11 replies to this topic

[QQ_lin]

I am now involving a study regarding soil bacteria. Is it compulsory for me to do serial dilutions for the soil sample before pouring the aliquot onto the agar medium? And what is the difference between mixing the soil sample with Phosphate Buffer Saline (PBS) instead of using sterile distilled water? Thanks.

Edited by QQ_lin, 16 July 2009 - 05:31 AM.

[pito]

 QQ_lin, on Jul 16 2009, 03:28 PM, said:

I am now involving a study regarding soil bacteria. Is it compulsory for me to do serial dilutions for the soil sample before pouring the aliquot onto the agar medium? And what is the difference between mixing the soil sample with Phosphate Buffer Saline (PBS) instead of using sterile distilled water? Thanks.

You do not really need to make a dilution, it all depends on how many cells you have in your sample.
You could make a dilution and plate out every dilution and see till what dilution you still have a good growth on your plates.

I you have almost no cells in your sample, then you do not want to dilute.

And PBS could favor your cells(its isotonic), but all depends on what cells you have.
You could try one sample with PBS and another one with distilled water. If both works out the same then simply use the cheapest one.

Edited by pito, 16 July 2009 - 06:08 AM.

[Dominic]

pbs - lucozade for cells

[GeorgeWolff]

Pito's resonse was a good one but Lucozade? PBS is hardly an energy drink. The idea is that it offers isotonic stability to cells that might otherwise by prone to lyse under hypotonic conditions. There's no sugar or signficant additonal nutrients.

Gratuitous comparison to Lucozade aside, what do you want to understand about the microbes in the soil sample? Total culturable count? Many of the bacterial species in sopil won't grow well or at all in culture and fungal biomass is not correctly estimated by culture counts.

Edited by GeorgeWolff, 16 July 2009 - 08:59 AM.

[Dominic]

didnt lucozade sell itself as being isotonic? or is that the wrong drink?

[pito]

 Dominic, on Jul 17 2009, 02:22 PM, said:

didnt lucozade sell itself as being isotonic? or is that the wrong drink?

I think it depends on what "kind" you buy because Lucozade Hydro Active is not isotonic but hypotonic.

Why do you come up with this Lucozade anyway?
Is it cheaper to buy then to make some PBS?

[GeorgeWolff]

Lucozade includes sugar syrup and other substrate whereas PBS will only offer dilute salt.

Ingredients in Lucozade
Carbonated Water, Glucose Syrup (26%), Citric Acid, Lactic Acid, Flavourings (including Caffeine), Preservatives (Sodium Benzoate, Sodium Bisulphate), Antioxidant (Ascorbic Acid), Colour (Sunset Yellow).

But back to the initial question - soil itself will offer more solutes than the little in PBS and what would be the purpose of buffering in dilution?
Stil - as pito said - try it both ways.

[QQ_lin]

Really thanks for your reply, Pito and also the others. I really appreciate it. Actually I want to see what types of bacteria colonies that I can get from the soil sample that I collected from different areas so that I can do isolation for those that I am interested in.

[pito]

 QQ_lin, on Jul 18 2009, 05:13 PM, said:

Really thanks for your reply, Pito and also the others. I really appreciate it. Actually I want to see what types of bacteria colonies that I can get from the soil sample that I collected from different areas so that I can do isolation for those that I am interested in.

If that is the case, then do not dilute.
See what you get first and then you can start thinking about making dilutions etc..

[GeorgeWolff]

I'm with pito of this. Try direct cultivation - you may otherwise miss some of the bugs that require cofactors (e.g. terragens factor).

[Tanja N]

I am working on cultivation of some soil bacteria also, and I use serial dilutions when plating and I am getting from 100 to 350 colonies per sample. Also, I would like to recommend TG medium for plating, since I have tried to use many different media, and this one gave me best results. It is a little bit time consuming process to make it, but as I have already said, it works. This is the paper where you can find recipe: Mierob Ecol (1989) 17:181-192 MICROBIAL ECOLOGY C) Springer-Verlag New York Inc. 1989. Phenotypical Divergences between Populations of Soil Bacteria Isolated on Different Media.
Roald Sorheim, Vigdis Lid Torsvik, and Jostein Goksoyr.

Good luck!
T.

[QQ_lin]

Thanks for your advices, Pito and GeorgeWolff. I will try that out. Hopefully I can get the best result for my cultivation. Also thank you, Tanja. Thanks for sharing useful information with me. I really appreciate it. Have a nice day, friends!