🚀 go-pugleaf

Trond Erik Vee Aune <trondaun@biotech.REMOVETHISBEFOREREPLYING.ntnu.no> writes:
> other different bacteria, I streaked it onto a new plate. For some
> reason it did not come up as a continous smear, but as distinct
> colonies, even where I had put a large amount of colony material. And
> more surprising, it even came up at places where I had not streaked. I
> tried it again, and this time I shook the needle before streaking to
> try to remove possible spores. When streaking, I made sure to keep the
> needle close to the surface of the agar. Despite of all these efforts,
> new colonies grew up up to 1,5 cm away from the area where I had put
> the colony material. And still no continous growth, almost like they
> are inhibiting themselves.

I presume you tried culturing one of your plates without streaking
anything on it...  If not, and you get colonies on it, then this
sounds like your autoclave needs fixing.

Tom Knight wrote:
> Trond Erik Vee Aune <trondaun@biotech.REMOVETHISBEFOREREPLYING.ntnu.no> writes:
> 
>>other different bacteria, I streaked it onto a new plate. For some
>>reason it did not come up as a continous smear, but as distinct
>>colonies, even where I had put a large amount of colony material. And
>>more surprising, it even came up at places where I had not streaked. I
>>tried it again, and this time I shook the needle before streaking to
>>try to remove possible spores. When streaking, I made sure to keep the
>>needle close to the surface of the agar. Despite of all these efforts,
>>new colonies grew up up to 1,5 cm away from the area where I had put
>>the colony material. And still no continous growth, almost like they
>>are inhibiting themselves.
> 
> 
> I presume you tried culturing one of your plates without streaking
> anything on it...  If not, and you get colonies on it, then this
> sounds like your autoclave needs fixing.

It's not contamination. I have full control of my plates. And the 
colonies I get only come up around where I streak the bug. Som it's a 
polar effect.

Trond Erik

> 
> 

Trond Erik Vee Aune wrote:
> 
> 
> Tom Knight wrote:
> 
>> Trond Erik Vee Aune 
>> <trondaun@biotech.REMOVETHISBEFOREREPLYING.ntnu.no> writes:
>>
>>> other different bacteria, I streaked it onto a new plate. For some
>>> reason it did not come up as a continous smear, but as distinct
>>> colonies, even where I had put a large amount of colony material. And
>>> more surprising, it even came up at places where I had not streaked. I
>>> tried it again, and this time I shook the needle before streaking to
>>> try to remove possible spores. When streaking, I made sure to keep the
>>> needle close to the surface of the agar. Despite of all these efforts,
>>> new colonies grew up up to 1,5 cm away from the area where I had put
>>> the colony material. And still no continous growth, almost like they
>>> are inhibiting themselves.
>>
>>
>>
>> I presume you tried culturing one of your plates without streaking
>> anything on it...  If not, and you get colonies on it, then this
>> sounds like your autoclave needs fixing.
> 
> 
> It's not contamination. I have full control of my plates. And the 
> colonies I get only come up around where I streak the bug. Som it's a 
> polar effect.
> 
> Trond Erik
> 

I must admit that the first thing I thought of was contamination. I 
would incubate an uninoculates plate from the same batch too.

I assume your loop or wire (if that's what you're using) is not too hot 
when you streak out your bacteria? Otherwise, the media or conditions in 
which you're growing them may not be suitable. Maybe you could try some 
other combinations.

Do you dry your plates before using them? Lots of motile bugs will swim 
away from where you streak them (i.e. lots of spirochaetes, Proteus etc) 
but usually they dont swim away to form discrete colonies. But it may be 
possible. You're plate doesnt even have to be actually wet - even if it 
is moist enough they can do it.

How much bigger than E. coli are they? Are they bigger, but still 
bacteria sized (rather than yeast or fungus sized).



-- 
Scott J. Coutts
--------------------------------------------------------------------------
  Bacterial Pathogenesis Research Group		
  Department of Microbiology			Ph  [+61 3 9905 4838]
  PO Box 53					Fax [+61 3 9905 4811]	
  Monash University, 3800, Austrlalia
--------------------------------------------------------------------------

Scott J. Coutts wrote:

> I must admit that the first thing I thought of was contamination. I 
> would incubate an uninoculates plate from the same batch too.

When I streak the bacteria I only streak it at about one fifth of the 
total area of the plate. The colonies are showing up in the vicinity of 
where I have streaked, up to 1.5 cm away. On the other half of the 
plate, away from where I have streaked, no colonies are showing up. So 
if it is a contaminant (which we rarely see at our laboratory) it is 
dependent on something my new bacteria is producing and will only grow 
close to it. Otherwise I would not see this polar effect.

> I assume your loop or wire (if that's what you're using) is not too hot 
> when you streak out your bacteria? Otherwise, the media or conditions in 
> which you're growing them may not be suitable. Maybe you could try some 
> other combinations.

I use a loop of wire yes. When I streak other bacteria I never see the 
same strange behaviour, so I don't think it's too hot. Since this is a 
new bacteria I'm not sure of it's preferred media. I grow it on LB + 
glucose, it grows fine at 30 and 37 degrees, and after one night 
incubation I have nice sized colonies. It really seems like the in some 
way inhibit each others growth, so that they need a few mm of space 
between each colony. I know they inhibit growth of other bacteria, they 
are producing some sort of antibiotics.

> Do you dry your plates before using them? Lots of motile bugs will swim 
> away from where you streak them (i.e. lots of spirochaetes, Proteus etc) 
> but usually they dont swim away to form discrete colonies. But it may be 
> possible. You're plate doesnt even have to be actually wet - even if it 
> is moist enough they can do it.

I dry the plates at 37 before streaking, so they should be dry. I 
thought of this solution, but it just seems too weird that they should 
swim for over a cm before proliferating and forming colonies. But at the 
moment I have no other explanation... Either long distance swimming or 
some way of shooting out spores (even though I couldn't see any spores 
through the microscope).

> How much bigger than E. coli are they? Are they bigger, but still 
> bacteria sized (rather than yeast or fungus sized).

Difficult to say, I'm not very experienced with the microscope. Some 
people at my lab said they looked like Azotobacter vinelandii. At least 
double the size of E.coli, I would think. They were unicellular (I could 
see someone in the progress of dividing by binary fission) and showed no 
signs of differentiation, they all looked the same - slightly bent rods 
with polar flagella.

Thank for the input Scott!

Trond Erik

> 
> 
> 

newshound007 wrote:
> trondaun@biotech.REMOVETHISBEFOREREPLYING.ntnu.no (Trond Erik Vee Aune) 
> wrote in sci.bio.microbiology in <3E5F1556.7040703
> @biotech.REMOVETHISBEFOREREPLYING.ntnu.no> :
> 
> 
>>When I streak the bacteria I only streak it at about one fifth of the 
>>total area of the plate. The colonies are showing up in the vicinity of 
>>where I have streaked, up to 1.5 cm away. 
> 
> 
> Solunds like hot loop "agar pop" to me.

You mean that the loop is so hot that agar splatters around and spreads 
bacteria? No, this can't be the reason.

What I do is sterilize the loop in flame. Then I cool it by sticking it 
down into the agar at the plate from where I take the inoculum. After it 
is cooled down I scoop up some of the bacteria and gently smear them on 
the new plate.

Trond Erik

> I use a loop of wire yes. When I streak other bacteria I never see the
> same strange behaviour, so I don't think it's too hot. Since this is a
> new bacteria I'm not sure of it's preferred media. I grow it on LB +
> glucose, it grows fine at 30 and 37 degrees, and after one night
> incubation I have nice sized colonies. It really seems like the in some
> way inhibit each others growth, so that they need a few mm of space
> between each colony. I know they inhibit growth of other bacteria, they
> are producing some sort of antibiotics.

Biocins are fairly common, see e.g. biocins of E. fecalis :) Colicins can
kill specific substrains of coli, while allowing the host strain survive.

