Re: [DIYbio] Re: Contamination. Help needed!

We are working with mammalian cells and we observe the contamination above all in haematological cell lines (K-562, TF-1 and also in CD34+ cells isolated from cord blood).

Normally, these cells are round but, after contamination, we observe really big cells, cells that look like fibroblasts, cells with protrusions (pictures attached).

Particles do not seem to move a lot but it is difficult to determine whether this movement is "autonomous" or not. They also seem to adhere to the support.

We try to observe if something grow in fresh media but we did not observe anything. It is a good idea to remove the cell line but we do not know how to distinguish these particles from the debris.

We do not have the material to study bacteria but we sent some samples to several labs:
- Nothing grew on their media
- 2 labs dit not observe anything with gram staining but one found gram + bacteria (but the staining was not good). These Gram + bacteria are found inside and outside the cells (pictures attached)
- We hypothesised that coud be mycobacteria. Then, we sent samples to a another lab to perform Ziehl staining. However, this test appeared negative.
- We do not know exactly whether microbiological tests were done on our media our on cells.
- We did mycoplasma test in our lab and the results were negative.
- We do not have the material to perform the universal PCR.
- Concerning cross-contamination, we don't think so because we are also working on primary cells, which are also contaminated.


Le jeudi 30 juillet 2015 17:32:58 UTC+2, Mike Horwath a écrit :
A few questions to help with diagnosis:

What cell lines are they? (mammalian, insect, etc)
Are these particles actually motile/swimming, or just drifting?  It is hard to tell from the video.
If you remove the  cell line (straining, hypososmotic lysis, etc) is the contamination able to replicate by itself in fresh media?
If you streak the culture on agar, do you get colonies?

What did you mean by this: "At the beginning we thought that the particles we observed were mycobacteria. But the gram tests from the microbiological labs appeared negative."  Mycoplasma will NOT show up on a gram stain.  Was there another test the lab did for mycoplasma?

Dakota's suggestions to check for contamination in your media and check for 16S RNA are good ones.

One of the hardest kinds of contamination to detect and get rid of is contamination from another cell line. It's possible you could have a 2nd cell line mixed in, that has a different morphology and might be producing more apoptotic blebs...

Mike




On Thursday, July 30, 2015 at 10:54:26 AM UTC-4, Dakota wrote:

Check some controls.  Look at just the growth media fresh, then after a few days.  Aerosols from pipettes could be building up but I imagine you use filter tips. 

Also, just spin everything down, extract DNA, and run 16s or ITS bacteria/fungal barcoding to look for a band.  Sequence it if you have one and you could get the exact species

On Jul 30, 2015 1:35 PM, "Cindy GRANDJENETTE" <tupa...@gmail.com> wrote:
Thanks for your answer.
We have this problem for 4 months.  At the beginning we thought that the particles we observed were mycobacteria. But the gram tests from the microbiological labs appeared negative. Of note, our cells look grainy.
We are not able to determine whether these particles are the source of contamination or just a consequence. 
We use penicillin-streptomycin and fungizone (Amphotericin B)  in our culture media. 
We already cleaned the lab (laminar flows, incubators, etc), autoclaved everything, and threw our cells away. But the problem is still here. And we do not know how to solve it.

Cindy




Le mercredi 29 juillet 2015 23:06:07 UTC+2, Ravasz a écrit :
Hi,

No idea what this is, but I can tell you what its not, maybe that helps:

- It's definitely not yeast, that looks very different. Yeast would consist of small groups (4-10) of oval particles, smaller than a mammalian cell, with a well defined, smooth outline. They would also outgrow your cells and kill the culture in a matter of days. This is not like that.

- It's not mycoplasma or a virus as both are too small to resolve in a light microscope. They would simply not be visible even in a rampant infection.

- It is not usual cell debris as it is moving.

My best bet is that it is some form of bacteria. Were you using penicillin-streptomycin in your medium? If not, try adding that. You can also add some Fungizone too if you are desperate, as it might be some fungus.

However, such "resurrected" cultures are never really perfect as the cells were put under stress and they might be permanently altered, even if appearing completely cleaned at some point. I would just discard the whole culture along with any media and buffers that are in use, autoclave any equipment that came into contact with it and thoroughly wash incubators and culture hoods with Virkon, or your disinfectant of choice.

