How to perform bacterial ID (I am trying to determine a Bacillus species)

[Nathan McCorkle]

So in my 5th year studying Biotech I'm taking Microbiology, though its
a 2nd or 3rd year course!!!

I isolated a colony from the autoclave handle in the lab, using TSA
plates. It looked layered (like a medium sized pizza on top of a large
pizza) with rough edges.

Gram stain came out positive and rod shaped, malachite green stain
showed spores.

I subcultured this colony onto blood agar and lactobacillus MRS agar.
Blood agar showed clearing, but I can't remember if it was under the
colonies or around it as well (thats the difference between alpha and
beta hemolysis, right?), MRS showed very structured colonies, having a
few wormlike or vertical projections.

So I know (i think) its a Bacillus, but how do I determine what
species it is? We use B. subtilis in these labs, and I saw a picture
indicating the 3D/worm-like growth being attributed to B. subtilis.

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Avery louie]

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For bacterial ID's:

16srRNA sequencing is, the very best there is. When you absolutely, positively, have to ID every single bacteria in the room; accept no substitute

(adapted from Samuel L in Jackie Brown)

Get some forward/reverse 16srRNA primers and PCR-ify it, then sequencify it.  Then BLAST ify it.

--Avery

[Nathan McCorkle]

I guess I should have mentioned using common media selection,
screening, microscopic and macroscopic techniques. Sure 16S is the way
to go, but I can't do that for this class... though I could probably
manage to get it done, its not the aim of this course.

[Avery louie]

I would check The Prokaryotes, book 4, sections 1.2.16-19.

--Avery

[Cathal Garvey]

Blood clearing is based on colour rather than radius, I think.

Bacilli are really awkward to tell apart, I had a scanned id chart lying around on my HD, I'll search tomorrow. You'll be getting into anaerobic or not, sugar metabolism, etc..

[Giovanni Lostumbo]

Is Biolog useful?

http://www.biolog.com/

[Nathan McCorkle]

On Wed, Oct 5, 2011 at 6:43 PM, Giovanni Lostumbo
<giovanni...@gmail.com> wrote:
> Is Biolog useful?
>
> http://www.biolog.com/
>

Yes, but I think its out of the timeframe I have now... I could
subculture onto all the different media that we have, but I feel
that's a less intelligent/directed way to approach it, rather I still
wouldn't know /how/ to use those results for categorization.

[Nathan McCorkle]

On Wed, Oct 5, 2011 at 5:29 PM, Avery louie <inact...@gmail.com> wrote:
> I would check The Prokaryotes, book 4, sections 1.2.16-19.
>

Thanks, will try to find it.

[Nathan McCorkle]

On Wed, Oct 5, 2011 at 5:39 PM, Cathal Garvey <cathal...@gmail.com> wrote:
> Blood clearing is based on colour rather than radius, I think.
>
> Bacilli are really awkward to tell apart, I had a scanned id chart lying
> around on my HD, I'll search tomorrow. You'll be getting into anaerobic or
> not, sugar metabolism, etc..

Sweet, post here or email me if/when you find it!

[Patrik]

The old-fashioned approach would be to get out "Bergey's Manual", aka
Bergey's Manual of Determinative Bacteriology:

http://www.wikimedia.org/wikipedia/en/wiki/Bergey%27s_Manual_of_Determinative_Bacteriology
http://www.bergeys.org/pubinfo.html#anchor21298

Here's a great identification flowchart, extracted from the dozens and
dozens of tables in the book:

http://www.uiweb.uidaho.edu/micro_biology/250/IDFlowcharts.pdf

If you already have a good idea which organism it may be (e.g. B.
subtilis), you may want to get out the big guns, and check Bergey's
Manual of *Systematic* Bacteriology, which will have a lot more
information of specific species phenotypes, beyond merely what is
essential for identifying the species.

Of course, nowadays the gold standard would be to sequence the 16S
rDNA, and use that to identify the organism down to the strain level.

[IronGhost]

There are a number of other chemical tests you can do to help narrow it down some more, although DNA testing would really pin it.

