SEA-PHAGES | Contamination of top agar and possibly other components

Link to this post \| posted 31 Mar, 2021 02:55
kmaclea	So, this is going to sound insane, but we've been running into recurrent bacterial contamination that a couple of attempts to fix doesn't seem to be fixing. We are trying to use both A. globiformis and M. foliorum for phage discovery. We had a lot of luck getting plaques originally, then at some point most of our students' phages have been unable to propagate further (no plaques). We've done countless direct and enriched isolations. A few weeks ago we had a contamination issue and re-made components. Things got better for a time, but then we were back in problems again. Last week there was obvious rampant contamination again. We received new glycerol stocks for bacteria, remade all the PYCa, all the top agar, all the dextrose, CaCl2, and CHX. The last three were sterile filtered, the first two were autoclaved. Autoclaving was standardly done with 500 ml at a time and 45 minute autoclaving at 121C. By all indications, other media being made using the same autoclave is just fine although no biological testing of the autoclave has been done recently. Most recent tests seem to indicate it is the top agar in which we are seeing contamination. Even in freshly made top agar, freshly autoclaved, with the other components all sterile filtered in the hood. Attention being paid to sterile technique during the handling, in the hood. What could be making our top agar contaminated? We have been using a metal bead bath for incubation of the top agar, but we are seeing this even in freshly made top agar. Beads in the bead bath have also been recently decontaminated. Ideas of any kind would be most definitely welcomed! Kyle – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129

Link to this post | posted 31 Mar, 2021 02:55

So, this is going to sound insane, but we've been running into recurrent bacterial contamination that a couple of attempts to fix doesn't seem to be fixing.

We are trying to use both A. globiformis and M. foliorum for phage discovery. We had a lot of luck getting plaques originally, then at some point most of our students' phages have been unable to propagate further (no plaques). We've done countless direct and enriched isolations.

A few weeks ago we had a contamination issue and re-made components. Things got better for a time, but then we were back in problems again. Last week there was obvious rampant contamination again. We received new glycerol stocks for bacteria, remade all the PYCa, all the top agar, all the dextrose, CaCl2, and CHX. The last three were sterile filtered, the first two were autoclaved. Autoclaving was standardly done with 500 ml at a time and 45 minute autoclaving at 121C. By all indications, other media being made using the same autoclave is just fine although no biological testing of the autoclave has been done recently.

Most recent tests seem to indicate it is the top agar in which we are seeing contamination. Even in freshly made top agar, freshly autoclaved, with the other components all sterile filtered in the hood. Attention being paid to sterile technique during the handling, in the hood.

What could be making our top agar contaminated? We have been using a metal bead bath for incubation of the top agar, but we are seeing this even in freshly made top agar. Beads in the bead bath have also been recently decontaminated.

Ideas of any kind would be most definitely welcomed!

Kyle

–
Kyle MacLea
Associate Professor, University of New Hampshire at Manchester
kyle.maclea@unh.edu +1 603-641-4129