If this is showing up in multiple cultures, then the source of contamination is not within the culture itself.

Maybe this helps.

Mate



On Wednesday, 29 July 2015 14:21:08 UTC+1, Cindy GRANDJENETTE wrote:
Dear all, 

We have problems of contamination in several of our cell lines. Cell viability is pretty good, however, cell morphology is affected whatever the cell line. In addition and importantly, we observe a lot of small particles in the medium. Their number increase over time.
Mycoplasma tests were negative. We also sent our samples to 2 microbiological labs. Nevertheless, they were not able to identify the source of our contamination. We are wondering whether these contaminations are due to (myco)bacteria, viruses, or even yeast.
You may find attached movies showing how these particles "appear". The morphology of this cell line is normally homogeneous with round cells.


Does somebody have an idea concerning this contamination ? 

Thanks for your help,

Cindy

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[DIYbio] Re: Contamination. Help needed!

I was under the impression that the only real strong test for mycoplasma was PCR (and even it can miss if you dont use diverse enough primers as there are many species of mycoplasma).

On Wednesday, July 29, 2015 at 6:21:08 AM UTC-7, Cindy GRANDJENETTE wrote:
Dear all, 

We have problems of contamination in several of our cell lines. Cell viability is pretty good, however, cell morphology is affected whatever the cell line. In addition and importantly, we observe a lot of small particles in the medium. Their number increase over time.
Mycoplasma tests were negative. We also sent our samples to 2 microbiological labs. Nevertheless, they were not able to identify the source of our contamination. We are wondering whether these contaminations are due to (myco)bacteria, viruses, or even yeast.
You may find attached movies showing how these particles "appear". The morphology of this cell line is normally homogeneous with round cells.


Does somebody have an idea concerning this contamination ? 

Thanks for your help,

Cindy

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Re: [DIYbio] Re: Contamination. Help needed!

Thanks for your answer.
We already looked if something grow in the fresh media, but we did not observe anything after few days.
For the second point, we thought to do it but unfortunately we do not have the material to perform it in the lab.



Le jeudi 30 juillet 2015 16:54:26 UTC+2, Dakota a écrit :

Check some controls.  Look at just the growth media fresh, then after a few days.  Aerosols from pipettes could be building up but I imagine you use filter tips. 

Also, just spin everything down, extract DNA, and run 16s or ITS bacteria/fungal barcoding to look for a band.  Sequence it if you have one and you could get the exact species

On Jul 30, 2015 1:35 PM, "Cindy GRANDJENETTE" <tupa...@gmail.com> wrote:
Thanks for your answer.
We have this problem for 4 months.  At the beginning we thought that the particles we observed were mycobacteria. But the gram tests from the microbiological labs appeared negative. Of note, our cells look grainy.
We are not able to determine whether these particles are the source of contamination or just a consequence. 
We use penicillin-streptomycin and fungizone (Amphotericin B)  in our culture media. 
We already cleaned the lab (laminar flows, incubators, etc), autoclaved everything, and threw our cells away. But the problem is still here. And we do not know how to solve it.

Cindy




Le mercredi 29 juillet 2015 23:06:07 UTC+2, Ravasz a écrit :
Hi,

No idea what this is, but I can tell you what its not, maybe that helps:

- It's definitely not yeast, that looks very different. Yeast would consist of small groups (4-10) of oval particles, smaller than a mammalian cell, with a well defined, smooth outline. They would also outgrow your cells and kill the culture in a matter of days. This is not like that.

- It's not mycoplasma or a virus as both are too small to resolve in a light microscope. They would simply not be visible even in a rampant infection.

- It is not usual cell debris as it is moving.

My best bet is that it is some form of bacteria. Were you using penicillin-streptomycin in your medium? If not, try adding that. You can also add some Fungizone too if you are desperate, as it might be some fungus.

However, such "resurrected" cultures are never really perfect as the cells were put under stress and they might be permanently altered, even if appearing completely cleaned at some point. I would just discard the whole culture along with any media and buffers that are in use, autoclave any equipment that came into contact with it and thoroughly wash incubators and culture hoods with Virkon, or your disinfectant of choice.

If this is showing up in multiple cultures, then the source of contamination is not within the culture itself.

Maybe this helps.

