Air Sampling & Analysis, LAB III

INDUSTRIAL HYGIENE SAMPLING STRATEGIES
MICROSCOPY

OSH 626, LAB III

MICROSCOPY

Purpose
In this experiment students will become familiar with microscopes and
will observe, count, and determine the size distribution of airborne
particles collected from a generated dust cloud.

DO NOT TOUCH OR ATTEMPT TO CLEAN ANY MICROSCOPE LENS

Part I
Familiarizing yourself with the microscope

Examine the microscope and become familiar with its parts.
Notice the condenser height adjustment, phase ring-iris-darkfield
adjustment, objective changing, focusing and stage manipulation.

View a prepared slide under brightfield illumination with both the
10X and 40X objectives.

View the same sample under darkfield and phase contrast with the 40X
objective. Be sure to use the appropriate illumination (Kohler) and
phase ring.

Calculate the theoretical resolution for each objective on the
microscope.

Part II
Calibration of a Porton reticule with a stage micrometer

Under the brightfield illumination, observe the stage micrometer with
10X and 40X objectives. Observe the lines and determine the distance
between the stage micrometer lines.

Move the microscope stage to superimpose the micrometer image on the
long side of the reticule. Determine the length of this side from the
stage micrometer. From this length all the dimensions of the Porton
graticule can be calculated as the length of this side is twice the
width of the graticule. The length is also equal to 100 times the
diameter of circle #2. The area of each circle is twice that of the
preceding circle. Thus, the diameter of each circle is larger than the
preceding circle by the amount equal to the square root of 2.
Perform this calibration for the 10X and 40X objectives.

View the stage micrometer with the 100X objective using immersion oil.
Take care to contact only the cover slip & the 100X objective with oil.
Do not attempt to clean the objective. Calibrate the graticule at 100X
magnification.

Prepare a calibration for each objective as shown in Table I.

Part III
Sizing of collected particles, (density 2.65 gm/cc)

Prepare a slide from the open face filter sample collected for
microscopic analysis as follows:

1. Cut a pie shaped wedge of the filter with an arc of about 1 cm and
place it on the end of a clean glass slide.

2. Place a drop of mounting medium on the center of the slide with the
applicator. Do not allow the applicator to touch the slide or
anything else. With a toothpick, smear the oil into a shape and
size similar to the filter wedge.

3. With forceps, place the wedge on top of the oil and allow it to
soak through. There should not be too much oil on the slide that
it washes across the filter surface. The oil should be completely
absorbed by the filter.

4. Place a cover slip on top of the filter and wait few minutes for the
filter to become transparent. If the slide is not satisfactory, try
again.

Determine the size distribution of the collected particles with the 40X
objective as discussed below:

1. Focus on the filter and choose a field at random (eyes away from the
microscope). One by one, compare all the particles whose centers fall

within the field (the six rectangles on the left side of the
reticule) to the nine Porton circles. For each particle, select the
circle which has the same projected area or a projected area
slightly larger than the particle's projected area. Indicate your
selection by pressing the appropriate lever on the counter or by a
tally matrix.

2. There should be 3-7 particles in each small rectangle. Fewer
particles will require more time for sizing and more particles means
that it may not be possible to keep track of which particles have
been sized and which have not.

3. When all particles on a given field have been sized, select a new
field at random. Continue until you have sized 200 particles.
Present the size distribution data as shown in Table II and plot
the data on the provided log-normal distribution graph paper.
Include all information related to the optical system used in the
experiment (objective type, magnification, numerical aperture, etc...).
Repeat the size distribution measurement with the 100X objective under
oil immersion. Prepare a new data Table II. Plot the data on the same
log-normal graph sheet used for the 40X sizing.

Part IV
Answer the following questions

1. Describe the appearance of the particles. Include such things as
shape, color and other identifying characteristics.

2. Describe the difference in appearance of the particles in the
brightfield & phase contrast. Describe the relative advantages of
each technique for viewing and sizing particles.

3. Roughly what weight fraction of the particles would you expect to be
respirable? Does this agree with the results found by the gravimetric
analysis in the previous experiment?

4. What are the mass and count median diameters (MMD & CMD) of the
distributions measured with the 40X and 100X objectives? Explain
any differences.

5. Are the distributions approximately log-normal? If so, what is the
geometric standard deviation?

NOTES
Table II is used for sizing particles counted under 40X and 100X
objectives under part III. Make sure that you have 2 blank tables
before you start writing your lab report.
- Wavelength of white light is 0.55 mm
- NA for 10X = 0.25
- NA for 40X = 0.65
- NA for 100X = 1.30

- Mass of a particle = [(3.1416)(dp3)(particle density)]/6

TABLE I, Diameter Represented by Circle, (mm)
Circle #: 1 2 3 4 5 6 7 8 9
10X Objective:________________________________________________________
40X Objective:________________________________________________________
100X Objective:_______________________________________________________

TABLE II, FOR 40X SIZING

Circle #: 1 2 3 4 5 6 7 8 9
Circle Diam. (mm):____________________________________________________
Mean Diam. (mm):______________________________________________________
Particles Counted:____________________________________________________
Cum. % by #:__________________________________________________________
Mass Particle (gm):___________________________________________________
Mass Size (gm):_______________________________________________________
Cum. Mass (gm):_______________________________________________________
Cum. % Wt.:___________________________________________________________
Total No. of Particles Counted:______________

TABLE II, FOR 100X SIZING