Link to this post \| posted 31 Mar, 2021 11:52
viknesh	kmaclea So, this is going to sound insane, but we've been running into recurrent bacterial contamination that a couple of attempts to fix doesn't seem to be fixing. We are trying to use both A. globiformis and M. foliorum for phage discovery. We had a lot of luck getting plaques originally, then at some point most of our students' phages have been unable to propagate further (no plaques). We've done countless direct and enriched isolations. A few weeks ago we had a contamination issue and re-made components. Things got better for a time, but then we were back in problems again. Last week there was obvious rampant contamination again. We received new glycerol stocks for bacteria, remade all the PYCa, all the top agar, all the dextrose, CaCl2, and CHX. The last three were sterile filtered, the first two were autoclaved. Autoclaving was standardly done with 500 ml at a time and 45 minute autoclaving at 121C. By all indications, other media being made using the same autoclave is just fine although no biological testing of the autoclave has been done recently. Most recent tests seem to indicate it is the top agar in which we are seeing contamination. Even in freshly made top agar, freshly autoclaved, with the other components all sterile filtered in the hood. Attention being paid to sterile technique during the handling, in the hood. What could be making our top agar contaminated? We have been using a metal bead bath for incubation of the top agar, but we are seeing this even in freshly made top agar. Beads in the bead bath have also been recently decontaminated. Ideas of any kind would be most definitely welcomed! Kyle Hi Kyle, Looks like there ate at least two problems: 1) not being abe to propoagate a phage and 2) contaminated materials. For #1, are you seeing this for both hosts, or just one? If the latter, which one. Also, will the control phage also not plaque? For #2, are you able to share images of the contamination? I can;t tell if the top agar is getting contaminated as you use it or whether it was never actually fully sterilized. For example, what happens to the freshly autoclaved top agar, still molten in the bottle with the lid closed, if left in the beadbath for a few days - never opened? Do you see microbial growth in the bottle, or only when you use it? Vic

Link to this post | posted 31 Mar, 2021 11:52

viknesh

kmaclea
So, this is going to sound insane, but we've been running into recurrent bacterial contamination that a couple of attempts to fix doesn't seem to be fixing.

We are trying to use both A. globiformis and M. foliorum for phage discovery. We had a lot of luck getting plaques originally, then at some point most of our students' phages have been unable to propagate further (no plaques). We've done countless direct and enriched isolations.

A few weeks ago we had a contamination issue and re-made components. Things got better for a time, but then we were back in problems again. Last week there was obvious rampant contamination again. We received new glycerol stocks for bacteria, remade all the PYCa, all the top agar, all the dextrose, CaCl2, and CHX. The last three were sterile filtered, the first two were autoclaved. Autoclaving was standardly done with 500 ml at a time and 45 minute autoclaving at 121C. By all indications, other media being made using the same autoclave is just fine although no biological testing of the autoclave has been done recently.

Most recent tests seem to indicate it is the top agar in which we are seeing contamination. Even in freshly made top agar, freshly autoclaved, with the other components all sterile filtered in the hood. Attention being paid to sterile technique during the handling, in the hood.

What could be making our top agar contaminated? We have been using a metal bead bath for incubation of the top agar, but we are seeing this even in freshly made top agar. Beads in the bead bath have also been recently decontaminated.

Ideas of any kind would be most definitely welcomed!

Kyle

Hi Kyle,

Looks like there ate at least two problems:
1) not being abe to propoagate a phage and
2) contaminated materials.

For #1, are you seeing this for both hosts, or just one? If the latter, which one. Also, will the control phage also not plaque?
For #2, are you able to share images of the contamination? I can;t tell if the top agar is getting contaminated as you use it or whether it was never actually fully sterilized. For example, what happens to the freshly autoclaved top agar, still molten in the bottle with the lid closed, if left in the beadbath for a few days - never opened? Do you see microbial growth in the bottle, or only when you use it?

Vic

Link to this post \| posted 03 Apr, 2021 03:51
kmaclea	Dear Vic, So, for #1: We have been having this problem with both hosts. The last time we tried a control, and previous phages we isolated against A. globiformis, none of them worked. But we have at various times this semester gotten plenty of plaques on direct isolations, failed on enriched isolations, gotten through multiple rounds of purifications on some phages, and then had them stop propagating. For #2: We're performing this exact experiment right now: Freshly autoclaved top agar is now sitting in the bead bath and we should know more soon. We tested the autoclave this week using biological indicators and it passes. Others making media for other classes without antibiotics do not seem to be getting substantial contamination. While the PYCa top agar has been the main contaminated piece (gram positive rods), the PYCa broth has been contaminated a couple of times. We remade all ingredients including new dextrose and CaCl2 with new filter sterilization and were extremely careful with adding these components in the hood but it was still, and very quickly, contaminated. Some of the PYCa broth from this has been contaminated, and some not. We also tried making the media with the dextrose/CaCl2/CHX in the media all together through the autoclave and it still became contaminated. Walking through our process it seemed like a possible explanation could be that the students were in taking the media/agar out of the autoclave were putting the bottles in a water bath to cool down before use, with the caps loosened. Could there be something getting into the threads of the bottle caps during this process, some water splash up? So today we set up some freshly autoclaved bottles and put them directly into the beadbath without this step. We should know more early next week. We are trying to get through these steps before we revisit the plaquing problem–it seemed like contamination could be a prime contributor to this issue. At one point before we started having substantial problems, we did have a plate with clearly visible plaques that nonetheless appears to also be covered with a layer of bacteria, which suggested to us the presence of two different bacteria, only one of which was a host. Kyle – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129