Mate



On Wednesday, 29 July 2015 14:21:08 UTC+1, Cindy GRANDJENETTE wrote:
Dear all, 

We have problems of contamination in several of our cell lines. Cell viability is pretty good, however, cell morphology is affected whatever the cell line. In addition and importantly, we observe a lot of small particles in the medium. Their number increase over time.
Mycoplasma tests were negative. We also sent our samples to 2 microbiological labs. Nevertheless, they were not able to identify the source of our contamination. We are wondering whether these contaminations are due to (myco)bacteria, viruses, or even yeast.
You may find attached movies showing how these particles "appear". The morphology of this cell line is normally homogeneous with round cells.


Does somebody have an idea concerning this contamination ? 

Thanks for your help,

Cindy

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Re: [DIYbio] Re: Contamination. Help needed!

A few questions to help with diagnosis:

What cell lines are they? (mammalian, insect, etc)
Are these particles actually motile/swimming, or just drifting?  It is hard to tell from the video.
If you remove the  cell line (straining, hypososmotic lysis, etc) is the contamination able to replicate by itself in fresh media?
If you streak the culture on agar, do you get colonies?

What did you mean by this: "At the beginning we thought that the particles we observed were mycobacteria. But the gram tests from the microbiological labs appeared negative."  Mycoplasma will NOT show up on a gram stain.  Was there another test the lab did for mycoplasma?

Dakota's suggestions to check for contamination in your media and check for 16S RNA are good ones.

One of the hardest kinds of contamination to detect and get rid of is contamination from another cell line. It's possible you could have a 2nd cell line mixed in, that has a different morphology and might be producing more apoptotic blebs...

Mike




On Thursday, July 30, 2015 at 10:54:26 AM UTC-4, Dakota wrote:

Check some controls.  Look at just the growth media fresh, then after a few days.  Aerosols from pipettes could be building up but I imagine you use filter tips. 

Also, just spin everything down, extract DNA, and run 16s or ITS bacteria/fungal barcoding to look for a band.  Sequence it if you have one and you could get the exact species

On Jul 30, 2015 1:35 PM, "Cindy GRANDJENETTE" <tupa...@gmail.com> wrote:
Thanks for your answer.
We have this problem for 4 months.  At the beginning we thought that the particles we observed were mycobacteria. But the gram tests from the microbiological labs appeared negative. Of note, our cells look grainy.
We are not able to determine whether these particles are the source of contamination or just a consequence. 
We use penicillin-streptomycin and fungizone (Amphotericin B)  in our culture media. 
We already cleaned the lab (laminar flows, incubators, etc), autoclaved everything, and threw our cells away. But the problem is still here. And we do not know how to solve it.

Cindy




Le mercredi 29 juillet 2015 23:06:07 UTC+2, Ravasz a écrit :
Hi,

No idea what this is, but I can tell you what its not, maybe that helps:

- It's definitely not yeast, that looks very different. Yeast would consist of small groups (4-10) of oval particles, smaller than a mammalian cell, with a well defined, smooth outline. They would also outgrow your cells and kill the culture in a matter of days. This is not like that.

- It's not mycoplasma or a virus as both are too small to resolve in a light microscope. They would simply not be visible even in a rampant infection.

- It is not usual cell debris as it is moving.

My best bet is that it is some form of bacteria. Were you using penicillin-streptomycin in your medium? If not, try adding that. You can also add some Fungizone too if you are desperate, as it might be some fungus.

However, such "resurrected" cultures are never really perfect as the cells were put under stress and they might be permanently altered, even if appearing completely cleaned at some point. I would just discard the whole culture along with any media and buffers that are in use, autoclave any equipment that came into contact with it and thoroughly wash incubators and culture hoods with Virkon, or your disinfectant of choice.

If this is showing up in multiple cultures, then the source of contamination is not within the culture itself.

Maybe this helps.

Mate



On Wednesday, 29 July 2015 14:21:08 UTC+1, Cindy GRANDJENETTE wrote:
Dear all, 

We have problems of contamination in several of our cell lines. Cell viability is pretty good, however, cell morphology is affected whatever the cell line. In addition and importantly, we observe a lot of small particles in the medium. Their number increase over time.
Mycoplasma tests were negative. We also sent our samples to 2 microbiological labs. Nevertheless, they were not able to identify the source of our contamination. We are wondering whether these contaminations are due to (myco)bacteria, viruses, or even yeast.
You may find attached movies showing how these particles "appear". The morphology of this cell line is normally homogeneous with round cells.


