Microbiology: Total Cell Counts & Viable Cell Counts PDF

Microbiology: Total cell counts and viable cell counts Estimating cell numbers in a microbial Using a Haemocytometer for a total cell count population 1. Place the cover slip on the slide and introduce a sample...

Microbiology: Total cell counts and viable cell counts Estimating cell numbers in a microbial Using a Haemocytometer for a total cell count population 1. Place the cover slip on the slide and introduce a sample of the cell culture into the central chamber of the haemocytometer by using a pipette. Count type Definition Techniques used 2. View the haemocytometer under a microscope and then count the number of microorganisms in 16 Total cell Gives the total Haemocytometer small squares (one medium square). Cells that touch the top and right lines of a square should not be count number of cells Turbidimetry counted. While the cells on the bottom and left side should be counted. present, whether living or dead. 3. In the example shown there are 19 cells in the medium square which has a known volume of Viable cell Considers only living Serial dilution with 0.004mm3. Therefore, in 1mm3 of the sample there will be 19/0.004 cells = 4750 cells. (Note: if the count cells, since these plating sample being investigated had been serially diluted then the dilution factor needs to be taken into are the only ones consideration when calculating the number of cells in the original sample.) Several squares need to be capable of dividing. counted and an average calculated to improve the accuracy of the result. Limitations The sample must be carefully prepared so that the cells are evenly suspended in the solution; and to ensure the sample is sufficiently Turbidimetry dilute to enable counting. Also, there can be counting errors. Living cells, dead cells and particles are counted, therefore overestimation of The turbidity of a culture is a quick and efficient way to total number of cells is likely. estimate the number of bacteria in a liquid medium. This method is much faster than using serial dilution and Serial dilution with plating A haemocytometer slide plating. However, the measurements must be correlated with cell number. This is achieved by determining the A known volume of organism is added to an agar plate, incubated and the colonies are counted. This is a thick slide etched with an turbidity of different concentrations of a given species and It is assumed that one cell gives rise to one colony. This technique makes no allowance for accurate grid of squares. When the constructing and using a standard curve to determine the clumping of cells in the initial inoculum so may lead to an underestimate of the numbers of cover slip is in position, the volume number of cells per ml of sample. microorganisms. Serial dilution is used to provide a final number within a countable range. To of liquid in each square of the grid is A colorimeter or spectrophotometer can be used for this calculate the number of cells in the original inoculum: known. technique. Number of colonies on plate x reciprocal of dilution factor = number of bacteria/mm3 1 ml 1 ml 1 ml 1 ml 1 ml Haemocytometer grid Original Inoculum Notice there is one large square in the middle of the Dilutions grid. This is subdivided into 1:10 1:100 1:1000 1:10.000 1:100.000 25 medium sized squares. Each medium sized square 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml is subdivided into 16 small squares. Plating 1 mm 1:10 1:100 1:1000 1:10.000 1:100.000