u can determine the total load by determining cfu count by making serial dilutions of your sample & then spreading them , this method will give u cfu/ml , which u can use to estimate the total load depending upon the total volume/weight of the sample , ofcourse these methods will give u approximate numbers ,there are no methods to detect exact accurate number of organisms in a sample . u have to combine two -three methods , u can take bacterial count by plating on nutrient agar , plating same sample for fungal cultures on sabroaud's agar , at the same time take microscopic counts ,because the number u will get on plates will be of culturable bacteria , nonculturable will be seen only under microscope ,

What is the purpose and facility available.

Can do the counting by Petroff Hauser chamber or simply using breed count method.But actually if you intend to do the viable cell count then go for estimation of CFU.

actually, we Determine using CFU's for any analysis....

but is there any possibility to determine the number of cells present ?

Hi Sudheer

I guess you can also use a hematocytometer for counting the number of cells present..see the link for details

http://en.wikipedia.org/wiki/Hemocytometer

Dear Ajay Saini,

bacterial cells need to be stained prior to it..but with wat do u stain them before counting in that chamber...??

Hi Sudheer

yes you are right..the bacterial cells need sto be stained and there are several options available as this is an established method...Infact you can even distinguish between 'live' and 'dead' cells using certain stains..

For example 'Trypan blue' is commonly used and some fluorescence stains are aslo able to differentiate between live and dead cells...(check on WWW for this)..

One link for you: http://www.amresco-inc.com/media.acux/c40e26e1-9c2d-4bf4-9f39-7d242551680e

Dear Ajay Saini,

My dear friend, you are getting it wrong...am not speaking about gram staining for microbes or tryphan blue staining for human cells....

I am in need of information about the count of microbial cells in an sample..!! for example, say, water sample..!! by using the above technique, we cannot determine the total bacterial load..!!

u can determine the total load by determining cfu count by making serial dilutions of your sample & then spreading them , this method will give u cfu/ml , which u can use to estimate the total load depending upon the total volume/weight of the sample , ofcourse these methods will give u approximate numbers ,there are no methods to detect exact accurate number of organisms in a sample . u have to combine two -three methods , u can take bacterial count by plating on nutrient agar , plating same sample for fungal cultures on sabroaud's agar , at the same time take microscopic counts ,because the number u will get on plates will be of culturable bacteria , nonculturable will be seen only under microscope ,

Dear Apeksha Mohile,

perfect answer ....!!

Traditionally, you can made a vital staining (or "in vivo staining"), using, for example, methylene blue of an aliquot of bacterial growth media and count live bacteria number in a Neubauer camera.Other alternative is the use of liquid particle counter, to detect bacterial growth in liquid systems.

Hi Sudheer

I am sorry for the confusion...but as per your query it seems you are interested in methods of cell counting other than 'estimating CFUs'....in this context methods using such chambers as suggested by some people above seems appropriate....and 'hematocytometer' has certainly evolved a long way .....

You are best person to decide the method to use for your scientific problem

Nicole Trombert,

Thank you for your response...i wud be happy if u cud send me some references if u hav any...!!

One has to be clear conceptually regarding the question.What is CFU (colony forming units)?

the organisms would produce colonies corresponding to its no. therefore we can directly say that whatever is the no. of organisms present is equal to the no. of colonies.Besides it is the best method for determination of cells that are alive.

Secondly the "Petroff Hauser method/haemocytometer or breed count method is used for counting the no. of microorganisms directly through the microscope."

You have to read the books to make yourself clear on the technique.

Besides as others have mentioned the use of vital stains,just mix the culture with appropriate amount of these stains and then observe in microscope you can distinguish between live and dead cells.

You can also do the direct counting in haemocytometer (without staining) you just need to close the diaphragm to maximum as this would make the transparent cells visible due to refraction of light.

You can use flow cytometer or rely on turbidometeric methods to develop the corelation between O.D and no. of cells(this again needs the use of haemocytometer).

As far as the counting of cells in water sample is concerned,we perform MPN determination to count the no. of E.coli present per 100ml of sample.

Just read these and apply to formulate your owns methodology.Most of the good Basic Microbiology books covers these.Moreover for understanding the techniques you can read the practical books(Specifically for Haemocytometer counting)-Kani Mukherjee-Three volumes(DMLT books).You can get sufficient information on the net too.

For protocols you can see the site- www.vadlo.com,you tube, biosolutionsblog.com &

kbiotech.com(for notes,books online etc.)

Direct microscopic counts can be done using the BactLight system for live and dead cells. You can also use flow cytometry for the direct counts using this stain. For total counts without distinguishing between live or dead, acridine orange is sometimes used. You will still have to perform a dilution series to ensure that you have the optimum number of bacteria per field. This method does require a good eye and an epifluorescent microscope.

Sudheer Aluru,

vital stain protocols are too old and really simples. For example, Ruziicka recommends the use of a staining solution

made by mixing equal volumes of 0.05 per cent neutral red and 0.05 per cent methylene blue solutions in distilled water. A few drops of this mixture are spread over the surface of a clean slide and allowed to evaporate at 350C. This leaves a thin, dry film of dye on which a drop of the bacterial suspension to be investigated is placed and examined under a cover-glass in a Petroff-Hauser counting chamber. Within a short time, living cells take on a violet color in which the red tone predominates. Dead cells show a predominance of blue tone.

Respect liquid particle counter or, specifically, coulter counter, a reference that could helps you is http://www.ncbi.nlm.nih.gov/pubmed/3536882 and http://www.ncbi.nlm.nih.gov/pubmed/16282312