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| Zwolver |
 Posted: Thu Jun 26, 2008 1:25 am Post subject: Weird bacteria found. |
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Forum Senior
Joined: 17 May 2006
Posts: 352
Location: Netherlands, Limburg, Ospel
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At my school, we are brewing beer. As an assignment ofcourse. But while doing so, we found a bacteria, that we can't classify.
It is a thick gram positive rod, with spores. Probably in the middle. Katalase negative, Oxidase negative. Grows aerobic with haemolytic effects on blood agar, and anaerobic it grows greenish, with a dark green dot, on blood agar.
Our API 20A showed a 99,5% positive id on Clostridium septicum. The problem is. That bacteria is strictly anaerobic, and ours grew better aerobic then most aerobic bacteria. It did grew better anaerobic, like a swarming bacteria. So it appears immobile with oxigen.
Anyone faced this problem before?
Anything would be helpfull, thanks
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If your can't get exactly what you want, get the next best.
Alles wat men wilt kan men krijgen, het probleem is dat men niet weet wat men wilt. Gelukkig zijn met wat is gekregen is belangrijker dan streven naar meer.
All we want we can get, the only problem is that nobody knows what they want. Being happy with what we got is more important than wanting more.
~Zwolver... |
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| i_feel_tiredsleepy |
| Posted: Thu Jun 26, 2008 2:30 am Post subject: |
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Forum Ph.D.
Joined: 21 Mar 2008
Posts: 601
Location: Montreal
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Well an alpha hemolytic, catalase negative rod really isn't much to go on. You need something more like which sugars it ferments if any, and a spore stain would be a huge help, if it does form spores the shape and position can sometimes help determine the genus/species. Maybe check for H2S also.
Edit: Also, C. septicum is a pretty rare soil bacteria and causes gangrene so it wouldn't be a likely contaminant, and definitely won't grow aerobically. No Clostridium should grow aerobically.
Edit 2: Just to make sure you used proper procedure, did you streak for isolation and do your test in triplicet to assure no mistakes were made? |
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| free radical |
| Posted: Thu Jun 26, 2008 7:53 am Post subject: |
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Forum Masters Degree
Joined: 24 Sep 2007
Posts: 503
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How do you rule out a member of Bacillus? They are quite abundant in classroom-type samples (typically more so than Clostridium as Clostridium tends to be anaerobic) and I don't see any immediate indications from your description (aside from the API test but isn't that designed for enterics) that would rule out a member of bacillus. In particular, I would look up Bacillus cereus as this is a common contaminant of grains.
The easiest direct means of identification is to amplify the 16S gene and sequence it. This can be done for a small cost by various facilities. |
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| Robbie |
| Posted: Thu Jun 26, 2008 8:14 am Post subject: |
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Forum Masters Degree
Joined: 08 Aug 2006
Posts: 584
Location: Dublin, Ireland
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Yeah I agree, unless you were to rule out bacillus, I'm not particularly astounded by this.
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| i_feel_tiredsleepy |
| Posted: Thu Jun 26, 2008 12:30 pm Post subject: |
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Forum Ph.D.
Joined: 21 Mar 2008
Posts: 601
Location: Montreal
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I think Bacillus cereus is catalase positive, and my first guess would have been Bacillus subtilis, but it is catalase positive also.
And ya the API is for enterobacter.
Ideally as FR said, PCR is the best way to identify, but it's kind of a waste of money if you don't care lol. |
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| Zwolver |
| Posted: Thu Jun 26, 2008 1:41 pm Post subject: |
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Forum Senior
Joined: 17 May 2006
Posts: 352
Location: Netherlands, Limburg, Ospel
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Well, the school said that we weren't allowed any more tests. And i am not sure about the H2S, i think it was on the API, but i did not read it out, someone from my workgroup did.