Does somebody have an idea concerning this contamination ? 

Thanks for your help,

Cindy

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Re: [DIYbio] Re: Contamination. Help needed!

Check some controls.  Look at just the growth media fresh, then after a few days.  Aerosols from pipettes could be building up but I imagine you use filter tips. 

Also, just spin everything down, extract DNA, and run 16s or ITS bacteria/fungal barcoding to look for a band.  Sequence it if you have one and you could get the exact species

On Jul 30, 2015 1:35 PM, "Cindy GRANDJENETTE" <tupariqc@gmail.com> wrote:
Thanks for your answer.
We have this problem for 4 months.  At the beginning we thought that the particles we observed were mycobacteria. But the gram tests from the microbiological labs appeared negative. Of note, our cells look grainy.
We are not able to determine whether these particles are the source of contamination or just a consequence. 
We use penicillin-streptomycin and fungizone (Amphotericin B)  in our culture media. 
We already cleaned the lab (laminar flows, incubators, etc), autoclaved everything, and threw our cells away. But the problem is still here. And we do not know how to solve it.

Cindy




Le mercredi 29 juillet 2015 23:06:07 UTC+2, Ravasz a écrit :
Hi,

No idea what this is, but I can tell you what its not, maybe that helps:

- It's definitely not yeast, that looks very different. Yeast would consist of small groups (4-10) of oval particles, smaller than a mammalian cell, with a well defined, smooth outline. They would also outgrow your cells and kill the culture in a matter of days. This is not like that.

- It's not mycoplasma or a virus as both are too small to resolve in a light microscope. They would simply not be visible even in a rampant infection.

- It is not usual cell debris as it is moving.

My best bet is that it is some form of bacteria. Were you using penicillin-streptomycin in your medium? If not, try adding that. You can also add some Fungizone too if you are desperate, as it might be some fungus.

However, such "resurrected" cultures are never really perfect as the cells were put under stress and they might be permanently altered, even if appearing completely cleaned at some point. I would just discard the whole culture along with any media and buffers that are in use, autoclave any equipment that came into contact with it and thoroughly wash incubators and culture hoods with Virkon, or your disinfectant of choice.

If this is showing up in multiple cultures, then the source of contamination is not within the culture itself.

Maybe this helps.

Mate



On Wednesday, 29 July 2015 14:21:08 UTC+1, Cindy GRANDJENETTE wrote:
Dear all, 

We have problems of contamination in several of our cell lines. Cell viability is pretty good, however, cell morphology is affected whatever the cell line. In addition and importantly, we observe a lot of small particles in the medium. Their number increase over time.
Mycoplasma tests were negative. We also sent our samples to 2 microbiological labs. Nevertheless, they were not able to identify the source of our contamination. We are wondering whether these contaminations are due to (myco)bacteria, viruses, or even yeast.
You may find attached movies showing how these particles "appear". The morphology of this cell line is normally homogeneous with round cells.


Does somebody have an idea concerning this contamination ? 

Thanks for your help,

Cindy

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[DIYbio] Re: Contamination. Help needed!

Thanks for your answer.
We have this problem for 4 months.  At the beginning we thought that the particles we observed were mycobacteria. But the gram tests from the microbiological labs appeared negative. Of note, our cells look grainy.
We are not able to determine whether these particles are the source of contamination or just a consequence. 
We use penicillin-streptomycin and fungizone (Amphotericin B)  in our culture media. 
We already cleaned the lab (laminar flows, incubators, etc), autoclaved everything, and threw our cells away. But the problem is still here. And we do not know how to solve it.

Cindy



Le mercredi 29 juillet 2015 23:06:07 UTC+2, Ravasz a écrit :
Hi,

No idea what this is, but I can tell you what its not, maybe that helps:

- It's definitely not yeast, that looks very different. Yeast would consist of small groups (4-10) of oval particles, smaller than a mammalian cell, with a well defined, smooth outline. They would also outgrow your cells and kill the culture in a matter of days. This is not like that.

- It's not mycoplasma or a virus as both are too small to resolve in a light microscope. They would simply not be visible even in a rampant infection.