Artem

Trond Erik Vee Aune wrote:
> 
> 
> Scott J. Coutts wrote:
> 
>> I must admit that the first thing I thought of was contamination. I 
>> would incubate an uninoculates plate from the same batch too.
> 
> 
> When I streak the bacteria I only streak it at about one fifth of the 
> total area of the plate. The colonies are showing up in the vicinity of 
> where I have streaked, up to 1.5 cm away. On the other half of the 
> plate, away from where I have streaked, no colonies are showing up. So 
> if it is a contaminant (which we rarely see at our laboratory) it is 
> dependent on something my new bacteria is producing and will only grow 
> close to it. Otherwise I would not see this polar effect.
> 
>> I assume your loop or wire (if that's what you're using) is not too 
>> hot when you streak out your bacteria? Otherwise, the media or 
>> conditions in which you're growing them may not be suitable. Maybe you 
>> could try some other combinations.
> 
> I use a loop of wire yes. When I streak other bacteria I never see the 
> same strange behaviour, so I don't think it's too hot. Since this is a 
> new bacteria I'm not sure of it's preferred media. I grow it on LB + 
> glucose, it grows fine at 30 and 37 degrees, and after one night 
> incubation I have nice sized colonies. It really seems like the in some 
> way inhibit each others growth, so that they need a few mm of space 
> between each colony. I know they inhibit growth of other bacteria, they 
> are producing some sort of antibiotics.

Where did it come from? You might be able to find a better medium for it 
depending on where you found it. You could try a more enriched media, 
maybe HBA or something like that. Maybe also try growing them 
anaerobically. It's just that often, if they dont grow very well on a 
medium, then instead of getting confluent growth at the point of the 
primary innoculation, you'll just get light growth or a few colonies.

>> Do you dry your plates before using them? Lots of motile bugs will 
>> swim away from where you streak them (i.e. lots of spirochaetes, 
>> Proteus etc) but usually they dont swim away to form discrete 
>> colonies. But it may be possible. You're plate doesnt even have to be 
>> actually wet - even if it is moist enough they can do it.
> 
> I dry the plates at 37 before streaking, so they should be dry. I 
> thought of this solution, but it just seems too weird that they should 
> swim for over a cm before proliferating and forming colonies. But at the 
> moment I have no other explanation... Either long distance swimming or 
> some way of shooting out spores (even though I couldn't see any spores 
> through the microscope).
> 

Remember that often bacterial spores will not look like fungal spores do 
- they will be refractile or differentially staining bodies inside the 
bacterial cell. They may or may not distend the cell. Have done any 
staining of these cells?

I dont know of any bacteria that can 'shoot' their spores. The swimming 
certainly sounds plausible, but not as discrete colonies. Proteus will 
often swim a few cm away, then proliferate, then swim again. But usually 
this shows as concentric rings of growth away from the innoculation point.

What do the colonies look like, and how big are they? Would it be 
possible for you to post some pictures on the web somewhere, or in 
alt.test (I dont think people appreciate binaries on this newsgroup). 
Otherwise, I'd be happy to take a look at them if you want to email them 
to me. It sounds interesting!

>> How much bigger than E. coli are they? Are they bigger, but still 
>> bacteria sized (rather than yeast or fungus sized).
> 
> Difficult to say, I'm not very experienced with the microscope. Some 
> people at my lab said they looked like Azotobacter vinelandii. At least 
> double the size of E.coli, I would think. They were unicellular (I could 
> see someone in the progress of dividing by binary fission) and showed no 
> signs of differentiation, they all looked the same - slightly bent rods 
> with polar flagella.
 >

Do you know they have polar flagella just because of the motion of their 
movement, or can you see them? If you can see them, then I dont think 
they're bacteria. But the cell size sounds like bacteria.

> 
> Thank for the input Scott!
> 

Anytime.

Ok, everything you replied to rules out what i said in my first post... 
hm... well, I dont know of any bacteria that exhibit this behaviour. I 
doubt that the cells are inhibiting their own growth. I cant see any 
reason for them to have evolved such a trait (although many eukaryotic 
cells will have contact inhibition that stops them from growing when in 
close proximity). Certainly many bacteria can inhibit or kill other 
species, but never usually their own.

I'd definitely recommend 16S sequencing if you have it available. It 
will give you a quick and definitive answer (unless it's completely 
unknown). Do you know about the RDP (ribosomal database project)? It is 
a database of all the known (read: submitted) species ribosomal 
sequences. You can view them as a (huge) tree and add your sequence to 
it to see visially where it fits. If yours is unique, it will simply 
join all the others (if you choose to submit it)- if you look at the 
full tree there's plenty of entries that have names like "unidentified 
organism from swamp water" hehehe.

Anyhow, here's the link if you're intersted:

http://rdp.cme.msu.edu/html/

PS: Do you mind me asking how you came across this bug? I've sequenced a 
few contaminants from plates we've found in our storage in the fridge, 
just for the fun of it. Sometimes it's interesting (:



-- 
Scott J. Coutts
--------------------------------------------------------------------------
  Bacterial Pathogenesis Research Group		
  Department of Microbiology			Ph  [+61 3 9905 4838]
  PO Box 53					Fax [+61 3 9905 4811]	
  Monash University, 3800, Austrlalia
--------------------------------------------------------------------------

Scott J. Coutts wrote:

> Where did it come from? 

Found in a water sample taken from the Ganges, India. I just testet a 
little of the water on LB + glucose to see the microflora. It caught my 
attention since it inhibited other microorganisms (a few mm of 
inhibition zone).

> You might be able to find a better medium for it 
> depending on where you found it. You could try a more enriched media, 
> maybe HBA or something like that. Maybe also try growing them 
> anaerobically. It's just that often, if they dont grow very well on a 
> medium, then instead of getting confluent growth at the point of the 
> primary innoculation, you'll just get light growth or a few colonies.

The strange thing is that they seem to be growing fine. Fat, nice 
colonies after one night incubation. But of course, maybe they could 
grow even better on a different media.

> Remember that often bacterial spores will not look like fungal spores do 
> - they will be refractile or differentially staining bodies inside the 
> bacterial cell. They may or may not distend the cell. Have done any 
> staining of these cells?

No, I haven't stained them. So maybe I missed spores while looking at 
them through the microscope.

> What do the colonies look like, and how big are they? Would it be 
> possible for you to post some pictures on the web somewhere, or in 
> alt.test (I dont think people appreciate binaries on this newsgroup). 
> Otherwise, I'd be happy to take a look at them if you want to email them 
> to me. It sounds interesting!

I'll post pictures tomorrow. I've had a plate with the bug laying here 
for a few days now, and every day they extend their growth about 1 cm. 
Now they've almost covered the plate. But still they only show this 
"distict colony" phenotype. Even at the place of inoculation there are 
only separated colonies. But I can now barely see signs og growth 
somewhere inbetween distinct colonies, so I believe they spread by 
swimming on top of the agar.

> Do you know they have polar flagella just because of the motion of their 
> movement, or can you see them? If you can see them, then I dont think 
> they're bacteria. But the cell size sounds like bacteria.

No, I couldn't see the flagella. But I could see one end of the cells 
twiggling as they whirled around ;)

> I'd definitely recommend 16S sequencing if you have it available.

I'll probably order primers next week, then do the sequence reaction on 
whole cell sample (instead of trying to isolate chromosomal DNA).

Thanks for your help, I'll keep you informed!

Trond Erik

> Found in a water sample taken from the Ganges, India. I just testet a
> little of the water on LB + glucose to see the microflora. It caught my
> attention since it inhibited other microorganisms (a few mm of
> inhibition zone).

This is very interesting ! I would bet these bugs secrete biocins of some
sort. If biocin secretion is associated with e.g. glucose in the medium, and
if the bug is slightly sensitive to the biocins as well, perhaps this would
explain the weird colony formation.

Alternatively, you might have a silent gene that gets triggered every so
often, so that from time to time you get cells that 'feel like exploring' so
to speak, and swim off to generate new colonies. This won't explain why you
do not get a colony at the initial streak site, *unless* the 'exploratory'
behavior is triggered every time you move the bug from one plate to the
other - either through mechanical stimulus or minor differences in oxygen
levels, nutrients, etc.

Alternatively, you might have contact stimulation instead of contact
inhibition and the colonies form when sufficient number of cells
accidentally stay in one area long enough to secrete the chemicals necessary
for rapid division. The rest of the cells keep wandering around or die.

It would be interesting to see how this bug grows in liquid medium, because
if this is chemical stimulus kind of thing you might get very very slow
growth (large volume of liquid to saturate), followed by burst of division
after a long slow growth period. Kind of exaggerated version of normal lag
period->growth behavior.

Artem

Artem Evdokimov wrote:

> > Found in a water sample taken from the Ganges, India. I just testet a
> > little of the water on LB + glucose to see the microflora. It caught my
> > attention since it inhibited other microorganisms (a few mm of
> > inhibition zone).
>
> This is very interesting ! I would bet these bugs secrete biocins of some
> sort. If biocin secretion is associated with e.g. glucose in the medium, and
> if the bug is slightly sensitive to the biocins as well, perhaps this would
> explain the weird colony formation.