Link to this post | posted 03 Apr, 2021 03:51

kmaclea

Dear Vic,

So, for #1:

We have been having this problem with both hosts. The last time we tried a control, and previous phages we isolated against A. globiformis, none of them worked. But we have at various times this semester gotten plenty of plaques on direct isolations, failed on enriched isolations, gotten through multiple rounds of purifications on some phages, and then had them stop propagating.

For #2:

We're performing this exact experiment right now: Freshly autoclaved top agar is now sitting in the bead bath and we should know more soon. We tested the autoclave this week using biological indicators and it passes. Others making media for other classes without antibiotics do not seem to be getting substantial contamination. While the PYCa top agar has been the main contaminated piece (gram positive rods), the PYCa broth has been contaminated a couple of times. We remade all ingredients including new dextrose and CaCl2 with new filter sterilization and were extremely careful with adding these components in the hood but it was still, and very quickly, contaminated. Some of the PYCa broth from this has been contaminated, and some not. We also tried making the media with the dextrose/CaCl2/CHX in the media all together through the autoclave and it still became contaminated.

Walking through our process it seemed like a possible explanation could be that the students were in taking the media/agar out of the autoclave were putting the bottles in a water bath to cool down before use, with the caps loosened. Could there be something getting into the threads of the bottle caps during this process, some water splash up? So today we set up some freshly autoclaved bottles and put them directly into the beadbath without this step. We should know more early next week.

We are trying to get through these steps before we revisit the plaquing problem–it seemed like contamination could be a prime contributor to this issue. At one point before we started having substantial problems, we did have a plate with clearly visible plaques that nonetheless appears to also be covered with a layer of bacteria, which suggested to us the presence of two different bacteria, only one of which was a host.

Kyle

–
Kyle MacLea
Associate Professor, University of New Hampshire at Manchester
kyle.maclea@unh.edu +1 603-641-4129

Link to this post \| posted 03 Apr, 2021 16:10
kmaclea	So here's an update. https://docs.google.com/document/d/1294ZwYilXMbQA6n0eFrWouodmvBk1M4ZOL4AsPiUsX8/ The contaminated bottle with the long chains is from the small bottle of top agar that was made with dextrose and CaCl2 added before autoclaving and NO opening or placement in a water bath. To quote the student assistant: "Ok so that smaller bottle is the pyca top agar that i made with the exact same recipe as the protocol EXCEPT i added the dextrose and calcium chloride before autoclaving. I autoclaved for 45 minutes, closed it immediately and placed it in the 55C bead bath (AND i handled it with fresh gloves on) It had absolutely NO contact with any water and was not opened, even in the hood, after autoclaving." So, that suggests to me that even 45 minutes in the autoclave is not killing what's in there?? Kyle – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129

Link to this post | posted 03 Apr, 2021 16:10

kmaclea

So here's an update.

https://docs.google.com/document/d/1294ZwYilXMbQA6n0eFrWouodmvBk1M4ZOL4AsPiUsX8/

The contaminated bottle with the long chains is from the small bottle of top agar that was made with dextrose and CaCl2 added before autoclaving and NO opening or placement in a water bath.

