. The area of that sphere is 4*pi*r2, which is 20.37 m2. You use the area of that sphere to calculate the amount of light on each square centimeter by simply dividing the 3.6 Watts over 20.37 m2, which is 17.67 µW. The figure of 10,000 µWSec/cm2 to kill M. tuberculosis was gotten from a medical UV sterilization site, which listed many other microorganisms that were easier to kill. I've found that figure for M. tuberculosis listed in a few different documents. This bacterium seems to be the gold standard for being hard to kill.

17.67 µW over one square centimeter is not much power. But remember that it's accumulated over time. When you are exposed to x-rays, it is the accumulated dose over time that matters. Similarly for sunburn and skin cancer. To find out the necessary time in this case, you take 10,000 µWSec/cm2 and divide it by the 17.67 µW/cm2. Notice that the µW and the cm2 cancel, so you are left with just seconds -- 565.9 of them. That's 9.43 minutes to kill more than 99% of M. tuberculosis.


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Invisiblefastfred
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Re: Don't trust UV lights for lab sterilization [Re: Terry M]
    #18705267 - 08/14/13 04:48 PM (10 years, 6 months ago)

Quote:

Terry M said:
17.67 µW over one square centimeter is not much power. But remember that it's accumulated over time. When you are exposed to x-rays, it is the accumulated dose over time that matters. Similarly for sunburn and skin cancer.




Remember that there will be a minimum intensity required though.  UV induced DNA damage is very quickly repaired.

There will be a minimum intensity required to penetrate the cell wall and you'll need to have enough intensity to damage the DNA faster than it can be repaired.

UV DNA repair is activated by light, so make sure to have no lights on during the exposure and for awhile afterwards.

You should do a few test plates and place them around the room.  You can also pick a bacterial contam and take a few loopfuls of it to some liquid media or water, shake, then plate out a ml or two on some plates to do some tests to see what distances the UV is effective at.

The vast majority of dust and contams are within a foot of the floor, so you might try lowering the lights so they expose the floor a little more.


-FF

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OfflineRogerRabbitM
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Re: Don't trust UV lights for lab sterilization [Re: Terry M]
    #18706330 - 08/14/13 09:04 PM (10 years, 6 months ago)

Quote:

Terry M said:

My contamination rates shot up to nearly 100%.




This got me thinking it's probably nothing to do with lab sterilization, but rather that one of your master slants or dishes got contaminated and you've been spreading it. I've seen contaminants not show up until the second grain to grain transfer, but they were obviously there since leaving the the petri dish to inoculate the grain master. Go back to a different culture and start over to see if that fixes things.
RR


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InvisibleOeric McKenna
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Re: Don't trust UV lights for lab sterilization [Re: RogerRabbit]
    #18707312 - 08/15/13 12:24 AM (10 years, 6 months ago)

That's true^


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OfflineTerry M
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Re: Don't trust UV lights for lab sterilization [Re: Oeric McKenna]
    #18707686 - 08/15/13 03:14 AM (10 years, 6 months ago)

Except that my test for lab contamination is to pour a bunch of Petri dishes and just let them sit undisturbed for at least 10 days. They were contaminating like crazy, mostly from an edge mycelium spot (strangely, always closest to the flow hood), though sometimes scattered spots of bacteria. I used to have zero contamination no matter how long they sat. I've been going over and over my sterile technique, looking for anything that might have changed, or was a weak spot all the time but has just now fallen over the hairy edge.

Perhaps the specific location of the edge contamination rings a bell with someone? I haven't changed my pouring procedures, and have been consistently scrupulous about hand positioning when lifting plate stacks to pour. I just poured 60 plates about 3 days ago, a day after bleach bombing. They're sitting undisturbed for several more days to see if they stay clean.


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OfflineRogerRabbitM
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Re: Don't trust UV lights for lab sterilization [Re: Terry M]
    #18707875 - 08/15/13 05:57 AM (10 years, 6 months ago)

Are you using a rack so the petri dishes are elevated and totally surrounded by the flow, or are they sitting on a table in front of the hood?

Remember, as air leaves the filter, it causes negative pressure all around the flowhood, creating a draft into the edges of the flow.  If the dishes are sitting on a table, contaminants from the table/flowhood interface can be sucked into the work area and hit your dishes.  Lift the flowhood and clean under it.  Some guys caulk the hood to the table, but I just lift the flowhood each time before sterile work and clean under and around it with alcohol or whatever I'm sanitizing with that day.
RR


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OfflineTerry M
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Re: Don't trust UV lights for lab sterilization [Re: RogerRabbit]
    #18707898 - 08/15/13 06:09 AM (10 years, 6 months ago)

Quote:

RogerRabbit said:
Are you using a rack so the petri dishes are elevated and totally surrounded by the flow, or are they sitting on a table in front of the hood?