[snip]

Since all of the bacteria in the culture should carry the plasmid that codes for
production of the biocin, as well as the immunity protein that protects the
cells that carry the plasmid from the effects of the biocin, this is not a very
likely explanation.  One *could* propose a very high rate of spontaneous curing
but that is not consistent with maintenance of the plasmid and would have been
selected against.

--
Larry D. Farrell, Ph.D.
Professor of Microbiology
Idaho State University

Scott J. Coutts wrote:

> What do the colonies look like, and how big are they? Would it be 
> possible for you to post some pictures on the web somewhere, or in 
> alt.test

Here's a large picture I took yesterday of the buggers:

http://www.biotech.ntnu.no/~trondaun/Bug.JPG

Let me explain the picture (a 9 cm plate). If you look carefully at the 
part of the plate furthest away from the camera you'll be able to see a 
thin red line written on the bottom of the plate. This was the boundary 
of the original streaking. I put a fair amount of colony material in 
this area, but the day after only distinct colonies had appeared, and 
too my surprise single colonies also had grown outside this area. For 
each day after this, the colonies spread further down the plate. At 
first they appear to have no connection with the other colonies, but 
after a few days, thin lines seem to connect them (you can probably see 
this between the oldest colonies. The newest colonies (those closest to 
the lens) seem to have no connection to the others). Some of the 
colonies also look like spirals, almost as if they have been built by 
bacteria swimming in a spiral.

So there are two strange things with this bug:

1) They seem not to like growing close. Where I started streaking, only 
distinct colonies appeared. Only occasionally to they grow so close that 
they touch each other. But as time progresses new colonies are forming 
inbetween the older.

2) They spread over the plate. Probably by swimming, but they seem to 
try to reach quite far away before proliferating.

These two points are probably related. Maybe they really are sensitive 
to their own antibiotic, but why would they then survive in colonies?

Btw, I have grown the bug in liquid media, I did not follow the growth, 
but the day after, the culture looked quite ordinary, like E.coli.

Trond Erik

"Trond Erik Vee Aune" <trondaun@chembio.NOSPAM.ntnu.no> wrote in message
news:3E625E1F.2050809@chembio.NOSPAM.ntnu.no...
>
>
> Scott J. Coutts wrote:
>
> > What do the colonies look like, and how big are they? Would it be
> > possible for you to post some pictures on the web somewhere, or in
> > alt.test
>
> Here's a large picture I took yesterday of the buggers:
>
> http://www.biotech.ntnu.no/~trondaun/Bug.JPG
>
> Let me explain the picture (a 9 cm plate). If you look carefully at the
> part of the plate furthest away from the camera you'll be able to see a
> thin red line written on the bottom of the plate. This was the boundary
> of the original streaking. I put a fair amount of colony material in
> this area, but the day after only distinct colonies had appeared, and
> too my surprise single colonies also had grown outside this area. For
> each day after this, the colonies spread further down the plate. At
> first they appear to have no connection with the other colonies, but
> after a few days, thin lines seem to connect them (you can probably see
> this between the oldest colonies. The newest colonies (those closest to
> the lens) seem to have no connection to the others). Some of the
> colonies also look like spirals, almost as if they have been built by
> bacteria swimming in a spiral.

An opinion - you will probably get several more.

It looks to me like a mixed culture including a highly motile species
(Proteus for example) which has actually swarmed over the entire surface of
the plate and produced confluent growth. The colonies you are getting are
from other species.  It is very difficult to separate swarming species from
others - if you take samples from the colonies you will get both. Try taking
a sample from a clear area of the plate and inoculating into broth. If
anything grows it's going to be the swarmer. Subculture from the broth back
on to agar and see what it looks like. If I'm right, you'll get confluent
growth without colonies.

Separating the colony formers from the swarmers is going to require special
media, selective agents or other selective methods (eg temperature).

This is why microbiologists use stains and stuff.

GS

"Graham Shepherd" <muhero.nospam@globalnet.co.uk> wrote in message
news:b3tt06$b4i$1@knossos.btinternet.com...
> "Trond Erik Vee Aune" <trondaun@chembio.NOSPAM.ntnu.no> wrote in message
> news:3E625E1F.2050809@chembio.NOSPAM.ntnu.no...
> >
> >
> > Scott J. Coutts wrote:
> >
> > > What do the colonies look like, and how big are they? Would it be
> > > possible for you to post some pictures on the web somewhere, or in
> > > alt.test
> >
> > Here's a large picture I took yesterday of the buggers:
> >
> > http://www.biotech.ntnu.no/~trondaun/Bug.JPG
> >
> > Let me explain the picture (a 9 cm plate). If you look carefully at the
> > part of the plate furthest away from the camera you'll be able to see a
> > thin red line written on the bottom of the plate. This was the boundary
> > of the original streaking. I put a fair amount of colony material in
> > this area, but the day after only distinct colonies had appeared, and
> > too my surprise single colonies also had grown outside this area. For
> > each day after this, the colonies spread further down the plate. At
> > first they appear to have no connection with the other colonies, but
> > after a few days, thin lines seem to connect them (you can probably see
> > this between the oldest colonies. The newest colonies (those closest to
> > the lens) seem to have no connection to the others). Some of the
> > colonies also look like spirals, almost as if they have been built by
> > bacteria swimming in a spiral.
>
> An opinion - you will probably get several more.
>
> It looks to me like a mixed culture including a highly motile species
> (Proteus for example) which has actually swarmed over the entire surface
of
> the plate and produced confluent growth. The colonies you are getting are
> from other species.  It is very difficult to separate swarming species
from
> others - if you take samples from the colonies you will get both. Try
taking
> a sample from a clear area of the plate and inoculating into broth. If
> anything grows it's going to be the swarmer. Subculture from the broth
back
> on to agar and see what it looks like. If I'm right, you'll get confluent
> growth without colonies.
>
> Separating the colony formers from the swarmers is going to require
special
> media, selective agents or other selective methods (eg temperature).
>
> This is why microbiologists use stains and stuff.
>
> GS
>

What about subculturing the swarmer onto a plate with a stronger agar
concentration? We used to use one (6% agar if I remember correctly) which
would stop the swarmers.

> > This is very interesting ! I would bet these bugs secrete biocins of
some
> > sort. If biocin secretion is associated with e.g. glucose in the medium,
and
> > if the bug is slightly sensitive to the biocins as well, perhaps this
would
> > explain the weird colony formation.
>
> [snip]
>
> Since all of the bacteria in the culture should carry the plasmid that
codes for
> production of the biocin, as well as the immunity protein that protects
the
> cells that carry the plasmid from the effects of the biocin, this is not a
very
> likely explanation.  One *could* propose a very high rate of spontaneous
curing
> but that is not consistent with maintenance of the plasmid and would have
been
> selected against.

It doesn't have to be a plasmid, though - it could be in the chromosome :)

I do agree that this explanation is stretching the limits of plausibility,
though.

Artem

Artem Evdokimov wrote:

> > > This is very interesting ! I would bet these bugs secrete biocins of
> some
> > > sort. If biocin secretion is associated with e.g. glucose in the medium,
> and
> > > if the bug is slightly sensitive to the biocins as well, perhaps this
> would
> > > explain the weird colony formation.
> >
> > [snip]
> >
> > Since all of the bacteria in the culture should carry the plasmid that
> codes for
> > production of the biocin, as well as the immunity protein that protects
> the
> > cells that carry the plasmid from the effects of the biocin, this is not a
> very
> > likely explanation.  One *could* propose a very high rate of spontaneous
> curing
> > but that is not consistent with maintenance of the plasmid and would have
> been
> > selected against.
>
> It doesn't have to be a plasmid, though - it could be in the chromosome :)
>
> I do agree that this explanation is stretching the limits of plausibility,
> though.
>
> Artem

That would make loss of immunity protein production at such a high rate even
more difficult to explain.


