bacterial cell count by counting chamber

Bacterial Cell count by counting chamber

 AIM: To count the number of microorganisms in a given sample by using counting chamber.

 INTRODUCTION: For unicellular microorganisms, such as bacteria, the reproduction of the cell reproduces the entire organism. Therefore, microbial growth is essentially synonymous with microbial reproduction. To determine rates of microbial growth and death, it is necessary to enumerate microorganisms, that is, to determine their numbers. It is also often essential to determine the number of microorganisms in a given sample. For example, the ability to determine the safety of many foods and drugs depends on knowing the levels of microorganisms in those products. A variety of methods has been developed for the enumeration of microbes.  Direct Count of Cells by counting chamber: Direct microscopic counts are performed by spreading a measured volume of sample over a known area of a slide, counting representative microscopic fields, and relating the averages back to the appropriate volume-area factors. Specially constructed counting chambers has the ability to count a defined area and convert the numbers observed directly to volume makes the direct enumeration procedure relatively easy. Direct counting procedures are rapid but have the disadvantage that they do not discriminate between living and dead cells. This method is used to assess the sanitation level of a food product and in performing blood cell counts in hematology and microbial cells in microbiology.

Materials required:

Direct Count Using a Counting Chamber, Bacterial suspension, lab Counting chamber, Pipettes.

Calculating cell count

The total number of cells per microliter of sample can be calculated from the number of cell counted and area counted. This is because the ruled areas of the chamber contain an exact volume of diluted sample. Since only a small volume of diluted sample is counted, a general formula must be used to convert the count into the number of cells/microliter.

The dilution factor used in the formula is determined by the blood dilution used in the cell count. The depth used in the formula is always 0.1. The area counted will vary for each type of cell count and is calculated using the dimensions of the ruled area.

The smallest square has an area of 0.0025 mm² therefore each main square has an area of 0.04 mm² (0.0025 x 16 = 0.04).

The depth of the chamber is 0.100 mm (space between the glass slide and cover slip) then the volume is calculated as:

(0.04) x (0.100) = 0.004 mm³ = 0.004 μl

To calculate the amount of cells in 1μl a rule of three is applied. If we have X amount of cells in 0.004μl, how many cells are in 1μl?:

# cells -------------0.004 μl

? --------------1μl

Then:

Cells in 1μl = (number of cells in a main square)(1μl) /0.004

Cells in 1 ml = (cells in 1μl) x (1000)

Example:

Lets calculate total WBC count by using Neubauer counting chamber.
Number of cells counted = N = 150 (suppose)

Area Counted = 1 mm² x 4 = 4 mm² (area of four large corner squares)

Depth = 1/10 mm

Dilution = 1:20

Hence WBC/Cubic mm of Whole Blood = N x 50 = 150 x 50 = 7,500

 Procedure:

 1. Clean a counting chamber with lens paper and then place it on the microscope stage.

2. Using the 40X objective find the ruled area and arrangements of larger squares and their small square subdivisions.

 3. Shake the sample suspension to distribute the cells evenly. Take out the counting chamber without changing the focus on the 40X objective. Place a coverslip over the counting chamber.

4. Using a transfer pipette, transfer some of the suspension to the groove of the counting chamber to fill the chamber by capillary action.

5. Carefully place the counting chamber back onto the microscopic stage and observe the cells under 40X.

6. Count the number of cells in at least 50 of the small squares. If cells fall on a line, include in your count those on the top and left-hand lines and exclude those on the bottom and right-hand lines.

7. Calculate the average number of cells per Small Square. Then calculate the number of per ml by dividing the average number of cells per Small Square by the volume of each small square which is 0.00025 µl. If diluted the sample, also multiply the results by the dilution factor to determine the concentration of cells in the original sample. Record the calculations and results.

Result:

Direct Counts Using A Counting Chamber

Total number of yeast cells counted __________________

Average number of cells per counting squares _____________

Volume associated with each counting square __________________

Dilution factor (if any)_____________

Concentration of yeasts in original sample____________________