Optical microscopy

Protocol for measuring the red blood cell diameter using the optical microscope

Materials:

  • Optical microscope with a transformer
  • Blood smear (red blood cells/ RBC = pink/ eosin from May-Grunwald- Giemsa staining)
  • Two scales:
    • ocular micrometer (microscope may be equipped with a scale that is built into one eyepiece); a typical scale consists of 50 - 100 divisions
    • stage micrometer (a microscope slide with a scale etched on the surface); length of the scale = 1 mm; the distance between two scale marks/ interval = 0.01 mm = 10 µm

Procedure:

A. Obtaining a clear image (without breaking the sample!):

1. place the blood smear on the microscope stage (the part of the slide glass with the blood smear should be up!)

2. always start adjusting the microscope with the lowest magnification objective! select the lowest objective (4x or 10x)

3. bring down the objective at the closest point to the blood smear, using the gross adjustment knob (the microscope has a security space between the lowest objective and the sample/ the sample will not be destroyed); during focusing on the specimen you check the process watching from the lateral side of the microscope

4. carefully, watching through the eyepieces, slowly move the objective away from the slide until the object comes into the focus (use the gross adjustment knob)

5. carefully by rotation, replace the lowest objective by a higher objective (20x or 40x) Attention! Do not touch the sample with the objective during this activity!

6. use only the fine adjustment knob to clarify the image in the visual field. Attention! Using the gross adjustment knob will break the sample!

7. adjust the intensity of the light using the condenser knob and the diaphragm of the condenser.

B. Measuring the diameter of the red blood cells:

1. obtain a clear image of the RBC using the blood smear (see point A)

2. identify the ocular micrometer

3. measure the diameter of nine RBC using the ocular micrometer (number of division on the ocular micrometer) and write the results in a table

4. calculate the mean, standard deviation and standard error mean for your results

C. Calibration of the ocular micrometer:

1. replace the blood smear with the stage micrometer

2. place carefully the stage micrometer onto the microscope stage

3. obtain a clear image of the scale for the stage micrometer

4. overlay and line up the ocular micrometer with the stage micrometer (see fig)

http://www.professionalmicroscopes.com/imageHND.JPG

5. select two scale marks on each scale where it is visible a proper overlaying; the larger distance between the scale marks the lowest errors

6. count the scale marks on the ocular micrometer between the selected marks

7. count the intervals (the distances between two marks) on the stage micrometer between the selected marks; the size of an interval = 10 micrometers

8. reorganize your working place

D. Calculation of the diameter of the RBC

1. use the rule of three (cross-multiplication) to calculate the diameter of the RBC - according to the calibration values

E.g.:

The eyepiece micrometer: ØRBC = 8.3 ± 0.7 scale marks (n = 9)

Overlaying of the marks on the both scales: to 70 scale marks on the ocular micrometer correspond 6 intervals on the stage micrometer

The rule of three:

70 scale marks ------------- 6 intervals (6 x 10 µm = 60 µm)

8.3 scale marks ------------- x

x = 7.1 µm

70 scale marks ------------- 6 intervals (60 µm)

0.7 scale marks ------------- y

y = 0,6 µm

ØRBC = 7.1 ± 0,6 µm (n = 9)

2. state one-two conclusions according to your results