16 replies to this topic

[Curtis]

my colonies on agar plates are sometimes small and very close to each other, or sometimes big and separate?

why does this happen? I'm using TOP10 and incubate 16-18 h at 37 C. It's very hard to pick small colonies.

[jiajia1987]

I do get big and small colonies at times. And I believe it is due to how you spread your dilution on the plates. But it is okay. It is very simply to pick small colonies. Here is one trick. Buy some toothpicks and place them in a beaker. Cover it with aluminium and send it for autoclaving. AFter that, you can always pick your tiny colonies with the toothpick and drink the toothpick into your culture tube containing LB broth and the appropriate antibiotics!

[swanny]

I do get big and small colonies at times. And I believe it is due to how you spread your dilution on the plates. But it is okay. It is very simply to pick small colonies. Here is one trick. Buy some toothpicks and place them in a beaker. Cover it with aluminium and send it for autoclaving. AFter that, you can always pick your tiny colonies with the toothpick and drink the toothpick into your culture tube containing LB broth and the appropriate antibiotics!

When you do that, remember to get cheap toothpicks. The more expensive brands are often impregnated with disinfectants... not goo if you're wanting to keep the bugs alive!!

[jiajia1987]

I do get big and small colonies at times. And I believe it is due to how you spread your dilution on the plates. But it is okay. It is very simply to pick small colonies. Here is one trick. Buy some toothpicks and place them in a beaker. Cover it with aluminium and send it for autoclaving. AFter that, you can always pick your tiny colonies with the toothpick and drink the toothpick into your culture tube containing LB broth and the appropriate antibiotics!

When you do that, remember to get cheap toothpicks. The more expensive brands are often impregnated with disinfectants... not goo if you're wanting to keep the bugs alive!!

Good one!

[eldon]

I do get big and small colonies at times. And I believe it is due to how you spread your dilution on the plates. But it is okay. It is very simply to pick small colonies. Here is one trick. Buy some toothpicks and place them in a beaker. Cover it with aluminium and send it for autoclaving. AFter that, you can always pick your tiny colonies with the toothpick and drink the toothpick into your culture tube containing LB broth and the appropriate antibiotics!

When you do that, remember to get cheap toothpicks. The more expensive brands are often impregnated with disinfectants... not goo if you're wanting to keep the bugs alive!!

Good one!

has nothing to do with how you spread your cells...and everything to do with plasmid copy number and the insert (size, complexity, etc.).

think about it.

cells with fewer plasmid copies will grow slower when challenged with same concentration of antibiotic over cells with a very high number of copies...toxicity of insert will affect growth in the same manner, and longer inserts take a little longer to replicate.

I do not know what TOP10 means or stands for, I suppose its the way how you spread your dilution on the plates?

how can you not know what TOP10 is? if you don't know something, don't offer bunk advice or at least look it up. good luck with that.

Edited by eldon, 03 July 2009 - 06:07 AM.

[Curtis]

lol @ eldon

thank you!

[Dominic]

how can you not know what TOP10 is? if you don't know something, don't offer bunk advice or at least look it up. good luck with that.

ooooooohhhh - angry

d

[klinmed]

my colonies on agar plates are sometimes small and very close to each other, or sometimes big and separate?

why does this happen? I'm using TOP10 and incubate 16-18 h at 37 C. It's very hard to pick small colonies.

I´m sure the answer is very simple. Not due to copy number or anything else "fancy". You describe small colonies when they are close together and large well isolate ones.

Colonies close together compete for nutrients and poison each other with their waste products. Hence slow growing bugs and small colonies. Well isolated colonies don´t have this problem (lots of space to grow, plenty of food and a large area for waste to diffuse away from colony.

[jiajia1987]

how can you not know what TOP10 is? if you don't know something, don't offer bunk advice or at least look it up. good luck with that.
[/quote]

I don't think the person means that he don't know what TOP10 cells are. I guess he/she doesnt know what TOP10 stands for; what the abbreviation is. Don't scare him/her off like this!!

