SEA-PHAGES | All posts created by viknesh

Link to this post \| posted 08 Sep, 2018 02:16
viknesh	Hi Evan, We've posted a modification that we know works for extracting DNA from M. foliorum phages. The modification is listed as an optional step in the DNA extraction protocol, and involves adding ProteinaseK and SDS. Vic

Posted in: Phage Discovery/Isolation → DNA Isolation from M. foliorum - any updates?

Link to this post \| posted 27 Aug, 2018 18:13
viknesh	kaylafast P2FF streak from stock #1 Incubation: 37 degrees C, 2 days (1 day photo unavailable) I would be suspicious of this streak plate (and the culture from which the streak was setup). There appears to be more growth than expected. Go ahead and test your latest culture, but also setup streak plates (and mock streaks) from your glycerol stocks. Let me know what you see. You can then go ahead and setup cultures.

Link to this post | posted 27 Aug, 2018 18:13

viknesh

kaylafast
P2FF streak from stock #1
Incubation: 37 degrees C, 2 days (1 day photo unavailable)

I would be suspicious of this streak plate (and the culture from which the streak was setup). There appears to be more growth than expected. Go ahead and test your latest culture, but also setup streak plates (and mock streaks) from your glycerol stocks. Let me know what you see. You can then go ahead and setup cultures.

Posted in: Phage Discovery/Isolation → D29 plaques barely visible on M. smeg

Link to this post \| posted 27 Aug, 2018 15:59
viknesh	kaylafast P2FF streak from stock #1 Kayla, can you include the incubation duration before the pictures were taken, for each plate?

Posted in: Phage Discovery/Isolation → D29 plaques barely visible on M. smeg

Link to this post \| posted 27 Aug, 2018 15:43
viknesh	kaylafast I have read the suggestion to streak out the liquid culture and if there are any colonies in 24 hrs, it is not smeg - or the smeg is contaminated. Does this mean any growth at all within 24 hours or just individual colonies? Streaks from our P1FF and P2FF do show growth within 24 hours, but just smears not individual colonies. Hi Kayla, A few things: 1 - Would you be able to share pictures of your streak plates (both from glycerol but also from liquid culture)? You'd expect them to behave similarly, with growth perhaps faster for the plate streaked from the liquid culture. 2 - From the pic you attached, it is hard to tell if a) there is a robust bacterial lawn but turbid plaques, b)light/non-robust lawn with turbid plaques, or c)light/non-robust lawn with clear plaques. If the plaques are turbid, it is likely that you have a contaminating microbe in your liquid culture that is not lysed by D29. When you setup liquid cultures, it maybe worth setting up a mock culture. For this, you do exactly what you do as you are setting up your liquid culture, but instead of picking up a colony with an inoculating stick/loop and dipping in into the liquid media, you just dip a sterile inoculating stick/loop directly into the media. This way you can determine if your flask, media, or inoculating loop is contaminated. Vic

Link to this post | posted 27 Aug, 2018 15:43

viknesh

kaylafast
I have read the suggestion to streak out the liquid culture and if there are any colonies in 24 hrs, it is not smeg - or the smeg is contaminated. Does this mean any growth at all within 24 hours or just individual colonies? Streaks from our P1FF and P2FF do show growth within 24 hours, but just smears not individual colonies.

Hi Kayla,

A few things:

1 - Would you be able to share pictures of your streak plates (both from glycerol but also from liquid culture)? You'd expect them to behave similarly, with growth perhaps faster for the plate streaked from the liquid culture.

2 - From the pic you attached, it is hard to tell if a) there is a robust bacterial lawn but turbid plaques, b)light/non-robust lawn with turbid plaques, or c)light/non-robust lawn with clear plaques. If the plaques are turbid, it is likely that you have a contaminating microbe in your liquid culture that is not lysed by D29. When you setup liquid cultures, it maybe worth setting up a mock culture. For this, you do exactly what you do as you are setting up your liquid culture, but instead of picking up a colony with an inoculating stick/loop and dipping in into the liquid media, you just dip a sterile inoculating stick/loop directly into the media. This way you can determine if your flask, media, or inoculating loop is contaminated.

