SEA-PHAGES | D29 plaques barely visible on M. smeg

Link to this post \| posted 27 Aug, 2018 15:28
kaylafast	We are having difficulty getting D29 and other control phages to appear on our M. smeg plates. If you look hard enough and get a lot of light coming through the plate, we can see clearings on spot tests and plaques on plaque assays. But, we have to look really hard, and most photographs cannot capture that the plaques were ever there. I've started multiple cultures from single colonies on a streak plate, and feel that all media components are good (many have been remade). The streak plates have come from different glycerol stocks. 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. Attached is a picture of a D29 plaque assay of the 10-3 dilution showing the plaques. I have many more pictures of streak plates and phage test plates that I can share if they will be helpful. 139Kb

Link to this post | posted 27 Aug, 2018 15:28

We are having difficulty getting D29 and other control phages to appear on our M. smeg plates. If you look hard enough and get a lot of light coming through the plate, we can see clearings on spot tests and plaques on plaque assays. But, we have to look really hard, and most photographs cannot capture that the plaques were ever there.

I've started multiple cultures from single colonies on a streak plate, and feel that all media components are good (many have been remade). The streak plates have come from different glycerol stocks. 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.

Attached is a picture of a D29 plaque assay of the 10-3 dilution showing the plaques. I have many more pictures of streak plates and phage test plates that I can share if they will be helpful.

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

Link to this post \| posted 27 Aug, 2018 15:51
kaylafast	P0FF Streak from stock #1 Incubation: 37 degrees C, 5 days Edited 27 Aug, 2018 16:01 1.95Mb

Link to this post \| posted 27 Aug, 2018 15:52
kaylafast	P1FF streak from stock #1 Incubation: 37 degrees C, 2 days (1 day photo unavailable) Edited 27 Aug, 2018 16:06 2.26Mb

Link to this post \| posted 27 Aug, 2018 15:55
kaylafast	P2FF streak from stock #1 Incubation: 37 degrees C, 2 days (1 day photo unavailable) Edited 27 Aug, 2018 16:07 2.23Mb

Link to this post \| posted 27 Aug, 2018 15:58
kaylafast	P0FF streak from stock #2 Incubation: 37 degrees C, 4 days Edited 27 Aug, 2018 16:08 1.75Mb

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?

Link to this post \| posted 27 Aug, 2018 15:59
kaylafast	P1FF streak from stock #2 Incubation: 37 degrees C, ~1 day Edited 27 Aug, 2018 16:10 2.23Mb

Link to this post \| posted 27 Aug, 2018 16:13
kaylafast	My newest M. smeg P2FF should be ready today so I can do another streak plate, spot test with D29, and the streak with no bacteria.

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.

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D29 plaques barely visible on M. smeg