--
Larry D. Farrell, Ph.D.
Professor of Microbiology
Idaho State University

Graham Shepherd wrote:
> "Trond Erik Vee Aune" <trondaun@chembio.NOSPAM.ntnu.no> wrote in message
> news:3E625E1F.2050809@chembio.NOSPAM.ntnu.no...
> 
>>
>>Scott J. Coutts wrote:
>>
>>
>>>What do the colonies look like, and how big are they? Would it be
>>>possible for you to post some pictures on the web somewhere, or in
>>>alt.test
>>
>>Here's a large picture I took yesterday of the buggers:
>>
>>http://www.biotech.ntnu.no/~trondaun/Bug.JPG
>>
>>Let me explain the picture (a 9 cm plate). If you look carefully at the
>>part of the plate furthest away from the camera you'll be able to see a
>>thin red line written on the bottom of the plate. This was the boundary
>>of the original streaking. I put a fair amount of colony material in
>>this area, but the day after only distinct colonies had appeared, and
>>too my surprise single colonies also had grown outside this area. For
>>each day after this, the colonies spread further down the plate. At
>>first they appear to have no connection with the other colonies, but
>>after a few days, thin lines seem to connect them (you can probably see
>>this between the oldest colonies. The newest colonies (those closest to
>>the lens) seem to have no connection to the others). Some of the
>>colonies also look like spirals, almost as if they have been built by
>>bacteria swimming in a spiral.
> 
> 
> An opinion - you will probably get several more.
> 
> It looks to me like a mixed culture including a highly motile species
> (Proteus for example) which has actually swarmed over the entire surface of
> the plate and produced confluent growth. The colonies you are getting are
> from other species.  It is very difficult to separate swarming species from
> others - if you take samples from the colonies you will get both. Try taking
> a sample from a clear area of the plate and inoculating into broth. If
> anything grows it's going to be the swarmer. Subculture from the broth back
> on to agar and see what it looks like. If I'm right, you'll get confluent
> growth without colonies.
>

But if it's a swarmer and a non-swarmer, why would the discrete colonies 
appear all over the plate? Can swarmers 'carry along' non-swarmers?! If 
the plate is inoculated at only one point, then the swarmers will 
spread, and single colonies of the other one will only be at the 
inoculation point, surely?

> 
> Separating the colony formers from the swarmers is going to require special
> media, selective agents or other selective methods (eg temperature).
> 

To seperate out our highly motile spirochaetes from other contaminants, 
we cut a slice in the agar, and inoculate the slice. Only our 
spirochaetes are motile enough to get out of there. I'm sure there's 
lots of others that could get out too, but thankfully, we never get them 
as contaminants (: But then, we also have the 'cheats' method of 
separating our spirochaetes - they get through a 0.2um filter, others 
dont (:

>
> This is why microbiologists use stains and stuff.
> 

I agree that it should be stained.


-- 
Scott J. Coutts
--------------------------------------------------------------------------
  Bacterial Pathogenesis Research Group		
  Department of Microbiology			Ph  [+61 3 9905 4838]
  PO Box 53					Fax [+61 3 9905 4811]	
  Monash University, 3800, Austrlalia
--------------------------------------------------------------------------

Anne Thomas wrote:

> What about subculturing the swarmer onto a plate with a stronger agar
> concentration? We used to use one (6% agar if I remember correctly) which
> would stop the swarmers.

Does this mean that swarming bacteria spreads through the agar and not 
on top of it?

Trond Erik

"Trond Erik Vee Aune" <trondaun@biotech.REMOVETHISBEFOREREPLYING.ntnu.no>
wrote in message
news:3E636981.9080808@biotech.REMOVETHISBEFOREREPLYING.ntnu.no...
>
>
> Anne Thomas wrote:
>
> > What about subculturing the swarmer onto a plate with a stronger agar
> > concentration? We used to use one (6% agar if I remember correctly)
which
> > would stop the swarmers.
>
> Does this mean that swarming bacteria spreads through the agar and not
> on top of it?
>
> Trond Erik
>

No, Proteus mirabilis, for example, grows as a discrete colony onstead of
swarming.

Annie ;o)

"Scott J. Coutts" <scott.coutts@med.monash.edu.au> wrote in message
news:3E62F0A3.4050407@med.monash.edu.au...
>
<snip>

>
> But if it's a swarmer and a non-swarmer, why would the discrete colonies
> appear all over the plate? Can swarmers 'carry along' non-swarmers?! If
> the plate is inoculated at only one point, then the swarmers will
> spread, and single colonies of the other one will only be at the
> inoculation point, surely?

I'm not sure from Trond Erik's description exactly what plating technique
he's using, and the appearance of the plate doesn't help much with that. I
would speculate that it might be possible for a non-motile organism to be
carried some distance but a motile one. I hope some of you are going to look
at the plate (especially the magnified view) and tell us what you think.

We don't know much about how dry the plates are, what incubation temperature
and time are being used and so on. They're geneticists who are presumably
handling a small range of well characterised bugs. They're seeing something
they haven't seen before, and are not geared up for isolating pure strains
from difficult mixtures.

>
> To seperate out our highly motile spirochaetes from other contaminants,
> we cut a slice in the agar, and inoculate the slice. Only our
> spirochaetes are motile enough to get out of there. I'm sure there's
> lots of others that could get out too, but thankfully, we never get them
> as contaminants (: But then, we also have the 'cheats' method of
> separating our spirochaetes - they get through a 0.2um filter, others
> dont (:

My suggestion was based on the types of procedure we know they have
available - I have assumed that their media are obtained ready to use - ie
they don't pour plates or make broth tubes. The ways that microbiologists
would use to do these separations (including Anne's 6% agar) may be
inaccessible to them.

I've only ever worked with dead spirochaetes, but since they were T.
pallidum, it was a satisfactory arrangement.

GS

"Larry Farrell" <farrlarr@isu.edu> wrote in message
news:778ec8936eb5de880c3ff8fd89f3a03e@news.teranews.com...
> That would make loss of immunity protein production at such a high rate
even
> more difficult to explain.

Unless immunity (ore secretion of the biocin, or of the chemotaxis molecule)
is directly linked to some sort of exhaustible supply in the medium - via
either positive or negative feedback. I am not saying that this is very
likely, however barring the obvious contaminant or mixed-culture type
arguements, all the other explanations are about equally improbable.

There are bugs that naturally form very weird, fractal-like colonies (not
Nocardia otitidiscaviarum, and no, I do not refer to starvation-grown
E.coli, like here http://classes.yale.edu/99-00/math190a/Bacteria.html or
here http://polymer.bu.edu/ogaf/html/chp51exp3.htm). I don't remember the
species, but I used to know a guy who tried to figure out what caused this
and how this sort of colony shape was regulated. I am not sure if he found
out or not.

http://star.tau.ac.il/~inon/wisdom1/preprint.html ?

Artem

However, one colony of Proteus mirabilis (or P. vulgaris) can eventually 
swarm over an entire plate. Getting bacteria subcultured and away from 
Proteus sp. is a constant problem with mixed cultures of pathogens that 
include it. This is why most clinical specimens are also plated on 
MacConkey agar, which inhibits Proteus's swarming proclivities for the 
most part. MacConkey, however, only grows gram negative rods. To split a 
gram positive away from a Proteus one must subculture onto a CNA or 
other plate that includes antibiotics. CNA stands for Colistin/Naladixic 
Acid, so anything that is sensitive to this will not grow on CNA. It 
inhibits most gram negatives with the exception of very resistant ones.

I looked at the picture of the colony and it looks like you've picked up 
a swarmer that's growing all over everything. Without using some sort of 
selective medium, you're kind of stuck as to splitting the organisms.

Judy Dilworth, M.T. (ASCP)
Microbiology (Clinical, 29 years)

 
> No, Proteus mirabilis, for example, grows as a discrete colony onstead of
> swarming.
> 
> Annie ;o)
> 
> 
> 
> 

Anne Thomas wrote:

>>>What about subculturing the swarmer onto a plate with a stronger agar
>>>concentration? We used to use one (6% agar if I remember correctly)
>>
> which
> 
>>>would stop the swarmers.
>>
>>Does this mean that swarming bacteria spreads through the agar and not
>>on top of it?
>>
>>Trond Erik
>>
> 
> 
> No, Proteus mirabilis, for example, grows as a discrete colony onstead of
> swarming.

I don't get this. Why would increasing the agar concentration help 
prevent swarming if they don't move through the agar?

Trond Erik

Graham Shepherd wrote:
> "Scott J. Coutts" <scott.coutts@med.monash.edu.au> wrote in message
> news:3E62F0A3.4050407@med.monash.edu.au...
> 
> <snip>
> 
>>But if it's a swarmer and a non-swarmer, why would the discrete colonies
>>appear all over the plate? Can swarmers 'carry along' non-swarmers?! If
>>the plate is inoculated at only one point, then the swarmers will
>>spread, and single colonies of the other one will only be at the
>>inoculation point, surely?
> 
> 
> I'm not sure from Trond Erik's description exactly what plating technique
> he's using, and the appearance of the plate doesn't help much with that.