Vic

Posted in: Phage Discovery/Isolation → D29 plaques barely visible on M. smeg

Link to this post \| posted 07 Jul, 2018 14:53
viknesh	asprenkle Cohort 11, going to the 11B workshop tomorrow! Downloaded the app from Google Play to my android phone. Tried to create account using .edu email and nothing happens. Preparatory info for the workshop suggests that I should get a link confirming the account from my email? Tried restarting my phone, no effect. Any ideas? Hi Amy, The app is down at the moment. When you check-in at UMBC, we'll tell you how to go about sample collection without using the app. See you soon!

Link to this post | posted 07 Jul, 2018 14:53

viknesh

asprenkle
Cohort 11, going to the 11B workshop tomorrow! Downloaded the app from Google Play to my android phone. Tried to create account using .edu email and nothing happens. Preparatory info for the workshop suggests that I should get a link confirming the account from my email?
Tried restarting my phone, no effect. Any ideas?

Hi Amy,

The app is down at the moment. When you check-in at UMBC, we'll tell you how to go about sample collection without using the app. See you soon!

Posted in: SEA-PHAGES App → a work in progress

Link to this post \| posted 10 Nov, 2017 18:24
viknesh	Adaptive Phage Therapeutics (APT) is currently in need of experienced bacteriophage microbiologists to join our team. Attached to this post is a job description for a BS- or MS-level bacteriophage microbiologist. APT is a clinical-stage pharmaceutical company based in Gaithersburg, Maryland that is focused on developing an effective and personalized bacteriophage therapy in response to the global rise of multi-drug resistant (MDR) bacterial pathogens. Two key components to APT's approach include a large and dynamically growing collection of bacteriophages, or PhageBank™, and an innovative rapid system for matching bacteriophage to patient-specific bacterial infections called the Host Range Quick Test. See links below for more information about Adaptive Phage Therapeutics, Inc.: Company Website: http://www.aphage.com News Articles: 1) http://www.businesswire.com/news/home/20170426006880/en/Adaptive-Phage-Therapeutics---Terminally-Ill-Patient 2) https://www.buzzfeed.com/azeenghorayshi/navy-phage-viruses-for-antibiotics-crisis?utm_term=.gvym0OEXQ#.vdmjdPXNV 3) https://www.nbcnews.com/mach/science/post-antibiotic-apocalypse-can-be-prevented-here-s-how-ncna812576

Link to this post | posted 10 Nov, 2017 18:24

viknesh

Adaptive Phage Therapeutics (APT) is currently in need of experienced bacteriophage microbiologists to join our team. Attached to this post is a job description for a BS- or MS-level bacteriophage microbiologist.

APT is a clinical-stage pharmaceutical company based in Gaithersburg, Maryland that is focused on developing an effective and personalized bacteriophage therapy in response to the global rise of multi-drug resistant (MDR) bacterial pathogens. Two key components to APT's approach include a large and dynamically growing collection of bacteriophages, or PhageBank™, and an innovative rapid system for matching bacteriophage to patient-specific bacterial infections called the Host Range Quick Test.

See links below for more information about Adaptive Phage Therapeutics, Inc.:

Company Website: http://www.aphage.com
News Articles:
1) http://www.businesswire.com/news/home/20170426006880/en/Adaptive-Phage-Therapeutics---Terminally-Ill-Patient
2) https://www.buzzfeed.com/azeenghorayshi/navy-phage-viruses-for-antibiotics-crisis?utm_term=.gvym0OEXQ#.vdmjdPXNV
3) https://www.nbcnews.com/mach/science/post-antibiotic-apocalypse-can-be-prevented-here-s-how-ncna812576