To quote the student assistant:
"Ok so that smaller bottle is the pyca top agar that i made with the exact same recipe as the protocol EXCEPT i added the dextrose and calcium chloride before autoclaving. I autoclaved for 45 minutes, closed it immediately and placed it in the 55C bead bath (AND i handled it with fresh gloves on) It had absolutely NO contact with any water and was not opened, even in the hood, after autoclaving."

So, that suggests to me that even 45 minutes in the autoclave is not killing what's in there??

Kyle

–
Kyle MacLea
Associate Professor, University of New Hampshire at Manchester
kyle.maclea@unh.edu +1 603-641-4129

Link to this post \| posted 03 Apr, 2021 16:57
viknesh	kmaclea So here's an update. https://docs.google.com/document/d/1294ZwYilXMbQA6n0eFrWouodmvBk1M4ZOL4AsPiUsX8/ Thanks for sharing all this, Kyle. I can't say for sure, of course, but it looks like you might have multiple things going on. First - the media. Did you try imaging the most recent cloudy bottle of top agar (the one that your TA added calcium and dextrose before authoclaving?). I ask because CaCl2 precipitates when heated for prolonged preiods (i.e. autoclaving), and the amount of precipitation depends on the pH and other salts in solution. While I am not discounting your contamination (all those beautiful microscopy images), the cloudiness in the recent bottles might just be precipitate, and cloudy top agar may be sufficient for plaques to look cloudy - in which case your contamination problems might be isolated events and not a widespread problem. Second - I think those contaminated spots on the enriched plates are not coming from the top agar, but instead from the samples spotted themselves. This is fairly common when the enrichment is not properly filtered, typically because there has been a rupture in the filter membrane. I used to see this fairly often and so now buy the fairly "tough" GDX Whatmann filters that rupture less often (but which still occasionally do). Of course, maybe you have mycoplasma, which can pas through the filter). For your plaque assay plates that are very clearly contaminated, again, this might be a problem with the sample itself being contaminated since some other plates look great - are these also filtered soil samples? If you performed/perform a dilution series with these samples, my guess if that you'll see the contamination dilute out too. Kyle, regardless of what's happening, the fact hat you're engaging your students in trying to figure out this issue is pretty awesome! What a ride for them Vic Edited 03 Apr, 2021 16:59

Link to this post | posted 03 Apr, 2021 16:57

viknesh

kmaclea
So here's an update.
https://docs.google.com/document/d/1294ZwYilXMbQA6n0eFrWouodmvBk1M4ZOL4AsPiUsX8/

Thanks for sharing all this, Kyle. I can't say for sure, of course, but it looks like you might have multiple things going on.

First - the media. Did you try imaging the most recent cloudy bottle of top agar (the one that your TA added calcium and dextrose before authoclaving?). I ask because CaCl2 precipitates when heated for prolonged preiods (i.e. autoclaving), and the amount of precipitation depends on the pH and other salts in solution. While I am not discounting your contamination (all those beautiful microscopy images), the cloudiness in the recent bottles might just be precipitate, and cloudy top agar may be sufficient for plaques to look cloudy - in which case your contamination problems might be isolated events and not a widespread problem.

Second - I think those contaminated spots on the enriched plates are not coming from the top agar, but instead from the samples spotted themselves. This is fairly common when the enrichment is not properly filtered, typically because there has been a rupture in the filter membrane. I used to see this fairly often and so now buy the fairly "tough" GDX Whatmann filters that rupture less often (but which still occasionally do). Of course, maybe you have mycoplasma, which can pas through the filter).

For your plaque assay plates that are very clearly contaminated, again, this might be a problem with the sample itself being contaminated since some other plates look great - are these also filtered soil samples? If you performed/perform a dilution series with these samples, my guess if that you'll see the contamination dilute out too.