I could elaborate. The purpose of this plate was to grow Bug X on the 
area behind til red line. And to keep it there. The next thing I wanted 
to do was to streak different bacteria up to this line to see at the 
inhibition (a test I remembered doing at my microbiology lab course). So 
I picked up a fair amount of colony material from the original colony at 
mother plate (the original LB+glucose plate containing a myriad of 
different bacteria and fungus from the Ganges water sample) and streaked 
it into this area, making sure that I touched every square mm.

> I
> would speculate that it might be possible for a non-motile organism to be
> carried some distance but a motile one. I hope some of you are going to look
> at the plate (especially the magnified view) and tell us what you think.
> 
> We don't know much about how dry the plates are, what incubation temperature
> and time are being used and so on. They're geneticists who are presumably
> handling a small range of well characterised bugs. They're seeing something
> they haven't seen before, and are not geared up for isolating pure strains
> from difficult mixtures.

The plate (LB+glucose) was pre-heated at 37 degrees before use. I 
incubated it at 37 degrees about 16 hours. I am only familar with 
E.coli, but we do work with 5-6 different bacteria, both g+ and g-. But 
yes, this is completely new to us, at least to me ;)

> My suggestion was based on the types of procedure we know they have
> available - I have assumed that their media are obtained ready to use - ie
> they don't pour plates or make broth tubes. The ways that microbiologists
> would use to do these separations (including Anne's 6% agar) may be
> inaccessible to them.

I make all my plates myself, both mixing the components and pouring 
them. I guess we use about 20-30 different medias regularly (selective 
for Pseudomonas, M63, Burks and so on). So it would be possible for me 
to make plates for separating these organism, if we have the components.

What I'll do next is to stain them and to try to separate the swarmer from
the possible non-swarmer as suggested earlier.

Thanks for all your help, I'll keep you informed! And if you have any 
more suggestions, please tell me, this is fun!

Trond Erik

Graham Shepherd wrote:
> "Scott J. Coutts" <scott.coutts@med.monash.edu.au> wrote in message
> news:3E62F0A3.4050407@med.monash.edu.au...
> 
> <snip>
> 
>>But if it's a swarmer and a non-swarmer, why would the discrete colonies
>>appear all over the plate? Can swarmers 'carry along' non-swarmers?! If
>>the plate is inoculated at only one point, then the swarmers will
>>spread, and single colonies of the other one will only be at the
>>inoculation point, surely?
> 
> 
> I'm not sure from Trond Erik's description exactly what plating technique
> he's using, and the appearance of the plate doesn't help much with that. I
> would speculate that it might be possible for a non-motile organism to be
> carried some distance but a motile one. I hope some of you are going to look
> at the plate (especially the magnified view) and tell us what you think.
> 
> We don't know much about how dry the plates are, what incubation temperature
> and time are being used and so on. They're geneticists who are presumably
> handling a small range of well characterised bugs. They're seeing something
> they haven't seen before, and are not geared up for isolating pure strains
> from difficult mixtures.
> 

Yup, for sure. But the conditions of the plate and streaking technique 
were outlined briefly. I assumed that the bug was inoculated only behind 
the line.


>>To seperate out our highly motile spirochaetes from other contaminants,
>>we cut a slice in the agar, and inoculate the slice. Only our
>>spirochaetes are motile enough to get out of there. I'm sure there's
>>lots of others that could get out too, but thankfully, we never get them
>>as contaminants (: But then, we also have the 'cheats' method of
>>separating our spirochaetes - they get through a 0.2um filter, others
>>dont (:
> 
> 
> My suggestion was based on the types of procedure we know they have
> available - I have assumed that their media are obtained ready to use - ie
> they don't pour plates or make broth tubes. The ways that microbiologists
> would use to do these separations (including Anne's 6% agar) may be
> inaccessible to them.
> 
> I've only ever worked with dead spirochaetes, but since they were T.
> pallidum, it was a satisfactory arrangement.
> 

heheh fair enough. We only work with two types, one is non-infectious 
for humans, the other is. I dont have to work with that too much (:


-- 
Scott J. Coutts
--------------------------------------------------------------------------
  Bacterial Pathogenesis Research Group		
  Department of Microbiology			Ph  [+61 3 9905 4838]
  PO Box 53					Fax [+61 3 9905 4811]	
  Monash University, 3800, Austrlalia
--------------------------------------------------------------------------

Trond Erik Vee Aune wrote:
> 
> 
> Anne Thomas wrote:
> 
>>>> What about subculturing the swarmer onto a plate with a stronger agar
>>>> concentration? We used to use one (6% agar if I remember correctly)
>>>
>>>
>> which
>>
>>>> would stop the swarmers.
>>>
>>>
>>> Does this mean that swarming bacteria spreads through the agar and not
>>> on top of it?
>>>
>>> Trond Erik
>>>
>>
>>
>> No, Proteus mirabilis, for example, grows as a discrete colony onstead of
>> swarming.
> 
> 
> I don't get this. Why would increasing the agar concentration help 
> prevent swarming if they don't move through the agar?
> 

Because it decreases the availability of water at the surface of the 
agar. It's a 'dryer' surface for them to move across, so they move more 
slowly. Kind of like a slug trying to move across a wet surface versus a 
dry surface.


-- 
Scott J. Coutts
--------------------------------------------------------------------------
  Bacterial Pathogenesis Research Group		
  Department of Microbiology			Ph  [+61 3 9905 4838]
  PO Box 53					Fax [+61 3 9905 4811]	
  Monash University, 3800, Austrlalia
--------------------------------------------------------------------------

Trond Erik Vee Aune wrote:
> 
> 
> Graham Shepherd wrote:
> 
>> "Scott J. Coutts" <scott.coutts@med.monash.edu.au> wrote in message
>> news:3E62F0A3.4050407@med.monash.edu.au...
>>
>> <snip>
>>
>>> But if it's a swarmer and a non-swarmer, why would the discrete colonies
>>> appear all over the plate? Can swarmers 'carry along' non-swarmers?! If
>>> the plate is inoculated at only one point, then the swarmers will
>>> spread, and single colonies of the other one will only be at the
>>> inoculation point, surely?
>>
>>
>>
>> I'm not sure from Trond Erik's description exactly what plating technique
>> he's using, and the appearance of the plate doesn't help much with that.
> 
> 
> I could elaborate. The purpose of this plate was to grow Bug X on the 
> area behind til red line. And to keep it there. The next thing I wanted 
> to do was to streak different bacteria up to this line to see at the 
> inhibition (a test I remembered doing at my microbiology lab course). So 
> I picked up a fair amount of colony material from the original colony at 
> mother plate (the original LB+glucose plate containing a myriad of 
> different bacteria and fungus from the Ganges water sample) and streaked 
> it into this area, making sure that I touched every square mm.
> 

So that plate picture you showed on the web was one where you streaked 
only the 'mystery bug' onto the agar plate, behind the red line (at the 
top of the picture) only? Is that right? You havent streaked anywhere 
else on the plate, or any other bacteria on the plate?


>> would speculate that it might be possible for a non-motile organism to be
>> carried some distance but a motile one. I hope some of you are going 
>> to look
>> at the plate (especially the magnified view) and tell us what you think.
>>
>> We don't know much about how dry the plates are, what incubation 
>> temperature
>> and time are being used and so on. They're geneticists who are presumably
>> handling a small range of well characterised bugs. They're seeing 
>> something
>> they haven't seen before, and are not geared up for isolating pure 
>> strains
>> from difficult mixtures.
> 
> 
> The plate (LB+glucose) was pre-heated at 37 degrees before use. I 
> incubated it at 37 degrees about 16 hours. I am only familar with 
> E.coli, but we do work with 5-6 different bacteria, both g+ and g-. But 
> yes, this is completely new to us, at least to me ;)

I would dry the plates for maybe 30 minutes at 37oC or in a laminar flow 
cabinet before using them. Or until you can see 'drying marks' on the 
surface of the agar (sort of slight wrinkles or small dull patches).


>> My suggestion was based on the types of procedure we know they have
>> available - I have assumed that their media are obtained ready to use 
>> - ie
>> they don't pour plates or make broth tubes. The ways that microbiologists
>> would use to do these separations (including Anne's 6% agar) may be
>> inaccessible to them.
> 
> I make all my plates myself, both mixing the components and pouring 
> them. I guess we use about 20-30 different medias regularly (selective 
> for Pseudomonas, M63, Burks and so on). So it would be possible for me 
> to make plates for separating these organism, if we have the components.
> 
> What I'll do next is to stain them and to try to separate the swarmer from
> the possible non-swarmer as suggested earlier.
> 

Look carefully at your stain to see if there is more than one type 
there. That may well be all you need to tell you if you have more than 
one type of bug in the colonies, if you look carefully enough. I assume 
you're going to using Gram's stain?