1. Indole test with Kovac's & Ehrlich's reagents.
2. Carbohydrate tests (using Glucose, Lactose, and Sucrose)
3. Citrate test
4. Gelatin test
5. Starch Hydrolosis test
6. MRVP test
7. TSI & H2S test
8. Urea test
9. catalase test
10. Oxidase test

[Nathan McCorkle]

On Thu, Oct 6, 2011 at 1:51 AM, Patrik <pat...@gmail.com> wrote:
> The old-fashioned approach would be to get out "Bergey's Manual", aka
> Bergey's Manual of Determinative Bacteriology:
>
> http://www.wikimedia.org/wikipedia/en/wiki/Bergey%27s_Manual_of_Determinative_Bacteriology
> http://www.bergeys.org/pubinfo.html#anchor21298
>

My roommate (also in class with me) found (some of?) these, thanks Patrik!

> Here's a great identification flowchart, extracted from the dozens and
> dozens of tables in the book:
>
> http://www.uiweb.uidaho.edu/micro_biology/250/IDFlowcharts.pdf
>

Yeah I found this my first or second google search in lab this past
Sunday, the first chart basically got me to think its a Bacillus... I
think we'll proceed to the more in-depth biochemical tests this week
and next.

> If you already have a good idea which organism it may be (e.g. B.
> subtilis), you may want to get out the big guns, and check Bergey's
> Manual of *Systematic* Bacteriology, which will have a lot more
> information of specific species phenotypes, beyond merely what is
> essential for identifying the species.
>
> Of course, nowadays the gold standard would be to sequence the 16S
> rDNA, and use that to identify the organism down to the strain level.
>

I was thinking that colony PCR is really quite easy, are there
universal primers for 16S? I could get some in the mail in a few days
for not more than $5-$15... not sure if I really need that level
though, since I think this Prof is a "very very fair" grader.

>
> On Oct 5, 1:34 pm, Nathan McCorkle <nmz...@gmail.com> wrote:
>> So in my 5th year studying Biotech I'm taking Microbiology, though its
>> a 2nd or 3rd year course!!!
>>
>> I isolated a colony from the autoclave handle in the lab, using TSA
>> plates. It looked layered (like a medium sized pizza on top of a large
>> pizza) with rough edges.
>>
>> Gram stain came out positive and rod shaped, malachite green stain
>> showed spores.
>>
>> I subcultured this colony onto blood agar and lactobacillus MRS agar.
>> Blood agar showed clearing, but I can't remember if it was under the
>> colonies or around it as well (thats the difference between alpha and
>> beta hemolysis, right?), MRS showed very structured colonies, having a
>> few wormlike or vertical projections.
>>
>> So I know (i think) its a Bacillus, but how do I determine what
>> species it is? We use B. subtilis in these labs, and I saw a picture
>> indicating the 3D/worm-like growth being attributed to B. subtilis.
>>
>> --
>> Nathan McCorkle
>> Rochester Institute of Technology
>> College of Science, Biotechnology/Bioinformatics
>

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[Nathan McCorkle]

Yeah we'll go through there, as well as decarboxylase test, and maybe
some others

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[Patrik]

On Oct 6, 12:00 am, Nathan McCorkle <nmz...@gmail.com> wrote:
> I was thinking that colony PCR is really quite easy, are there
> universal primers for 16S? I could get some in the mail in a few days
> for not more than $5-$15... not sure if I really need that level
> though, since I think this Prof is a "very very fair" grader.

JGI typically uses universal primers 27F/1391R for bacterial 16S rRNA:

http://my.jgi.doe.gov/general/protocols/SOP_16S18S_rRNA_PCR_Library_Creation.pdf

The Wikipedia page actually has a table with universal primers as
well:

http://en.wikipedia.org/wiki/16S_ribosomal_RNA#Universal_Primers

16S is such a common target in microbiology, that chances are somebody
at your lab may have some primers available that you could use. Ask
around among the grad students and postdocs...

[digitalbio]

When I taught microbiology, we identified Bacillus species by their
ability to form spores, and the fact that they are aerobic (unlike
Clostridium).

You can boil a sample that contains Bacillus for 10 minutes. This
kills all vegetative cells. Cool the liquid, then plate it on
nutrient agar. The spores will germinate and you'll get aerobic gram
+ spore forming bacteria - i.e. Bacillus.