- It is not usual cell debris as it is moving.

My best bet is that it is some form of bacteria. Were you using penicillin-streptomycin in your medium? If not, try adding that. You can also add some Fungizone too if you are desperate, as it might be some fungus.

However, such "resurrected" cultures are never really perfect as the cells were put under stress and they might be permanently altered, even if appearing completely cleaned at some point. I would just discard the whole culture along with any media and buffers that are in use, autoclave any equipment that came into contact with it and thoroughly wash incubators and culture hoods with Virkon, or your disinfectant of choice.

If this is showing up in multiple cultures, then the source of contamination is not within the culture itself.

Maybe this helps.

Mate



On Wednesday, 29 July 2015 14:21:08 UTC+1, Cindy GRANDJENETTE wrote:
Dear all, 

We have problems of contamination in several of our cell lines. Cell viability is pretty good, however, cell morphology is affected whatever the cell line. In addition and importantly, we observe a lot of small particles in the medium. Their number increase over time.
Mycoplasma tests were negative. We also sent our samples to 2 microbiological labs. Nevertheless, they were not able to identify the source of our contamination. We are wondering whether these contaminations are due to (myco)bacteria, viruses, or even yeast.
You may find attached movies showing how these particles "appear". The morphology of this cell line is normally homogeneous with round cells.


Does somebody have an idea concerning this contamination ? 

Thanks for your help,

Cindy

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[DIYbio] Re: Contamination. Help needed!

Hi,

No idea what this is, but I can tell you what its not, maybe that helps:

- It's definitely not yeast, that looks very different. Yeast would consist of small groups (4-10) of oval particles, smaller than a mammalian cell, with a well defined, smooth outline. They would also outgrow your cells and kill the culture in a matter of days. This is not like that.

- It's not mycoplasma or a virus as both are too small to resolve in a light microscope. They would simply not be visible even in a rampant infection.

- It is not usual cell debris as it is moving.

My best bet is that it is some form of bacteria. Were you using penicillin-streptomycin in your medium? If not, try adding that. You can also add some Fungizone too if you are desperate, as it might be some fungus.

However, such "resurrected" cultures are never really perfect as the cells were put under stress and they might be permanently altered, even if appearing completely cleaned at some point. I would just discard the whole culture along with any media and buffers that are in use, autoclave any equipment that came into contact with it and thoroughly wash incubators and culture hoods with Virkon, or your disinfectant of choice.

If this is showing up in multiple cultures, then the source of contamination is not within the culture itself.

Maybe this helps.

Mate



On Wednesday, 29 July 2015 14:21:08 UTC+1, Cindy GRANDJENETTE wrote:
Dear all, 

We have problems of contamination in several of our cell lines. Cell viability is pretty good, however, cell morphology is affected whatever the cell line. In addition and importantly, we observe a lot of small particles in the medium. Their number increase over time.
Mycoplasma tests were negative. We also sent our samples to 2 microbiological labs. Nevertheless, they were not able to identify the source of our contamination. We are wondering whether these contaminations are due to (myco)bacteria, viruses, or even yeast.
You may find attached movies showing how these particles "appear". The morphology of this cell line is normally homogeneous with round cells.


Does somebody have an idea concerning this contamination ? 

Thanks for your help,

Cindy

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Required || QlikView Developer || NYC,NY || 6+ Months || Phone And F2F

Dear Partner

Hope you are doing great!!

Please go through the requirement and let me know if you have any consultant for the same position.

 

Please do share me your hotlist also at Arpit@tresourceinc.com

 

QlikView Developer

NYC,NY

6+ Months

Candidates must be local and available for a F2F interview

Description

We are seeking an experienced IT professional that will be responsible for gathering requirements, architecting, designing, developing, and supporting QlikView applications. The candidate must have experience working on QlikView scripting, data analytics, data modeling and dashboard design using QlikView. Candidate must also be well versed in full software development lifecycle.