 >
> Thanks for all your help, I'll keep you informed! And if you have any 
> more suggestions, please tell me, this is fun!
> 
> Trond Erik
> 


-- 
Scott J. Coutts
--------------------------------------------------------------------------
  Bacterial Pathogenesis Research Group		
  Department of Microbiology			Ph  [+61 3 9905 4838]
  PO Box 53					Fax [+61 3 9905 4811]	
  Monash University, 3800, Austrlalia
--------------------------------------------------------------------------

Scott J. Coutts wrote:

> So that plate picture you showed on the web was one where you streaked 
> only the 'mystery bug' onto the agar plate, behind the red line (at the 
> top of the picture) only? Is that right? You havent streaked anywhere 
> else on the plate, or any other bacteria on the plate?

Correct.

> Look carefully at your stain to see if there is more than one type 
> there. That may well be all you need to tell you if you have more than 
> one type of bug in the colonies, if you look carefully enough. I assume 
> you're going to using Gram's stain?

Yes. I'll do that later today

Trond Erik

They swarm on top of the medium. The harder agar means less moisture.

Judy Dilworth, M.T. (ASCP)
Microbiology


> Anne Thomas wrote:
>>>> What about subculturing the swarmer onto a plate with a stronger agar
>>>> concentration? We used to use one (6% agar if I remember correctly)
>>>which would stop the swarmers.
>>>

"Trond Erik Vee Aune" <trondaun@biotech.REMOVETHISBEFOREREPLYING.ntnu.no>
wrote in message
news:3E64867F.9010805@biotech.REMOVETHISBEFOREREPLYING.ntnu.no...
>
>
> Scott J. Coutts wrote:
>
> > So that plate picture you showed on the web was one where you streaked
> > only the 'mystery bug' onto the agar plate, behind the red line (at the
> > top of the picture) only? Is that right? You havent streaked anywhere
> > else on the plate, or any other bacteria on the plate?
>
> Correct.
>
Just a thought, which way round did you incubate your plate, agar on top or
bottom?

Dave

Dave L wrote:

> Just a thought, which way round did you incubate your plate, agar on top or
> bottom?

Agar on top.

By the way, why do we incubate this way? Why not agar on bottom?

Trond Erik

> 
> Dave
> 
> 

So that when condensation forms on the lid of the dish, it doesnt drip 
down onto our carefully streaked out and well seperated colonies, 
spreading them everywhere! If you incubate them agar side up, no 
condensation forms there because it is already more moist than the air 
inside the petri dish.

Trond Erik Vee Aune wrote:
> 
> 
> Dave L wrote:
> 
>> Just a thought, which way round did you incubate your plate, agar on 
>> top or
>> bottom?
> 
> 
> Agar on top.
> 
> By the way, why do we incubate this way? Why not agar on bottom?
> 
> Trond Erik
> 
>>
>> Dave
>>
>>
> 


-- 
Scott J. Coutts
--------------------------------------------------------------------------
  Bacterial Pathogenesis Research Group		
  Department of Microbiology			Ph  [+61 3 9905 4838]
  PO Box 53					Fax [+61 3 9905 4811]	
  Monash University, 3800, Austrlalia
--------------------------------------------------------------------------

Scott J. Coutts wrote:

> I agree that it should be stained.

It took me some time to do the staining, but now it's done. I compared 
it with E.coli. It looks to be gram negative.

I've also tried to separate the "swarmer" from the "colony former" by 
inoculating from an area without any obvious colony growth and from a 
colony, but without success. In both cases I get the same growth as 
described earlier. So maybe it is one strain after all?

I'm still eager to do 16s sequencing, but first I need to separate them 
if there's more than one strain. Do you microbiologists have any idea to 
how I could separate them?

Actually a 16s sequencing would tell me if I have more than one 
genotype. But it would be better to establish this before ordering the 
primers. At least if there's an easy way to do this.

Thanks again for all your helpful suggestions!

Trond Erik

Trond Erik Vee Aune wrote:
 >
 > Scott J. Coutts wrote:
 >
 >> I agree that it should be stained.
 >
 >
 > It took me some time to do the staining, but now it's done. I
 > compared it with E.coli. It looks to be gram negative.
 >
 > I've also tried to separate the "swarmer" from the "colony former" by
 >  inoculating from an area without any obvious colony growth and from
 > a colony, but without success. In both cases I get the same growth as
 >  described earlier. So maybe it is one strain after all?
 >

Did you see more than one cell shape in the stain? Could you post a pic 
of it from the microscope on the web (if you have the facilities)?

 >
 > I'm still eager to do 16s sequencing, but first I need to separate
 > them if there's more than one strain. Do you microbiologists have any
 > idea to how I could separate them?
 >

I'd recommend the 6% agar trick first of all. It will also make those 
colonies smaller, too. Spread them onto a plate. I'm still not convinced 
that you have two types. Do you have a dark field or phase contrast 
microscope? (probably not if you're a genetics lab). You may see a 
difference in the motility if you can look at them directly.

 >
 > Actually a 16s sequencing would tell me if I have more than one
 > genotype. But it would be better to establish this before ordering
 > the primers. At least if there's an easy way to do this.
 >

Well, even if you separate them (assuming there is actually two to be 
separated), you're still going to need to order the primers... why not 
order them anyhow (:

 >
 > Thanks again for all your helpful suggestions!
 >
 > Trond Erik
 >


-- 
Scott J. Coutts
--------------------------------------------------------------------------
  Bacterial Pathogenesis Research Group		
  Department of Microbiology			Ph  [+61 3 9905 4838]
  PO Box 53					Fax [+61 3 9905 4811]	
  Monash University, 3800, Austrlalia
--------------------------------------------------------------------------

Oh, wait, did you say you inoculated from a site on the plate with no 
apparent colonies? In that case, I'd say definitely there is two types. 
If you could post a pic of the stain, that'd probably help, maybe using 
the 40x and 100x (with oil) objectives if possible.

Scott J. Coutts wrote:
> Trond Erik Vee Aune wrote:
>  >
>  > Scott J. Coutts wrote:
>  >
>  >> I agree that it should be stained.
>  >
>  >
>  > It took me some time to do the staining, but now it's done. I
>  > compared it with E.coli. It looks to be gram negative.
>  >
>  > I've also tried to separate the "swarmer" from the "colony former" by
>  >  inoculating from an area without any obvious colony growth and from
>  > a colony, but without success. In both cases I get the same growth as
>  >  described earlier. So maybe it is one strain after all?
>  >
> 
> Did you see more than one cell shape in the stain? Could you post a pic 
> of it from the microscope on the web (if you have the facilities)?
> 
>  >
>  > I'm still eager to do 16s sequencing, but first I need to separate
>  > them if there's more than one strain. Do you microbiologists have any
>  > idea to how I could separate them?
>  >
> 
> I'd recommend the 6% agar trick first of all. It will also make those 
> colonies smaller, too. Spread them onto a plate. I'm still not convinced 
> that you have two types. Do you have a dark field or phase contrast 
> microscope? (probably not if you're a genetics lab). You may see a 
> difference in the motility if you can look at them directly.
> 
>  >
>  > Actually a 16s sequencing would tell me if I have more than one
>  > genotype. But it would be better to establish this before ordering
>  > the primers. At least if there's an easy way to do this.
>  >
> 
> Well, even if you separate them (assuming there is actually two to be 
> separated), you're still going to need to order the primers... why not 
> order them anyhow (:
> 
>  >
>  > Thanks again for all your helpful suggestions!
>  >
>  > Trond Erik
>  >
> 
> 


-- 
Scott J. Coutts
--------------------------------------------------------------------------
  Bacterial Pathogenesis Research Group		
  Department of Microbiology			Ph  [+61 3 9905 4838]
  PO Box 53					Fax [+61 3 9905 4811]	
  Monash University, 3800, Austrlalia
--------------------------------------------------------------------------

Scott J. Coutts wrote:

> Did you see more than one cell shape in the stain? Could you post a pic 
> of it from the microscope on the web (if you have the facilities)?

I could only see one shape. I don't have a microscope with a camera 
attached. But I'll check with other departments.

>  > I'm still eager to do 16s sequencing, but first I need to separate
>  > them if there's more than one strain. Do you microbiologists have any
>  > idea to how I could separate them?
> 
> I'd recommend the 6% agar trick first of all. It will also make those 
> colonies smaller, too. Spread them onto a plate. 

