Measuring and Counting with a Light Microscope David

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Measuring and Counting with a Light Microscope David R. Caprette, Ph. D. Bio. Ed

Measuring and Counting with a Light Microscope David R. Caprette, Ph. D. Bio. Ed Online

Stage Micrometer 0. 01 mm/division 0. 1 mm/division 2 mm Scale on mirrored surface

Stage Micrometer 0. 01 mm/division 0. 1 mm/division 2 mm Scale on mirrored surface – left end with fine divisions Not all types are mirrored Bio. Ed Online www. Bio. Ed. Online. org

Estimating Field Diameter of a Microscope (1) Set up so that the finely divided

Estimating Field Diameter of a Microscope (1) Set up so that the finely divided part overlaps one edge of the field (2) Line up major division on opposite edge Field diameter = 0. 52 mm Bio. Ed Online www. Bio. Ed. Online. org

Estimating Number of Objects Per Unit Area Stomata from leaves of Japanese privet (Ligustrum

Estimating Number of Objects Per Unit Area Stomata from leaves of Japanese privet (Ligustrum japonicum) Reject Accept Reject Count: 13 stomata in field (4 overlapping stomata accepted, 3 rejected Bio. Ed Online www. Bio. Ed. Online. org

Calibrating an Eyepiece Reticule Ocular micrometer scale and appearance through the eyepiece Apparent field

Calibrating an Eyepiece Reticule Ocular micrometer scale and appearance through the eyepiece Apparent field of view Ocular scale Stage micrometer scale Line up ocular micrometer scale with part of stage micrometer scale. At left, 50 divisions cover 1 mm, giving calibration 0. 02 mm/division (20 µm/div). Bio. Ed Online www. Bio. Ed. Online. org

Measuring with an Eyepiece Reticule Determine width of a Spirogyra filament Before lining up

Measuring with an Eyepiece Reticule Determine width of a Spirogyra filament Before lining up Lined up on specimen Estimated 13 divisions at 20 µm/division gives width of 260 µm Bio. Ed Online www. Bio. Ed. Online. org

Resolving and Reporting Dimensions Sketches of Chlamydomonas reinhardi cell with paired flagella Image as

Resolving and Reporting Dimensions Sketches of Chlamydomonas reinhardi cell with paired flagella Image as it might appear with resolution to the nearest 10 nanometers Length can be estimated as 4. 1 divisions Image as it might appear in a light microscope with total magnification 400 x Length appears to be nearly 5 divisions Bio. Ed Online www. Bio. Ed. Online. org

Calibrating the Fine Focus Control Calibration marks on fine focus control 9 cm mark

Calibrating the Fine Focus Control Calibration marks on fine focus control 9 cm mark Distance traveled Use focus to lower the stage To calibrate a fine or coarse focus knob: • measure distance from top of stage to a fixed point above the stage; • lower stage and count number of full turns; • measure distance traveled; and • divide distance traveled by number of turns to get distance traveled per turn. Bio. Ed Online www. Bio. Ed. Online. org

Using the Fine Focus to Measure Depth Test Focus level at higher magnification Aperture

Using the Fine Focus to Measure Depth Test Focus level at higher magnification Aperture Pseudopodia Bio. Ed Online www. Bio. Ed. Online. org

Using Volume to Estimate Depth This cover slip is square, 22 mm on a

Using Volume to Estimate Depth This cover slip is square, 22 mm on a side. Depth of liquid under the cover slip must be 40 cu. mm ÷ 484 sq. mm = 0. 082 mm, or 80 µm. Its surface area, then, is 484 sq. mm. Drop of volume 40 microliters has volume of 40 cubic millimeters. Bio. Ed Online www. Bio. Ed. Online. org

Counting Chamber (Hemacytometer) Neubauer type hemacytometer . . . mounted on a stage Hemacytometer

Counting Chamber (Hemacytometer) Neubauer type hemacytometer . . . mounted on a stage Hemacytometer design Cover glass Sample depth 0. 1 mm TOP VIEW SIDE VIEW Grid areas Sample introduction point Bio. Ed Online www. Bio. Ed. Online. org

Using a Hemacytometer grid (40 x) 1/25 sq. mm Views at 100 x Small

Using a Hemacytometer grid (40 x) 1/25 sq. mm Views at 100 x Small square = 1/400 sq. mm 1/25 sq. mm Bio. Ed Online www. Bio. Ed. Online. org

Example: Counting a Cell Suspension Count 4 corners of central square and the center

Example: Counting a Cell Suspension Count 4 corners of central square and the center (dots). Yeast cells on center part of grid, portion of field at 40 x. Suspension was diluted by a factor of 100 before charging or filling the hemacytometer. Bio. Ed Online Portion of boxed area at 100 x. Accept cells overlapping grid on top or right (red arrow); reject cells overlapping left or bottom (blue arrows). Count = 14 cells in 1/25 sq. mm. Count 4 corners of central square and the center (dots); 77 cells in 5/25 sq. mm. www. Bio. Ed. Online. org

Example: Calculating a Cell Concentration n Suspension was diluted 100 fold to make it

Example: Calculating a Cell Concentration n Suspension was diluted 100 fold to make it possible to count cells. n Counted 5 squares in symmetrical pattern to obtain a significant count. n n Count was 77 cells in 1/5 (0. 2) sq. mm, 0. 1 mm deep. n 77 ÷ 0. 2 gives number cells/sq. mm = 385. n 385 ÷ 0. 1 mm gives 3850 cells per cubic mm. n 3, 850 x 1000 gives 3. 85 x 106 cells per cubic centimeter = 3. 85 x 106 cells per ml. n 100 x 3. 85 x 106 = 3. 85 x 108 cells per ml undiluted. It would be realistic to report the concentration in undiluted sample as 3. 8 x 108 cells/ml (significant figures). Bio. Ed Online www. Bio. Ed. Online. org