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micro micro
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11 years ago
When a protocol instructs to transfer 10 mL of bacteria, what does it mean exactly? I imagine it is being assumed that bacteria is cultured in broth but then again, how is 10 mL a quantity for bacteria? Shouldn't the optical density be stated? I'm finding this in several protocols: "transfer 50 uL of cell solution to 2mL of medium" or "resuspend pellet of 10 mL cell culture".
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wrote...
#1
Donated
11 years ago
Quote from: micro (11 years ago)
When a protocol instructs to transfer 10 mL of bacteria, what does it mean exactly? I imagine it is being assumed that bacteria is cultured in broth but then again, how is 10 mL a quantity for bacteria?

It depends on your bacterial concentration. Have you done a cell count yet? Usually what you do is take a pipette, place it on a microscope slide that has a grid, and then count how many you see per area of the grid. There are other methods, and some are outlined in the source below.

The slide should look like this up-close:



and it fits on your microscope:



You can also get an idea of the number by reading the colony number, as mentioned below Downwards Arrow
Source  http://www.disknet.com/indiana_biolab/b038.htm
micro Author
wrote...
#2
11 years ago
But the protocols don't specify a cell count either. Its just "10 mL of cell sample" and thats it. That's what I find strange about it.
wrote...
#3
Donated
11 years ago
Then they are missing something in the protocol. Does the protocol include how to grow the bacteria? Does it tell you to dilute it?
micro Author
wrote...
#4
11 years ago
One starts: "in microfuge tubes, spin down 0.1 mL of uninduced cells grown to near saturation".

Second one: "place the tube of desired cells in the hood, spin down the cell solution to the bottom of the tube. Transfer 50 uL of the cell solution into 2 mL broth.

Third one: "Resuspend pellet of 10 mL cell culture in 1 mL lysis buffer".
wrote...
#5
Donated
11 years ago
Those instructions seem pretty clear. I'm guessing it's grown in a petri dish. Take a swab of the colony that's grown and place it in the microfuge tube. You can dilute it however you like, but don't go past the tube limit.

That's the only way I see it.
micro Author
wrote...
#6
11 years ago
These are the actual protocols I'm referring to; posted here by a forum member:

http://labs.fhcrc.org/hah...h/coli_total_protein.html
http://wolfson.huji.ac.il...ysis_Bacterial_Cells.html

I highly doubt you can take as many bacteria as you like since I'm sure you need to have a certain amount of protein (in these particular protocols) to have good results.
wrote...
#7
Donated
11 years ago
Quote from: micro (11 years ago)
http://labs.fhcrc.org/hah...h/coli_total_protein.html
http://wolfson.huji.ac.il...ysis_Bacterial_C ells.html

Undecided Both links didn't work? they were cut off for some reason.

Quote from: micro (11 years ago)
I highly doubt you can take as many bacteria as you like since I'm sure you need to have a certain amount of protein (in these particular protocols) to have good results.

The more bacteria, the more protein. The more protein, the better it is.
micro Author
wrote...
#8
11 years ago
The more protein, the better it is....not really when you are performing electrophoresis.

http://wolfson.huji.ac.il/purification/TagProteinPurif/Lysis_Bacterial_Cells.html
http://labs.fhcrc.org/hahn/Methods/biochem_meth/coli_total_protein.html
wrote...
#9
Donated
11 years ago
Quote from: micro (11 years ago)
The more protein, the better it is....not really when you are performing electrophoresis.

No, I meant, the more protein quantity you have, the better it is for you, because they have more wiggle room to make mistakes and create more replicates.

http://wolfson.huji.ac.il/purification/TagProteinPurif/Lysis_Bacterial_Cells.html

There's information missing in this protocol, starting with step 1. It could be that it's a continuation of some other protocol on their website.
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