Morfologically the bacterium looks HUGE, like Bacillus megaterium, that would be my first guess. The spores vary in size and shape. Some look like droplets, others like small coccoid rods, and others like orbs. And it was a clean colony i checked. From 3 days old (to be able to see the spores). But a 99,5% API 20A positivity isn't nothing. It is also possible that the catalase test was spoiled. I could only get a very weak positive reaction from my control strain (S.aureus).
I wish i had my own lab of some sort, school doesn't even keep their reactants in the cooler. and they bought the katalase last year. And it has been out since.
I know some morfologic properties, and it does not look like B.cereus on a PCA, the colonies are twice as big after 24 hours, and they don't form the destinctive lines on the colonies.
When i determine bacteria at my previous work, i use elisa, because i think PCR is to specific. Or is there an allround kit to identify all dna patterns of bacteria? Because i don't know that.
_________________
If your can't get exactly what you want, get the next best.
Alles wat men wilt kan men krijgen, het probleem is dat men niet weet wat men wilt. Gelukkig zijn met wat is gekregen is belangrijker dan streven naar meer.
All we want we can get, the only problem is that nobody knows what they want. Being happy with what we got is more important than wanting more.
~Zwolver... |
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| i_feel_tiredsleepy |
| Posted: Thu Jun 26, 2008 2:19 pm Post subject: |
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Forum Ph.D.
Joined: 21 Mar 2008
Posts: 601
Location: Montreal
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| Zwolver wrote: |
Well, the school said that we weren't allowed any more tests. And i am not sure about the H2S, i think it was on the API, but i did not read it out, someone from my workgroup did.
Morfologically the bacterium looks HUGE, like Bacillus megaterium, that would be my first guess. The spores vary in size and shape. Some look like droplets, others like small coccoid rods, and others like orbs. And it was a clean colony i checked. From 3 days old (to be able to see the spores). But a 99,5% API 20A positivity isn't nothing. It is also possible that the catalase test was spoiled. I could only get a very weak positive reaction from my control strain (S.aureus).
I wish i had my own lab of some sort, school doesn't even keep their reactants in the cooler. and they bought the katalase last year. And it has been out since.
I know some morfologic properties, and it does not look like B.cereus on a PCA, the colonies are twice as big after 24 hours, and they don't form the destinctive lines on the colonies.
When i determine bacteria at my previous work, i use elisa, because i think PCR is to specific. Or is there an allround kit to identify all dna patterns of bacteria? Because i don't know that. |
Catalase is very easy to test in bacteria go buy some peroxide, droplet on the bacteria, if you get oxygen bubbles there is catalase.
Edit: ELISA works if you have the antibodies for surface markers. |
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| Zwolver |
| Posted: Thu Jun 26, 2008 2:40 pm Post subject: |
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Forum Senior
Joined: 17 May 2006
Posts: 352
Location: Netherlands, Limburg, Ospel
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i know, but they didn't have any other peroxide at school. (really, a chemical/biological school, and no peroxide)... and i don't want to have bacteria at home. At least i don't want to grow them there.
But indeed, you still need the right antibodies in the cups...
_________________
If your can't get exactly what you want, get the next best.
Alles wat men wilt kan men krijgen, het probleem is dat men niet weet wat men wilt. Gelukkig zijn met wat is gekregen is belangrijker dan streven naar meer.
All we want we can get, the only problem is that nobody knows what they want. Being happy with what we got is more important than wanting more.
~Zwolver... |
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| free radical |
| Posted: Thu Jun 26, 2008 6:57 pm Post subject: |
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Forum Masters Degree
Joined: 24 Sep 2007
Posts: 503
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| Zwolver wrote: |
| Or is there an allround kit to identify all dna patterns of bacteria? Because i don't know that. |
More like all around sequencing services. I don't actually know if you can send a colony and have them amplify up and sequence for you, but google "ribosomal sequencing service" for starts. |
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| i_feel_tiredsleepy |
| Posted: Fri Jun 27, 2008 8:40 am Post subject: |
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Forum Ph.D.