But how with this help me separate them? It'll only prevent the swarmers 
from spreading. I'll still have the two strains together.

> I'm still not convinced 
> that you have two types. Do you have a dark field or phase contrast 
> microscope? (probably not if you're a genetics lab). You may see a 
> difference in the motility if you can look at them directly.

I don't know, I'll have to check it up.

Trond Erik

-- 
Trond Erik Vee Aune
Department of Biotechnology, NTNU
Sem Selandsvei 6/8
N-7491 TRONDHEIM
Tlf: +0047 73551386
Email: trondaun@biotech.ntnu.no
http://www.biotech.ntnu.no/molgen

Trond Erik Vee Aune wrote:
>> I'd recommend the 6% agar trick first of all. It will also make those 
>> colonies smaller, too. Spread them onto a plate. 
>> 
> 
> But how with this help me separate them? It'll only prevent the swarmers 
> from spreading. I'll still have the two strains together.
> 

Yup, that's the idea. I assume you know how to streak a plate to obtain 
single colonies? I.e. streak an initial inoculum over  a section of the 
plate, flame your loop, do a set of streakes coming from the first 
incolulation point, flame the loop, set of streaks from first set of 
streaks, flame etc etc.

Otherwise, do a set of serial dilutions of your broth, and plate out 
100uL of the dilutions on your plates. Some of them should end up with 
very few single colonies.

-- 
Scott J. Coutts
--------------------------------------------------------------------------
  Bacterial Pathogenesis Research Group		
  Department of Microbiology			Ph  [+61 3 9905 4838]
  PO Box 53					Fax [+61 3 9905 4811]	
  Monash University, 3800, Austrlalia
--------------------------------------------------------------------------

"Scott J. Coutts" <scott.coutts@med.monash.edu.au> wrote in message
news:3E6883CC.9040508@med.monash.edu.au...
>
>
> Trond Erik Vee Aune wrote:
> >> I'd recommend the 6% agar trick first of all. It will also make those
> >> colonies smaller, too. Spread them onto a plate.
> >>
> >
> > But how with this help me separate them? It'll only prevent the swarmers
> > from spreading. I'll still have the two strains together.
> >
>
> Yup, that's the idea. I assume you know how to streak a plate to obtain
> single colonies? I.e. streak an initial inoculum over  a section of the
> plate, flame your loop, do a set of streakes coming from the first
> incolulation point, flame the loop, set of streaks from first set of
> streaks, flame etc etc.
>


Doesn't it make you wish you could actually be there and show Trond what we
mean about separating colonies by streaking them out?

Hopefully Trond, if you have 2 organisms there, the colonies may look
different on 6% and then you could subculture them again separately.

Good Luck!

Annie :o)

Anne Thomas wrote:
> "Scott J. Coutts" <scott.coutts@med.monash.edu.au> wrote in message
> news:3E6883CC.9040508@med.monash.edu.au...
> 
>>
>>Trond Erik Vee Aune wrote:
>>
>>>>I'd recommend the 6% agar trick first of all. It will also make those
>>>>colonies smaller, too. Spread them onto a plate.
>>>>
>>>
>>>But how with this help me separate them? It'll only prevent the swarmers
>>>from spreading. I'll still have the two strains together.
>>>
>>
>>Yup, that's the idea. I assume you know how to streak a plate to obtain
>>single colonies? I.e. streak an initial inoculum over  a section of the
>>plate, flame your loop, do a set of streakes coming from the first
>>incolulation point, flame the loop, set of streaks from first set of
>>streaks, flame etc etc.
>>
> 
> 
> 
> Doesn't it make you wish you could actually be there and show Trond what we
> mean about separating colonies by streaking them out?
> 

Hehe yes, it's kind of hard to describe sometimes! Actually, I was half 
asleep when I wrote that, so it's not a very thorough description (:

> Hopefully Trond, if you have 2 organisms there, the colonies may look
> different on 6% and then you could subculture them again separately.
> 
> Good Luck!
> Annie :o)
> 




-- 
Scott J. Coutts
--------------------------------------------------------------------------
  Bacterial Pathogenesis Research Group		
  Department of Microbiology			Ph  [+61 3 9905 4838]
  PO Box 53					Fax [+61 3 9905 4811]	
  Monash University, 3800, Australia
--------------------------------------------------------------------------

Hi,
If you have a digital camera, just take a shot from the eye-piece.
Use timer and hold it still to avoid blur.
It will do for a discussion here.

Let us see it quick! :)

Hiroshi

"Trond Erik Vee Aune" <trondaun@biotech.REMOVETHISBEFOREREPLYING.ntnu.no> wrote in message
news:3E686EE1.9090806@biotech.REMOVETHISBEFOREREPLYING.ntnu.no...

> I could only see one shape. I don't have a microscope with a camera
> attached. But I'll check with other departments.

--
Hiroshi Hirotani
Osaka Kyoiku University, Osaka, Japan
hirotani@cc.osaka-kyoiku.ac.jp

It sounds like you're doing micro without a loop or wire to pick 
colonies or something to flame your loop in between touching colonies.

After you make your 6% agar (or use some differential media as I 
described in another posting) you need to streak for isolation. If you 
obtain two colony morphologies, each colony is a pure culture. You need 
to flame your loop, pick one colony and streak it to another plate. You 
then need to flame your loop again, pick the second colony type, plate 
to a separate plate and streak for isolation. Then flame again.

This is basic microbiology. If you're using bacteria, even in a genetics 
laboratory, I don't understand how you can do it without all of the 
basic equipment that you need. The ONLY way to separate bacteria is 
streak for isolation, using solid media. You put your inoculum down on 
about 1/3 of the plate. Then you turn the plate a little and streak out 
from this area. You then flame, and then streak another quadrant, then 
turn and streak the last quadrant. You then incubate the plate upside 
down, at least overnight, and look for different colony morphology.

A visit to your local clinical micro lab will help you immensely! We do 
this EVERY DAY. Gram negative rods don't look THAT different from each 
other upon staining, at least with Gram's stain. Some are smaller and 
thinner or thicker, but that doesn't help you separate them out.

Judy Dilworth, M.T. (ASCP)
Microbiology

Scott J. Coutts wrote:

>> But how with this help me separate them? It'll only prevent the 
>> swarmers from spreading. I'll still have the two strains together.
>>
> 
> Yup, that's the idea. I assume you know how to streak a plate to obtain 
> single colonies? I.e. streak an initial inoculum over  a section of the 
> plate, flame your loop, do a set of streakes coming from the first 
> incolulation point, flame the loop, set of streaks from first set of 
> streaks, flame etc etc.

Yes, of course.

So the idea is that as soon as the swarmer can't swim it'll form 
colonies? And the colonies I've seen so far is not from the swarmer, but 
the other strain, which is in some way travelling along with the swarmer 
and forms colonies a certain distance away from the others? This sounds 
abit far fetched ;) Isn't it more plausible that I only have _one_ 
strain which have all this traits? This is a more simple solution since 
we don't have to try to find a reason for how the non-swarmer are 
spreading (not carried on the back of the swarmer I guess ;).

I'll do it anyway and keep you informed on what's happening!

Thanks for all your input!

Trond Erik

Of course I know all this. Why do you think otherwise?

What I don't understand is:

1) Why do you think I have two strains when it's easier to explain what 
I see with one strain than with two?
2) According to your theory the swarmer is not forming colonies under 
the current condition, but would, maybe, do this if it is not allowed to 
spread out. Why is this?

Trond Erik

JEDilworth wrote:
> It sounds like you're doing micro without a loop or wire to pick 
> colonies or something to flame your loop in between touching colonies.
> 
> After you make your 6% agar (or use some differential media as I 
> described in another posting) you need to streak for isolation. If you 
> obtain two colony morphologies, each colony is a pure culture. You need 
> to flame your loop, pick one colony and streak it to another plate. You 
> then need to flame your loop again, pick the second colony type, plate 
> to a separate plate and streak for isolation. Then flame again.
> 
> This is basic microbiology. If you're using bacteria, even in a genetics 
> laboratory, I don't understand how you can do it without all of the 
> basic equipment that you need. The ONLY way to separate bacteria is 
> streak for isolation, using solid media. You put your inoculum down on 
> about 1/3 of the plate. Then you turn the plate a little and streak out 
> from this area. You then flame, and then streak another quadrant, then 
> turn and streak the last quadrant. You then incubate the plate upside 
> down, at least overnight, and look for different colony morphology.
> 
> A visit to your local clinical micro lab will help you immensely! We do 
> this EVERY DAY. Gram negative rods don't look THAT different from each 
> other upon staining, at least with Gram's stain. Some are smaller and 
> thinner or thicker, but that doesn't help you separate them out.
> 
> Judy Dilworth, M.T. (ASCP)
> Microbiology
> 

His picture looks like it is a pure plate more than a streak plate.  but the
growth on some of the colonies look odd?  Judy does the colonies look like a
GNR to you?

