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spot plate method - (Mar/12/2010 )

I'm starting a new experiment that involves microbiology...something that's been a while for me. Anyway, my basic question is how do you perform and quantify bacterial cells in the spot plate method? I've enumerated my dilutions by the spread plate, basically pipetting 100ul onto a plate and spreading it evenly to count the next day. Is it similar but you can count all your dilutions on fewer plates? A reference would be nice if you have one.

Thanks!

-archercr-

check the paper in the attechment.

However I do not really see what your problem is.
Attached File

-pito-

No reference but you just plate 10 ul spots - you can divide your plate into 8 sections, and put 3 spots of the same dilution into each section so that you have replication. A 10 ul spot of a -1 dilution = a -3 dilution. Then you average your 3 spots.

-microgirl-

Thanks. I've read the Gaudy paper, but I'm still unclear on the proper way to enumerate. It says to place 20ul of your dilutions on the plate, so should I use the same calculation to determine CFU/ml as in the standard plate method? (original cell density = #colonies/(volume plated)(dilution factor))

-archercr-

I think a 20 ul spot is a lot, if it takes too long to soak in/dry then your colonies tend to all be at the outside edge - a 10 ul spot also makes your calculation easier. Just multiply your colonies counted by your dilution. So if you made a -1 dilution and plated 10 ul of that, you've effectively made a -3 dilution. If you count an average of 4 colonies per spot (you've plated 3 spots of this dilution) then you have an original cell density of 4x10^3.

archercr on Mar 17 2010, 12:00 PM said:

Thanks. I've read the Gaudy paper, but I'm still unclear on the proper way to enumerate. It says to place 20ul of your dilutions on the plate, so should I use the same calculation to determine CFU/ml as in the standard plate method? (original cell density = #colonies/(volume plated)(dilution factor))

-microgirl-

microgirl on Mar 18 2010, 03:03 PM said:

I think a 20 ul spot is a lot, if it takes too long to soak in/dry then your colonies tend to all be at the outside edge - a 10 ul spot also makes your calculation easier. Just multiply your colonies counted by your dilution. So if you made a -1 dilution and plated 10 ul of that, you've effectively made a -3 dilution. If you count an average of 4 colonies per spot (you've plated 3 spots of this dilution) then you have an original cell density of 4x10^3.

archercr on Mar 17 2010, 12:00 PM said:

Thanks. I've read the Gaudy paper, but I'm still unclear on the proper way to enumerate. It says to place 20ul of your dilutions on the plate, so should I use the same calculation to determine CFU/ml as in the standard plate method? (original cell density = #colonies/(volume plated)(dilution factor))



Microgirl,

Can you explain me why a 10Ál spot of a dilution -1 is a -3 dilution?

In my opinion:
If you have a bottle(100Ál or whatever) with -1 diluted sample and you plate out 10Ál of that bottle on a plate and lets say you plated out 1 10ÁL sample (to make it easy) and you notice 4 colonies then you have 4 colonies per 10Ál wich is -1 diluted thus 40 colonies for the undeluted sample.

I really dont understand how a -1dilution becomes a -3 dilution ?


Can you clarify this?

-lucilius-

lucilius on Mar 18 2010, 08:52 AM said:

microgirl on Mar 18 2010, 03:03 PM said:

I think a 20 ul spot is a lot, if it takes too long to soak in/dry then your colonies tend to all be at the outside edge - a 10 ul spot also makes your calculation easier. Just multiply your colonies counted by your dilution. So if you made a -1 dilution and plated 10 ul of that, you've effectively made a -3 dilution. If you count an average of 4 colonies per spot (you've plated 3 spots of this dilution) then you have an original cell density of 4x10^3.

archercr on Mar 17 2010, 12:00 PM said:

Thanks. I've read the Gaudy paper, but I'm still unclear on the proper way to enumerate. It says to place 20ul of your dilutions on the plate, so should I use the same calculation to determine CFU/ml as in the standard plate method? (original cell density = #colonies/(volume plated)(dilution factor))



Microgirl,

Can you explain me why a 10Ál spot of a dilution -1 is a -3 dilution?

In my opinion:
If you have a bottle(100Ál or whatever) with -1 diluted sample and you plate out 10Ál of that bottle on a plate and lets say you plated out 1 10ÁL sample (to make it easy) and you notice 4 colonies then you have 4 colonies per 10Ál wich is -1 diluted thus 40 colonies for the undeluted sample.

I really dont understand how a -1dilution becomes a -3 dilution ?


Can you clarify this?

I'll try to explain! Let's say you don't dilute at all, just plate 10 ul straight from your culture and get 4 colonies. That would actually be 400 cfu/ml - you didn't plate the whole ml, just 10 ul of it - so it's a "plate dilution" of -2. So if you made a -1 "tube dilution" and plated the whole 1 ml of it, it would still only be a -1 dilution. But since you're plating 10 ul of it, it works out to be a -3 "plate dilution" - your -1 tube dilution plus your -2 plate dilution. Hope that helps.

-microgirl-

original cell density = #colonies/(volume plated)(dilution factor)

If you plug into that formula, it seems to work out and account for that -2 factor, regardless of the volume you plate. Makes sense to me

-archercr-

Lucilius is right and so is microgirl.

However you both use an another way or idea of working

Microgirl uses a strange idea of dilutions.


In my opinion a dilution is something you dilute.. not something you didnt put on a plate completely.

If you plate out 10Ál of a 1ml solution then you didnt dilute the 1ml, you just didnt plate it out completely.
But thas my opinion.
I would then state: you plated out

So I would not use a term like "plate dilution".

Mayeb you are taught to use that but for me the explenation lucilius used makes more sence.

If you have a sample of 1 ml and you plate out 10Ál then you plated out 1/100the of the total volume.
So if you have 4 colonies you have 4 colonies in 10ÁL what makes 4*100 (400) colonies in 1 ml.

So indeed you can say that "the 10Ál of 1ml" is a 10^-2 dilution however I find this a rather strange way of working. But I think it just depends on how you work the best or how you interpret it.


Its always dangerous working with term like "dilution" and losing sight of the why and what it is.

So archercr formula is ok if you regard the "partial" use of a sample as a dilution.



PS. lucilius and microgirl: the discrepancy between your calculation is just the fact that lucilius works with a 100Ál 'stock' solution and mircogirl, you use 1ml.

-pito-