How do you prepare a pure bacterial spore suspension?

[stbell]

I am attempting to prepare a spore suspension of Bacillus subtilis but am not having any luck geting rid of the vegetative cells... I have heat shocked at 70C for 15 minutes and centrifuged that heat shocked suspension at 5000rpm for 10 minutes twice in attempts to get rid of the vegetative cells but upon the confirmatory spore stain, several rods are still present (nearly as many as the control stain which was taken from the pre-heat shocked suspension). I have searched high and low for a little more guidance as how one separated vegetative cells from spores but haven't come across anything yet... any help would be greatly appreciated!

 

[CharonY]

There are a lot of methods for which you should be able to find literature rather easily. Note that most are older, so you may not find them by quick googling (unless you got the right keywords). However, in almost all methods you will find some extent of bacterial contamination, unless you repeat the purification steps.

 

Edit to remove details, although they should be easy enough to find in the literature,

Edited October 27, 2015 by CharonY

[John Cuthber]

If the guys in the black helicopters are not looking carefully at this thread I'm not sure they are doing their jobs properly.

Who needs the instructions for getting spores of the second cousin to anthrax, but is in an environment where they have to go and trawl the web for that information?

[CharonY]

Hmm, good point, actually...

[StringJunky]

If the guys in the black helicopters are not looking carefully at this thread I'm not sure they are doing their jobs properly.

Who needs the instructions for getting spores of the second cousin to anthrax, but is in an environment where they have to go and trawl the web for that information?

I wikied it and it's food safe and a commensal organism in humans. It is much used as a model, it seems.

[CharonY]

That is not the the point. B. subtilis is a harmless model organism for Gram+ bacteria. However, isolation of spores is also applicable (to some extent) to the pathogen. What John brought up is that a lab that is doing standard analyses should either already have protocols in place or have some sort of guidance on how to find them, as it is not precisely obscure knowledge for microbiologists.

It is quite possible that it is a legitimate question by a student, though as usual I wonder why the question is not directed to the adviser first. Note that there are a lot of other normally harmless microbiological techniques that can result in increased scrutiny due to their potential of abuse.

Edited October 27, 2015 by CharonY

[StringJunky]

That is not the the point. B. subtilis is a harmless model organism for Gram+ bacteria. However, isolation of spores is also applicable (to some extent) to the pathogen. What John brought up is that a lab that is doing standard analyses should either already have protocols in place or have some sort of guidance on how to find them, as it is not precisely obscure knowledge for microbiologists.

Right.

[John Cuthber]

I wikied it and it's food safe and a commensal organism in humans. It is much used as a model, it seems.

Yes, I know it's used as a model.

My colleagues use it as a model- for anthrax.

Now, imagine that I was looking to find out how to get anthrax to form spores.

I could simply ask my colleague- because I work in the sort of place where we do that sort of thing. (Don't worry- it's perfectly civilised- they even have a facebook page but, since my ideas don't always align with theirs I'm not saying whom I work for)

 

Now imagine I'm some deranged bomber who actually wants to know how to weaponise anthrax, but doesn't work in a lab.

Well, I guess I could post on a web site

"How do I kill lots of people?"

And, with just a little luck the people whose job it is to worry about that sort of loony would come and arrest me before I caused any trouble.

 

So, imagine I'm just slightly brighter than that- I realise I can't ask about anthrax because it would set off every alarm in the security services.

So I will ask about a different bug- but one which is similar enough that, if it works for B Subtilis, then there's every chance it will work (or at least be a good hint for) B anthracis.

 

I'm not saying that's what this guy is asking about.

But it's probably not a good idea to answer the question because

(1) he might be a terrorist and

(2) someone else, who is a terrorist might see it and cause havoc.

 

And, anyone with a reasonable need to do it would be able to ask one of their peers.

[StringJunky]

Yes, I know it's used as a model.

My colleagues use it as a model- for anthrax.

Now, imagine that I was looking to find out how to get anthrax to form spores.

I could simply ask my colleague- because I work in the sort of place where we do that sort of thing. (Don't worry- it's perfectly civilised- they even have a facebook page but, since my ideas don't always align with theirs I'm not saying whom I work for)

 

Now imagine I'm some deranged bomber who actually wants to know how to weaponise anthrax, but doesn't work in a lab.

Well, I guess I could post on a web site

"How do I kill lots of people?"

And, with just a little luck the people whose job it is to worry about that sort of loony would come and arrest me before I caused any trouble.

 

So, imagine I'm just slightly brighter than that- I realise I can't ask about anthrax because it would set off every alarm in the security services.

So I will ask about a different bug- but one which is similar enough that, if it works for B Subtilis, then there's every chance it will work (or at least be a good hint for) B anthracis.

 

I'm not saying that's what this guy is asking about.

But it's probably not a good idea to answer the question because

(1) he might be a terrorist and

(2) someone else, who is a terrorist might see it and cause havoc.

 

And, anyone with a reasonable need to do it would be able to ask one of their peers.

That makes sense.

[Micro.Pete]

Have you tried using old Bacillus colonies (over 3 days old)? From my experience, Bacillus spp. sporulare on the agar after 3 days fue to nutrient deprivation. Just wait 3-4 days and check the old colonies for endospores, I'm sure you'll see over 90% endospores.

Another thing, you don't always have to special stain your cells to see endospores. Just use Gram staining. After you Gram stain the cells you will see the endospores as a white area inside the bacterial cell (which stained Gram-positive).

[John Cuthber]

Have you tried ...

Did you read the bit about not answering this ?

[Micro.Pete]

Yes and decided to answer anyway. If you don't want people to answer just close the thread.

[Micro.Pete]

Yes and decided to answer anyway. If you don't want people to answer just close the thread.