Kyle, regardless of what's happening, the fact hat you're engaging your students in trying to figure out this issue is pretty awesome! What a ride for them smile

Vic

Edited 03 Apr, 2021 16:59

Link to this post \| posted 05 Apr, 2021 17:39
kmaclea	Vic We confirmed we are using the Whatman GD/X filters as well. We also confirmed that two of the three bottles of contaminated agar (TSA top agar, and PYCa top agar) were tested by simple staining and shown to contain bacteria. The third could be done as well, but had not been done. Kyle – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129

Link to this post | posted 05 Apr, 2021 17:39

kmaclea

Vic

We confirmed we are using the Whatman GD/X filters as well.

We also confirmed that two of the three bottles of contaminated agar (TSA top agar, and PYCa top agar) were tested by simple staining and shown to contain bacteria. The third could be done as well, but had not been done.

Kyle

–
Kyle MacLea
Associate Professor, University of New Hampshire at Manchester
kyle.maclea@unh.edu +1 603-641-4129

Link to this post \| posted 05 Apr, 2021 17:44
kmaclea	Just confirmed that all three cloudy bottles in that picture were indeed contaminated as shown by methylene blue simple staining. Only the broth bottle on the left in that picture is uncontaminated. Kyle – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129

Link to this post \| posted 06 Apr, 2021 03:46
kmaclea	And further work by the students shows some changing contamination. Some long rods, some shorter rods, and even dominance by cocci. Given that some of our top agar is contaminated after the autoclave with NO ingredients added after autoclave and with the bottle being never opened, it would seem to suggest some problem with our autoclave? Perhaps among other problems. Kyle – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129

Link to this post | posted 06 Apr, 2021 03:46

kmaclea

And further work by the students shows some changing contamination. Some long rods, some shorter rods, and even dominance by cocci. Given that some of our top agar is contaminated after the autoclave with NO ingredients added after autoclave and with the bottle being never opened, it would seem to suggest some problem with our autoclave?

Perhaps among other problems.

Kyle

–
Kyle MacLea
Associate Professor, University of New Hampshire at Manchester
kyle.maclea@unh.edu +1 603-641-4129

Link to this post \| posted 14 Apr, 2021 19:14
kmaclea	I wanted to send an update that we have conclusively proven the source of our contamination giving us so many problems. The agar we were using to make the top agar appears to have been contaminated with something that could survive 40 minutes of autoclaving (volume 500 ml). Now that we have switched out the agar, our contamination issues have gone away completely. Just an update for anyone following the thread. Kyle – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129

Link to this post | posted 14 Apr, 2021 19:14

kmaclea

I wanted to send an update that we have conclusively proven the source of our contamination giving us so many problems.

The agar we were using to make the top agar appears to have been contaminated with something that could survive 40 minutes of autoclaving (volume 500 ml).

Now that we have switched out the agar, our contamination issues have gone away completely.

Just an update for anyone following the thread.

Kyle

–
Kyle MacLea
Associate Professor, University of New Hampshire at Manchester
kyle.maclea@unh.edu +1 603-641-4129

Link to this post \| posted 14 Apr, 2021 19:25
viknesh	kmaclea I wanted to send an update that we have conclusively proven the source of our contamination giving us so many problems. The agar we were using to make the top agar appears to have been contaminated with something that could survive 40 minutes of autoclaving (volume 500 ml). Now that we have switched out the agar, our contamination issues have gone away completely. Just an update for anyone following the thread. Kyle Thrilled to hear it, Kyle. Hope your students had fun figuring this out.

Link to this post | posted 14 Apr, 2021 19:25

viknesh

kmaclea
I wanted to send an update that we have conclusively proven the source of our contamination giving us so many problems.

The agar we were using to make the top agar appears to have been contaminated with something that could survive 40 minutes of autoclaving (volume 500 ml).

Now that we have switched out the agar, our contamination issues have gone away completely.

Just an update for anyone following the thread.

Kyle

Thrilled to hear it, Kyle. Hope your students had fun figuring this out.

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Contamination of top agar and possibly other components