Problem in Determination of Total Bacterial Cells - (Nov/10/2013 )

Dear all, 

I  am looking for a protocol which can use to determine total number of both viable and non-viable Gram-positive bacterial cells. I have an idea by using crystal violet for the bacterial cells staining, incubate bacterial cells with 1% crystal violet and count under microscope. However, I notice not only bacterial cells were being stained, even other materials (such as sugar, maltodextrin) also being stained. Besides that, the bacterial cells are difficult to count using microscope, even is power 100x. 

What do you think? Did anyone have any other counting method can suggest to me? Please advice. 

Thank you. 

How about doing a conventional Gram stain - it won't tell you viable numbers, but you could perhaps stain with a dye that is live uptake and do a count before fixing and staining.

Thanks for your suggestion. Right now I am incubating the sample with crystal violet, follow by gram's iodine, however, iodine will form crystal when contact with crystal violet and make it a big trouble when doing cell count under microscope. Do you think I should include the step of ethanol washing? 

Secondly, in my sample contains sugar, citric acid, etc. Do you have any idea how can I extract all the bacteria from sample without remaining any other materials? 

Thanks. 

There are a number of methods used for counting total bacteria - one is to use a hemocytometer, another would be to do a conventional Gram stain (including the destain steps)  using a defined volume and count off that.  If you do a normal Gram stain, there shouldn't be problems with crystal formation.  The salt and sugar crystals should be removed by the washing steps.

For the living cells it is probably easiest to do viable plate counts (i.e. spread a defined volume and work out pfu/ml). 

Can you use fluorescence microscope? If so, try FDA/PI staining (fluorescein diacetate/propidium iodide). You will see viable cells green, and dead ones in red

Dear bob1,

do you mind to share the protocol? Different research papers have showed different incubation time, and I have tried off most of it but no one can provide me a reliable result. Besides that, do you think normal microscope with power 100x is suitable to use for cell counting? 

Thank you 

Protocol for which procedure?  Any basic microbiology techniques text will tell you how to do all 3 techniques I mentioned (google is your friend).  100x objective (1000x total mag) is about the max for light microscopy and should be fine for most bacteria.  Even mycoplasma are visible quite easily at 1000x if stained appropriately.

Yup. I have went through most of the protocol from internet. However most of it are guiding on the technique of gram stain on a slide, there is no protocol is mentioning about conventional gram stain for total bacterial count. Thank you.  

No there won't be, but if you spread a defined volume on the slide and then do a count of all the cells in that volume...

Suggest a hemocyometer - you could try a live dead stain.  Never seen spreading a volume on a slide and counting every stained field.  Doubt that would be practical.