 

Qualifications

Skill Required
- 5 plus years working with QlikView v9/10/11 design and development
- 3 plus years gathering requirements for QlikView
- Strong understanding of QlikView v11 architecture
- Good understanding of data modeling concepts
- Good at Dashboard and UI design
- Good data analysis skills
- Strong Database skills (DB2, Sybase)
- Excellent problem solving skills
- Excellent verbal and written communication skills
- Ability to multi-task

Skills Desired
- Experience working with offshore teams/ coordinating tasks.
- Experience with QlikSense / Tableau / SAP BusinessObjects
- Experience with Autosys

 

Thanks and Regards,

Arpit Arora | Sr.Technical Recruiter

Technology Resource Group Inc. 
3736 Hillsdale Court Santa Clara, CA 95051

Office:408-709-1760 Ext : 961, Cell: 408-502-5132

Gtalk: Arpittechwire | Fax: 408-884-2409

Arpit@tresourceinc.com | www.tresourceinc.com

 

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[DIYbio] Contamination. Help needed!

Dear all, 

We have problems of contamination in several of our cell lines. Cell viability is pretty good, however, cell morphology is affected whatever the cell line. In addition and importantly, we observe a lot of small particles in the medium. Their number increase over time.
Mycoplasma tests were negative. We also sent our samples to 2 microbiological labs. Nevertheless, they were not able to identify the source of our contamination. We are wondering whether these contaminations are due to (myco)bacteria, viruses, or even yeast.
You may find attached movies showing how these particles "appear". The morphology of this cell line is normally homogeneous with round cells.

https://www.dropbox.com/s/n1p711dwfxnzxgy/150724%20tf1%202h30%20mes%205%20min%202%28converti%29.mov?dl=0
https://www.dropbox.com/s/ggbt1u8i3piau0q/150724%20tf1%202h30%20mes%205%20min.avi?dl=0
https://www.dropbox.com/s/sp2ncal7szl2kkr/150726%20tf1%202%20min%20mes%20100ms.mov?dl=0
https://www.dropbox.com/s/c0okt4k3twgzg4z/150726%20tf1%204h%205%20min.avi?dl=0
https://www.dropbox.com/s/6ngjhzcy9fejzjn/TF-1%20big%20cell.avi?dl=0

Does somebody have an idea concerning this contamination ? 

Thanks for your help,

Cindy

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[DIYbio] DIYbio en Chile?

Hola!
¿Hay alguien interesado en hacer un espacio abierto en Valparaíso, Chile?

Saludos,
Romina

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[DIYbio] Re: diybio in buenos aires?

EKG as in electrocardiogram? I want to learn to program and I chose python because is a multi purpose language. Also MATLAB to use as a simulation tool, and to learn to program in a more math oriented language.

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[DIYbio] diyBio Salt Lake?

Hello,

   Is there a DIY bio group in Salt Lake City Utah?  Is there interest?  

Rgds,
  Mike Gruenhagen
  email mikegruenhagen@hotmail.com

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Re: [DIYbio] Hard-to-find paper request

It would be such a convenience. Especially since said book is not being sold anywhere in any form an no one is making money off of it. Oh well, I guess Ill just have to read the book, write down the recipe, and move on with life.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC


> On Jul 28, 2015, at 2:39 PM, Cathal Garvey <cathalgarvey@cathalgarvey.me> wrote:
>
> Wouldn't it be neat if a scan of that book were to appear on a torrent site in the next few days? ;)
>
>> On 28/07/15 19:33, Sebastian S Cocioba wrote:
>> I tried getting it electronically sent and apparently the 50 page upper limit they allow is suddenly too large for fair use copy. Its sitting at the NYPL for me. Going Thursday to copy the mysterious tome.
>>
>> Sebastian S. Cocioba
>> CEO & Founder
>> New York Botanics, LLC
>>
>>
>>>> On Jul 26, 2015, at 11:18 AM, John Griessen <john@industromatic.com> wrote:
>>>>
>>>> On 07/26/2015 04:48 AM, Sebastian S Cocioba wrote:
>>>> Most likely some salts based recipe with ammonium as the main nitrogen source.
>>>
>>> Hmmm.... That company with the dirt bacterial sprays must be expert on just that.
>>> They develop ammonia oxidizing bacteria strains plus ?more? ?b. subtilis? recipes.
>>>
>>> http://www.bloomberg.com/news/articles/2015-07-22/cosmetics-startup-aobiome-sells-bacteria-for-healthier-skin
>>>
>>> The founder started from a notice of how horses roll in dirt then researched
>>> many many papers, (including yours), then began building and testing.
>>> maybe he'd help -- you're not competing with his market...
>>>
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