Posted in: General Message Board → Bacteriophage Microbiologist Position

Link to this post \| posted 16 Oct, 2017 17:44
viknesh	mdgainey Hi all, hope your semester is going well. We are currently working with phages isolated from Microbacterium foliorum… Hi Marie, We've done some work with M. foliorum. The yields from the 8 or so preps were similar to what we typically get with mycobacteriophages, which is 60 - 80 ng/ul from 1 ml of a e9 pfu/ml lysate. When we PEG-precipitate phage, the DNA yields increased proportionately with the volume of lysate used. With regard to degraded DNA, we (including several SEA faculty) have noticed that some DNA preps that were isolated using the new isolation protocol (no PEG precipitation) is degraded upon mixing with restriction enzyme buffer. We think this may indicate that some of the nucleases added are coming through the DNA prep, and activated when mixed with the restriction enzyme buffer. This does not seem to happen when PEG precipitation in included, suggesting that the precipitation step is helpful with removing some/most of the nucleases that are added. In your case, where no nucleases are added, its surprising that you still see degraded DNA. Do you see degraded DNA when run on a gel without restriction enzyme buffer? One other possibility we've entertained is that when the Wizard denaturation mix is not properly mixed before use, you might not have full inactivation of nucleases that are added or host bacterial nucleases present in the lysate. You might have to give us more information about how you are performing your DNA isolation, and when and where you are seeing the degraded DNA. As for restriction enzymes, we tested a panel of enzymes and found NspI (also called XceI) is the most diagnostic enzyme. The majority of other enzymes tested did not cut DNA. SacII was able to digest one or two phage DNA preps, and HaeIII, as expected, cut so many times that unless a high percentage gel is used, is not informative. If you search for phage Eleri on pahgesDB, the restriction gel on the web includes several other M. foliorum phages for comparison.

Link to this post | posted 16 Oct, 2017 17:44

viknesh

mdgainey
Hi all, hope your semester is going well. We are currently working with phages isolated from Microbacterium foliorum…

Hi Marie,

We've done some work with M. foliorum. The yields from the 8 or so preps were similar to what we typically get with mycobacteriophages, which is 60 - 80 ng/ul from 1 ml of a e9 pfu/ml lysate. When we PEG-precipitate phage, the DNA yields increased proportionately with the volume of lysate used.

With regard to degraded DNA, we (including several SEA faculty) have noticed that some DNA preps that were isolated using the new isolation protocol (no PEG precipitation) is degraded upon mixing with restriction enzyme buffer. We think this may indicate that some of the nucleases added are coming through the DNA prep, and activated when mixed with the restriction enzyme buffer. This does not seem to happen when PEG precipitation in included, suggesting that the precipitation step is helpful with removing some/most of the nucleases that are added. In your case, where no nucleases are added, its surprising that you still see degraded DNA. Do you see degraded DNA when run on a gel without restriction enzyme buffer? One other possibility we've entertained is that when the Wizard denaturation mix is not properly mixed before use, you might not have full inactivation of nucleases that are added or host bacterial nucleases present in the lysate. You might have to give us more information about how you are performing your DNA isolation, and when and where you are seeing the degraded DNA.

As for restriction enzymes, we tested a panel of enzymes and found NspI (also called XceI) is the most diagnostic enzyme. The majority of other enzymes tested did not cut DNA. SacII was able to digest one or two phage DNA preps, and HaeIII, as expected, cut so many times that unless a high percentage gel is used, is not informative. If you search for phage Eleri on pahgesDB, the restriction gel on the web includes several other M. foliorum phages for comparison.

Posted in: Microbacterium → Restriction Enzyme Digests

Link to this post \| posted 10 Oct, 2017 15:17
viknesh	Yet another full-time position at a phage therapy company (same company as in the previous post) for a BS/MS with phage experience. Located in Gaithersburg, MD. 87Kb

Posted in: General Message Board → Bacteriophage Microbiologist Position

Link to this post \| posted 25 Sep, 2017 13:27
viknesh	Pollenz I am unable to find the 200-400 mesh carbon-formvar coated copper grids as specified in the ordering guide and listed as item #1813. Is there a typo or can someone help me to purchase the correct item as I have no experience with these materials. The cat # is 01813. The 0 was dropped from the original excel sheet (standard excel formatting), but has now been updated. Were you able to find the item? Vic

Link to this post | posted 25 Sep, 2017 13:27

viknesh

Pollenz
I am unable to find the 200-400 mesh carbon-formvar coated copper grids as specified in the ordering guide and listed as item #1813. Is there a typo or can someone help me to purchase the correct item as I have no experience with these materials.

The cat # is 01813. The 0 was dropped from the original excel sheet (standard excel formatting), but has now been updated. Were you able to find the item?

Vic

Posted in: General Message Board → EM GRIDS PURCHASE ITEM 1813 not showing up

Link to this post \| posted 17 Nov, 2016 20:58
viknesh	Hi Stephanie, I would be great if you could upload it here. Vic Edited 17 Nov, 2016 20:58

Posted in: Phage Discovery/Isolation → Cluster Specific Primers

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