Cell Counting with the Sedgewick-Rafter Chamber and Whipple Micrometer Disc
|Author: Stephen M. Gittleson and Mandanna Ganapathy|
|Affiliation: Fairleigh Dickinson University, School of Natural Sciences 1000 River Road, Teaneck, NJ 07666. E-mail: email@example.com|
|Source: Protocol Online|
|Date Added: Tue Mar 08 2011|
|Date Modified: Sat Apr 09 2011|
|Abstract: A step by step protocol to standardize the counting of cells using the Sedgewick-Rafter Chamber and Whipple Micrometer Disc. Statistically significant counts of protists in cultures of millions/ml may be obtained by directly counting less than 0.1% .|
Summary. A step by step protocol to standardize the counting of cells using the Sedgewick-Rafter Chamber and Whipple Micrometer Disc. Statistically significant counts of protists in cultures of millions/ml may be obtained by directly counting less than 0.1% .
Abbreviations: SRC-Sedgewick-Rafter Chamber, WMD-Whipple Micrometer Disc, P. papillata-Polytomella papillata, CC-Calibration Constant, Confidence Interval (CI),Introduction
We identify and control sources of error such as those related to geometry of the SRC, sample volume, method of dispensing the sample and settling behavior of the preserved organisms. The The flagellate P. papillata is referred to here but this cell counting technique should be applicable to other free-living protozoa, algae, and phytoplankton.Procedure
The SRC without grid with cover glasses is available from Wildlife Supply Company, 95 Botsford Place, Buffalo NY 14216 (www.wildco.com ). The WMD is available from Max Levy Autograph, Inc., 220 W. Roberts Ave, Philadelphia PA 19144 (www.maxlevy.com ). Each step of this protocol is supplemented with comments in an attempt to minimize confusion over ‘obvious’ manipulations that may influence the repeatability and accuracy of this technique. Sample data, calculations and statistics appear in the results and discussion.
Comments for step 1: This calibration procedure establishes the relationship of the number of organisms counted under the WMD grid to the total number of organisms in the sample within the SRC. A 10X ocular and 10X objective is used here, but other lens magnifications appropriate for counting organisms of different sizes, can be selected. A separate calibration is necessary for each different arrangement of lenses and for different microscopes . The WMD contains a grid composed of 100 equivalent squares. Dimensions of the squares are measured with a slide micrometer. In our arrangement, the side of each square measures 0.16 mm. Thus, each square has an area of 0.0256 mm2. . Since the SRC measures 20 mm by 50 mm it has an area of 1000 mm2 . The portion of the SRC covered by one square of the WMD grid is 1000 mm2 divided by 0.0256 mm2 . Therefore, the particular CC for our setup is 39,062 which is the number of WMD squares needed to cover the entire SRC.
Placement of the cover glass limits the volume to 1 ml.
The final calculation for cell count in the original culture is carried out using the following formula: C = (DF) (NC) (CC) / S
C-cells/ml, DF-dilution factor, NC-cells counted, CC-calibration constant, S-number of WMD squares containing NC
For example, using the mean NC between counts 1 and 2 in Tables 1 and 2 C = (4) (314) (39,062) / 50 C = 981,237
Thus, statistically significant counts of organism numbers in cultures of millions/ml may be obtained by directly counting less than 0.1%.