You can also stain them and see the spores under a microscope.

[Nathan McCorkle]

On Oct 6, 2011 5:55 PM, "Patrik" <pat...@gmail.com> wrote:
>
> On Oct 6, 12:00 am, Nathan McCorkle <nmz...@gmail.com> wrote:
> > I was thinking that colony PCR is really quite easy, are there
> > universal primers for 16S? I could get some in the mail in a few days
> > for not more than $5-$15... not sure if I really need that level
> > though, since I think this Prof is a "very very fair" grader.
>
> JGI typically uses universal primers 27F/1391R for bacterial 16S rRNA:
>
> http://my.jgi.doe.gov/general/protocols/SOP_16S18S_rRNA_PCR_Library_Creation.pdf
>

Thanks, will have a look

> The Wikipedia page actually has a table with universal primers as
> well:
>
> http://en.wikipedia.org/wiki/16S_ribosomal_RNA#Universal_Primers
>

Yeah wasn't sure if the amplicons were so long such that you needed to use each primer in that table, then assemble and BLAST.

Would I look for best hit with score < 10^-20?

> 16S is such a common target in microbiology, that chances are somebody
> at your lab may have some primers available that you could use. Ask
> around among the grad students and postdocs...
>

No grads or post-docs here in bio... But i'll ask a few profs, or just buy some from sigma, shouldn't be more than $15

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[Patrik]

On Oct 6, 9:09 pm, Nathan McCorkle <nmz...@gmail.com> wrote:
> Yeah wasn't sure if the amplicons were so long such that you needed to use
> each primer in that table, then assemble and BLAST.

Nah. 16S has highly conserved and more variable regions. They pick
primers in the highly conserved regions, spaced such that they span
the most variable regions of the gene. That way you have enough
sequence diversity in a single PCR product to identify pretty much all
bacteria down to the species or strain level.

> Would I look for best hit with score < 10^-20?

If what you have is really contamination with a B. subtilis lab
strain, there's a good chance you may find an identical hit. If it's
an environmental strain, a simple BLAST is not the best way to
estimate 16S homology, because it doesn't necessarily give you the
optimal alignment. Instead, you can use our knowledge of the 16S rRNA
structure and the huge library of 16S sequences to first figure out
the optimal alignment, and then estimate the evolutionary distance
based on that alignment.

The GreenGenes website provides some state-of-the-art tools to do all
of this:

http://greengenes.lbl.gov/cgi-bin/nph-index.cgi

[Gabriel Wallace]

It should be noted that 16srRNA is not actually good for identifying a
bacillus species. I did the same thing in microbiology and it can only
identify it down to the genus. For mine that was the case, also for a
few other people in that lab. The professor suggested some other
method but I forgot. I'll look through my lab notes to see if I can
find it.

[Patrik]

Some 16S primers will give you a shorter PCR product and therefore
less resolution in identifying the species. A nearly full length 16S
sequence should typically allow you to go below the genus level.

Alternatively, there are some phylogenetic marker genes which are
still well conserved, but less so than 16S, so they can give better
resolution. The tradeoff is that there typically do not exist any
universal primers that will work across all bacteria. But you should
be able to find a Bacillus-specific set or primers.

For example, here's a paper claiming 4.5 times better resolution using
the RNA polymerase beta subunit (rpoB) gene:

http://www.ncbi.nlm.nih.gov/pubmed/19166882

[Steve]

Hi Nathan,
Have you tried any of the API test strip products, you can get them
from Biomerieux:
http://www.biomerieux-diagnostics.com
http://www.biomerieux-usa.com
Various test strips for identification, they generate a scorecard id
which you then enter into apiweb (apiweb.biomerieux.com).
It has strips to identify thousands of microorganisms, if you already
know it is a Bacillus it narrows the search.
Accurate and fast. Also the test should cost around 5 USD or so.
Steve Geary

[Nathan McCorkle]

Steve,
Thanks,
I know we'll be using some sort of strips with several tests in them,
I think they're metabolism related, called enterotubes.

I'm waiting on my registration on biomerieux to see pricing now.

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[Nathan McCorkle]

Looks like the Bacillus ID strips cost about $20/each, and only come in
a pack of 10 :(