[eldon]

Colonies close together compete for nutrients and poison each other with their waste products. Hence slow growing bugs and small colonies. Well isolated colonies don´t have this problem (lots of space to grow, plenty of food and a large area for waste to diffuse away from colony.

So explain a bacterial lawn of growth. Nutrients on an LB plate are in excess and can support a lawn of bacteria for up to a month or longer.

[klinmed]

Colonies close together compete for nutrients and poison each other with their waste products. Hence slow growing bugs and small colonies. Well isolated colonies don´t have this problem (lots of space to grow, plenty of food and a large area for waste to diffuse away from colony.

So explain a bacterial lawn of growth. Nutrients on an LB plate are in excess and can support a lawn of bacteria for up to a month or longer.

Very easily!

A lawn is made up of a huge number of small colonies (due to the large number of cell plated). The small colonies are all suffering from nutrient starvation and the toxicity of waste products.

If, as you claim "nutrients on an LB plate are in excess....." the bacterial lawn would continue to grow for " a month or longer" and fill the dish and eventually flood over the top of the dish!

The lawn MAY survive for a some time in the dish, but the bugs are not growing actively...

[HomeBrew]

I agree with klinmed -- "not growing" is not the same as "dead". I believe the large vs small colony phenotype you're seeing is simply a matter of varying cell density on the plate, and thus is correctable by proper dilution.

You can test this for yourself easily enough -- carefully pick a single small colony and streak it to a plate in the usual manner, such that a quandrant of the plate will have well-isolated single colonies.

If the size of the colonies is a function of plasmid copy number or other such genotypic influence, the well-isolated colonies in this area of the plate will be "small". If it's just a matter of how crowded the neighborhood was on the original plate, these colonies will be "large".

My money's on "large"...

[jiajia1987]

I agree with klinmed -- "not growing" is not the same as "dead". I believe the large vs small colony phenotype you're seeing is simply a matter of varying cell density on the plate, and thus is correctable by proper dilution.

You can test this for yourself easily enough -- carefully pick a single small colony and streak it to a plate in the usual manner, such that a quandrant of the plate will have well-isolated single colonies.

If the size of the colonies is a function of plasmid copy number or other such genotypic influence, the well-isolated colonies in this area of the plate will be "small". If it's just a matter of how crowded the neighborhood was on the original plate, these colonies will be "large".

My money's on "large"...

my money is on large too.

[Functional Screens]

One more thing to add..............recombination and toxicity by gene expression
For example, if you transform a retro or lenti vector encoded hTERT into competent cells, you will see all sizes of colonies.
For example, if you transform a lentiviral vector into TOP10, the bigger colonies are recombinants in most of cases.
For example, if you transform pcDNA3.1 expressing a transcription factor, the bacteria cannot take it since the gene expression (leak expression from CMV promoter) makes the bacteria sick.
So, size matters, bigger colony might not be better sometimes.

Edited by Functional Screens, 08 July 2009 - 07:36 AM.

[klinmed]

One more thing to add..............recombination and toxicity by gene expression
For example, if you transform a retro or lenti vector encoded hTERT into competent cells, you will see all sizes of colonies.
For example, if you transform a lentiviral vector into TOP10, the bigger colonies are recombinants in most of cases.
For example, if you transform pcDNA3.1 expressing a transcription factor, the bacteria cannot take it since the gene expression (leak expression from CMV promoter) makes the bacteria sick.
So, size matters, bigger colony might not be better sometimes.

Totally agree with Functional Screens. At the far extreme, a leaky vector and a toxic expressed product = no colonies at all containing intact plasmid.

IF your colonies are well spaced, and they are of different sizes, ALWAYS pick a mix of small and large for subsequent screening.

Eldon (earlier) has suggested that different colony size on a single plate is dependent on copy number. This is nonsensical! After a short period of growth, all bugs containing a particular plasmid will contain roughly the same number of plasmid copies. How many depends on the origin of replication. For example with a ColE1 origin ca 400/ cell while with p15A ca 10.