Joined: 21 Mar 2008
Posts: 601
Location: Montreal
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| Zwolver wrote: |
i know, but they didn't have any other peroxide at school. (really, a chemical/biological school, and no peroxide)... and i don't want to have bacteria at home. At least i don't want to grow them there.
But indeed, you still need the right antibodies in the cups... |
Peroxide also spoils very quickly, as oxygen evolves naturally with time until you only have water left. So, you could always buy a bottle, they cost like 3 dollars. |
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| CShark |
| Posted: Fri Jun 27, 2008 8:45 am Post subject: |
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Forum Sophomore
Joined: 08 May 2008
Posts: 169
Location: East Coast of Canada
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| i_feel_tiredsleepy wrote: |
I think Bacillus cereus is catalase positive, and my first guess would have been Bacillus subtilis, but it is catalase positive also.
And ya the API is for enterobacter.
Ideally as FR said, PCR is the best way to identify, but it's kind of a waste of money if you don't care lol. |
B.cereus and B.subtilis are both catalase pos. (Used to use B.sub to test our clean-room media).
Problem with PCR is, you need a probe. Which means you need to know what you are looking for...and around we go!
ELISA has the same problem: you need an antibody coat and conjugate specific for your target, or you get non-specific binding (false positives).
Someone mentioned H2S...have you tried the IMVIC (apart from catalase) TSI and a sugar panel ? You can get API's for gram pos rods, btw. |
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| free radical |
| Posted: Fri Jun 27, 2008 10:12 am Post subject: |
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Forum Masters Degree
Joined: 24 Sep 2007
Posts: 503
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Cheers for the API info, Cshark!
There are universal primers for 16S sequences, and 16S sequence is the main standard for bacterial phylogeny and identification. A probe (primer) is probably not the limiting step here.
Zwolver, keep in mind that a good number (well over 10%, in my past experience) of environmental isolates are never accurately identified in coursework similar to what you are describing, due to the incredible diversity of bacteria in the environment and the limited number of tests available in the classroom. 16s sequence is the surest identification method, is provided by many commercial facilities (although many of these require a PCR product, some do not but the cost then becomes prohibitive) and the primers to accomplish this are well-characterised and widely used.
Bottom line, almost certainly not worth it, but if you want a definitive answer look at something like this:
http://www.baseclear.com/index.php
which was chosen for location and there are many others:
http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html
Cost can be requested at the website. |
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| CShark |
| Posted: Fri Jun 27, 2008 7:13 pm Post subject: |
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Forum Sophomore
Joined: 08 May 2008
Posts: 169
Location: East Coast of Canada
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| free radical wrote: |
Cheers for the API info, Cshark!
There are universal primers for 16S sequences, and 16S sequence is the main standard for bacterial phylogeny and identification. A probe (primer) is probably not the limiting step here.
Zwolver, keep in mind that a good number (well over 10%, in my past experience) of environmental isolates are never accurately identified in coursework similar to what you are describing, due to the incredible diversity of bacteria in the environment and the limited number of tests available in the classroom. 16s sequence is the surest identification method, is provided by many commercial facilities (although many of these require a PCR product, some do not but the cost then becomes prohibitive) and the primers to accomplish this are well-characterised and widely used.
Bottom line, almost certainly not worth it, but if you want a definitive answer look at something like this:
http://www.baseclear.com/index.php
which was chosen for location and there are many others:
http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html
Cost can be requested at the website. |
Ipersonally never used API strips, 'cept as a breif stint as micro head in a small private lab many years ago. However, I have seen a wide number of varying targets now for these, including anaerobes, obligates, cocci, gm POS rods, etc.
It so happens the past twelve years I have been working in a position that requires a fair bit of immunoserology and real-time PCR work. My micro days are well past I am afraid, although it was always my favourite place to work.
Cheers. |
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