Remember, as air leaves the filter, it causes negative pressure all around the flowhood, creating a draft into the edges of the flow.  If the dishes are sitting on a table, contaminants from the table/flowhood interface can be sucked into the work area and hit your dishes.  Lift the flowhood and clean under it.  Some guys caulk the hood to the table, but I just lift the flowhood each time before sterile work and clean under and around it with alcohol or whatever I'm sanitizing with that day.
RR



Yup, I always use a rack. Have never changed this procedure. BUT ...
Though I include the flowhood front framing when I give the filter a spray of Lysol, I've never cleaned under the flowhood!!

Thanks RR, I really hope this is it. It would explain the symptoms perfectly. Will be pouring more plates soon!


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Re: Don't trust UV lights for lab sterilization [Re: Terry M]
    #18709111 - 08/15/13 01:18 PM (10 years, 6 months ago)

Dishes are very cheap compared to the piece of mind of verifying everything is working as you expect.  I say that you should never trust any piece of equipment, always do a control or tests to verify it.  Flowhood, PC, and the UV shouldn't be blindly trusted.  There's lots of ways to screw up or have failures on any stuff in the lab.

Edge contamination usually indicates dirty dishes or incompletely sterilized media IME.  If your overall sterility is difficult then try adding some Lysol or generic spray to the mix.  Lysol leaves a residue that will continue to keep surfaces sterile better than just one-time surface sterilization methods like bleach or alcohol.  It's especially useful for hidden and hard to reach areas that might be less likely to get a good swabbing.


-FF

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OfflineTerry M
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Re: Don't trust UV lights for lab sterilization [Re: fastfred]
    #18709130 - 08/15/13 01:22 PM (10 years, 6 months ago)

Have made thousands of dishes in my lab over the last couple of years. Had zero problems until now.


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OfflineOICU812
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Re: Don't trust UV lights for lab sterilization [Re: Terry M]
    #18709333 - 08/15/13 02:14 PM (10 years, 6 months ago)

Quote:

Terry M said:

Yup, I always use a rack. Have never changed this procedure. BUT ...
Though I include the flowhood front framing when I give the filter a spray of Lysol, I've never cleaned under the flowhood!!

Thanks RR, I really hope this is it. It would explain the symptoms perfectly. Will be pouring more plates soon!




You could have compromised your filter spraying it with lysol.  When I was at Aloha, they NEVER sprayed their lab flowhood filter with anything.  John even warned us in class against touching the filter with anything.

Problem is, a particle counter is the only way I know of to tell for sure.




--------------------
--------------
"Democracy is two wolves and a lamb voting on what to have for lunch. Liberty is a well-armed lamb contesting the vote!" --Benjamin Franklin

"Those who give up liberty for security won't have, or deserve, either.". . . Benjamin Franklin
----> Read: The Fight of our Lives - Defeating the Ideological War Against the West - by Victor Davis Hanson

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Re: Don't trust UV lights for lab sterilization [Re: OICU812]
    #18709380 - 08/15/13 02:25 PM (10 years, 6 months ago)

Quote:

OICU812 said:
Quote:

Terry M said:

Yup, I always use a rack. Have never changed this procedure. BUT ...
Though I include the flowhood front framing when I give the filter a spray of Lysol, I've never cleaned under the flowhood!!

Thanks RR, I really hope this is it. It would explain the symptoms perfectly. Will be pouring more plates soon!




You could have compromised your filter spraying it with lysol.  When I was at Aloha, they NEVER sprayed their lab flowhood filter with anything.  John even warned us in class against touching the filter with anything.

Problem is, a particle counter is the only way I know of to tell for sure.







I use a light Lysol spray of the flow hood filter before turning it on as per RR's instructions on his video.


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Re: Don't trust UV lights for lab sterilization [Re: Terry M]
    #18721631 - 08/18/13 03:29 AM (10 years, 6 months ago)

> Problem is, a particle counter is the only way I know of to tell for sure.

Just use a petri dish.

Sure you have to wait a few days for them to develop, but what do you do if your $1,000 particle counter doesn't agree with the $0.50 petri dish test?  Then you've wasted $1k AND all the time and supplies wasted by operating with a broken filter.


-FF

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Re: Don't trust UV lights for lab sterilization [Re: fastfred]
    #18721723 - 08/18/13 04:50 AM (10 years, 6 months ago)

Quote:

fastfred said:
> Problem is, a particle counter is the only way I know of to tell for sure.

Just use a petri dish.