DenniS


"JEDilworth" <bactitech@nospamhortonsbay.com> wrote in message
news:3E6CBF70.90706@nospamhortonsbay.com...
> It sounds like you're doing micro without a loop or wire to pick
> colonies or something to flame your loop in between touching colonies.
>
> After you make your 6% agar (or use some differential media as I
> described in another posting) you need to streak for isolation. If you
> obtain two colony morphologies, each colony is a pure culture. You need
> to flame your loop, pick one colony and streak it to another plate. You
> then need to flame your loop again, pick the second colony type, plate
> to a separate plate and streak for isolation. Then flame again.
>
> This is basic microbiology. If you're using bacteria, even in a genetics
> laboratory, I don't understand how you can do it without all of the
> basic equipment that you need. The ONLY way to separate bacteria is
> streak for isolation, using solid media. You put your inoculum down on
> about 1/3 of the plate. Then you turn the plate a little and streak out
> from this area. You then flame, and then streak another quadrant, then
> turn and streak the last quadrant. You then incubate the plate upside
> down, at least overnight, and look for different colony morphology.
>
> A visit to your local clinical micro lab will help you immensely! We do
> this EVERY DAY. Gram negative rods don't look THAT different from each
> other upon staining, at least with Gram's stain. Some are smaller and
> thinner or thicker, but that doesn't help you separate them out.
>
> Judy Dilworth, M.T. (ASCP)
> Microbiology
>

sorry  I meant POUR PLATE.


"DenniS" <grtmilenko@mindspring.com> wrote in message
news:b4klgp$eeb$1@slb6.atl.mindspring.net...
> His picture looks like it is a pure plate more than a streak plate.  but
the
> growth on some of the colonies look odd?  Judy does the colonies look like
a
> GNR to you?

DenniS wrote:
> His picture looks like it is a pure plate more than a streak plate.

As I told in an earlier posting it _is_ a streak plate, but the colonies 
do not form continously, they sort of just pop up somewhere along where 
I've streaked. And they also pop up away from where I originally 
streaked because they are swarming. After a few days they have covered 
the whole plate, but they still do not seem to like growing close to 
eachother, so it is only sporadic colonies all over the plate.

The question now is whether this strange growth is caused by one strain 
or more.

Trond Erik

>  but the
> growth on some of the colonies look odd? 

They have an irregular border. And in some cases they develop in an 
spiral (you can see this on some of the closest colonies). But I 
wouldn't say that there is any reason to suspect multiple strains 
because of this, the colonies seem fairly homogenous to me.

> Judy does the colonies look like a
> GNR to you?

What's this then?

Trond Erik

> 
> DenniS
> 
> 
> "JEDilworth" <bactitech@nospamhortonsbay.com> wrote in message
> news:3E6CBF70.90706@nospamhortonsbay.com...
> 
>>It sounds like you're doing micro without a loop or wire to pick
>>colonies or something to flame your loop in between touching colonies.
>>
>>After you make your 6% agar (or use some differential media as I
>>described in another posting) you need to streak for isolation. If you
>>obtain two colony morphologies, each colony is a pure culture. You need
>>to flame your loop, pick one colony and streak it to another plate. You
>>then need to flame your loop again, pick the second colony type, plate
>>to a separate plate and streak for isolation. Then flame again.
>>
>>This is basic microbiology. If you're using bacteria, even in a genetics
>>laboratory, I don't understand how you can do it without all of the
>>basic equipment that you need. The ONLY way to separate bacteria is
>>streak for isolation, using solid media. You put your inoculum down on
>>about 1/3 of the plate. Then you turn the plate a little and streak out
>>from this area. You then flame, and then streak another quadrant, then
>>turn and streak the last quadrant. You then incubate the plate upside
>>down, at least overnight, and look for different colony morphology.
>>
>>A visit to your local clinical micro lab will help you immensely! We do
>>this EVERY DAY. Gram negative rods don't look THAT different from each
>>other upon staining, at least with Gram's stain. Some are smaller and
>>thinner or thicker, but that doesn't help you separate them out.
>>
>>Judy Dilworth, M.T. (ASCP)
>>Microbiology
>>
> 
> 
> 

> What I don't understand is:
>
> 1) Why do you think I have two strains when it's easier to explain what
> I see with one strain than with two?

For all it's worth, I personally do not think you have two discrete bugs. I
think you've got one species with some very weird traits.

If you have two species, than both of them probably are gram-negative rods
(according to your staining experiment). In theory, your sequencing should
tell you if you have more than one strain - the result would be totally
muddled.

Artem

Yup, I agree with this too.

Artem Evdokimov wrote:
>>What I don't understand is:
>>
>>1) Why do you think I have two strains when it's easier to explain what
>>I see with one strain than with two?
> 
> 
> For all it's worth, I personally do not think you have two discrete bugs. I
> think you've got one species with some very weird traits.
> 
> If you have two species, than both of them probably are gram-negative rods
> (according to your staining experiment). In theory, your sequencing should
> tell you if you have more than one strain - the result would be totally
> muddled.
> 
> Artem
> 
> 

"Trond Erik Vee Aune" <trondaun@biotech.NOSPAM.ntnu.no> wrote in message
news:3E6D881A.10905@biotech.NOSPAM.ntnu.no...
>
>
> Scott J. Coutts wrote:
>
> >> But how with this help me separate them? It'll only prevent the
> >> swarmers from spreading. I'll still have the two strains together.
> >>
> >
> > Yup, that's the idea. I assume you know how to streak a plate to obtain
> > single colonies? I.e. streak an initial inoculum over  a section of the
> > plate, flame your loop, do a set of streakes coming from the first
> > incolulation point, flame the loop, set of streaks from first set of
> > streaks, flame etc etc.
>
> Yes, of course.
>
> So the idea is that as soon as the swarmer can't swim it'll form
> colonies? And the colonies I've seen so far is not from the swarmer, but
> the other strain, which is in some way travelling along with the swarmer
> and forms colonies a certain distance away from the others? This sounds
> abit far fetched ;) Isn't it more plausible that I only have _one_
> strain which have all this traits? This is a more simple solution since
> we don't have to try to find a reason for how the non-swarmer are
> spreading (not carried on the back of the swarmer I guess ;).

It is very much possible. I remember many years ago a colleague of mine was
trying to isolate filamentous phototrophic bacterium, and could not get rid
of the associated microorganism that was much smaller and, as seen under the
microscope, attached to the photosynthetic host.
Emir

>
> I'll do it anyway and keep you informed on what's happening!
>
> Thanks for all your input!
>
> Trond Erik
>
>

"EK" <khatipovNO-SPAM@NO-SPAMuchicago.edu> wrote in message
news:_Pwba.89$M4.23827@news.uchicago.edu...
>
> It is very much possible. I remember many years ago a colleague of mine
was
> trying to isolate filamentous phototrophic bacterium, and could not get
rid
> of the associated microorganism that was much smaller and, as seen under
the
> microscope, attached to the photosynthetic host.
> Emir

I am not doubting that it's not possible - microbiology has examples of
similar errors, and if you want to dig deep, biology in general has a fairly
spotted history (remember Aristotle's comical insects, or the
salamander/axolotl confusion ?) - I just do not think that the symptoms that
Erik is describing are enough to unambiguously claim mixed culture.

More data is needed :)

Artem

An so do I, I'll order the primers today! :)

Trond Erik

Scott Coutts wrote:
> Yup, I agree with this too.
> 
> Artem Evdokimov wrote:
> 
>>> What I don't understand is:
>>>
>>> 1) Why do you think I have two strains when it's easier to explain what
>>> I see with one strain than with two?
>>
>>
>>
>> For all it's worth, I personally do not think you have two discrete 
>> bugs. I
>> think you've got one species with some very weird traits.
>>
>> If you have two species, than both of them probably are gram-negative 
>> rods
>> (according to your staining experiment). In theory, your sequencing 
>> should
>> tell you if you have more than one strain - the result would be totally
>> muddled.
>>
>> Artem
>>
>>
>