Sure you have to wait a few days for them to develop, but what do you do if your $1,000 particle counter doesn't agree with the $0.50 petri dish test?  Then you've wasted $1k AND all the time and supplies wasted by operating with a broken filter.
-FF




. . . and if you use poor sterile technique and contaminate the petri dish, you may needlessly replace a perfectly good filter.  Use what you have.  For me, since I have both, I'll use both.  I can't stop operations for 10 days waiting on a petri dish.  For the hobbyist, the petri dish is their logical choice.  More than one is a good idea too.


--------------------
--------------
"Democracy is two wolves and a lamb voting on what to have for lunch. Liberty is a well-armed lamb contesting the vote!" --Benjamin Franklin

"Those who give up liberty for security won't have, or deserve, either.". . . Benjamin Franklin
----> Read: The Fight of our Lives - Defeating the Ideological War Against the West - by Victor Davis Hanson

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Re: Don't trust UV lights for lab sterilization [Re: OICU812]
    #18724497 - 08/18/13 08:01 PM (10 years, 6 months ago)

But again, if you have your expensive counter and it DOESN'T agree with REAL tests then you are back to square one.

I wouldn't mind having one to play around with, but if you're serious then it's just a 2ndary test or quality control check.

For the average Joe it's too expensive and unnecessary.  For the serious scientist it doesn't provide the necessary proof of results.

I'm still hoping to hear if Terry tracks down his problems.  Hopefully he's put out dishes and will soon find out exactly how effective the UV is and where his contamination is coming from.  There's lots of sources and lots of fixes.  Nailing down your lab sterility is certainly a good feeling and well worthwhile.

Maybe every time the door opens so many contams are coming in that only a dusting of hypochlorite from excessive bleaching has protected him so far.

UV works.  It's proven and in wide use.  I'm sure Terry has felt the sheer nastiness of that harsh light.  It's no magic bullet, due to it's limitations, but I can't imagine anyone disputing that it doesn't kill things like nobody's business.


-FF

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Re: Don't trust UV lights for lab sterilization [Re: fastfred]
    #18724879 - 08/18/13 09:29 PM (10 years, 6 months ago)

Quote:


But again, if you have your expensive counter and it DOESN'T agree with REAL tests then you are back to square one.




Not necessarily, mycelium breath. The petri dish will tell you whether you have contaminants, the particle counter will allow you to pinpoint the location of leaks in a filter.  The petri dish only tells you if you had contaminants fall at that particular spot at that given time, if the contaminants flew over that petri dish and landed somewhere else, the petri dish could give a false reading.  Particle counters can't tell a contaminant from an elephant, but it can tell you, much more precisely than a petri dish, if you have an abnormally large volume of particles, and a change in the volume of particles for a given location versus another location or from one time to another.

Quote:

I wouldn't mind having one to play around with, but if you're serious then it's just a 2ndary test or quality control check.

For the average Joe it's too expensive and unnecessary.  For the serious scientist it doesn't provide the necessary proof of results.




Neither is secondary to the other.  They are very different tests.  Both are better than either individually.  Neither are superfluous.  The average Joe can absolutely get buy with just a petri dish, but it isn't necessarily proof positive either of the absolute presence or absence of contaminants.  I don't think the test exists that is absolute proof of the presence or absence of contaminants, however, the more data one has, the higher the probability of correlating results being accurate.



Quote:

I'm still hoping to hear if Terry tracks down his problems.  Hopefully he's put out dishes and will soon find out exactly how effective the UV is and where his contamination is coming from.  There's lots of sources and lots of fixes.  Nailing down your lab sterility is certainly a good feeling and well worthwhile.

Maybe every time the door opens so many contams are coming in that only a dusting of hypochlorite from excessive bleaching has protected him so far.




I have no doubt that Terry will track down his problems.  He is smart, logical and methodical and will eliminate probable sources until he is successful at discovering where his contamination source resides. 

Quote:

UV works.  It's proven and in wide use.  I'm sure Terry has felt the sheer nastiness of that harsh light.  It's no magic bullet, due to it's limitations, but I can't imagine anyone disputing that it doesn't kill things like nobody's business.




I agree, UV works.  I also agree that it is no magic bullet.  Those nasty contaminants can lurk in some pretty inconspicuous shadows and hide from many different forms of sanitization.


--------------------
--------------
"Democracy is two wolves and a lamb voting on what to have for lunch. Liberty is a well-armed lamb contesting the vote!" --Benjamin Franklin

"Those who give up liberty for security won't have, or deserve, either.". . . Benjamin Franklin
----> Read: The Fight of our Lives - Defeating the Ideological War Against the West - by Victor Davis Hanson

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