and Calculating Procedures
Student Learning Strategies
This activity is performed in two procedures:
counting the particles using a microscope, and calculating
the total count using a formula and a worksheet. It is important
to learn the correct method for counting and calculating the total
number, since the total count will lead us to the exact level of cleanliness.
The presence of one particle more than the maximum number allowed
for certain sizes, will result in a false level of cleanliness, which
may be costly in the long run.
Before counting particles, the ocular
lenses should be calibrated as follows:
After this activity, students will be able to:
- Choose the scanning lens first.
- Place the stage micrometer on the stage
- Through the right ocular lens, make sure
the micrometer and the ocular scales are aligned.
- Adjust the ocular lens until its scale
(0-100) is aligned with the micrometer.
- Calibrate the ocular lens.
- Use a microscope to count the particles
in different size ranges.
- Use a formula and worksheet to calculate
the total count for all sizes.
- Figure out the overall level of cleanliness
for their liquid.
Counting the particles
- Place the prepared slide under the selected
objective lens and focus on the grid lines of the membrane filter.
Make sure the light source is focused on the filter from the side,
so it would cast a shadow around the hard-to-see particles.
- Look throughout the filter to make sure
that the particles are randomly distributed; otherwise another sample
should be used or the total surface should be counted.
- If using statistical count, you need to
find the number of particles of a certain size on a series of squares
on the filter grid.
- Figure out your SCF ( Statistical Calibration
Factor) for calculating/statistical estimates. This is a factor
used in calculating the total number of particles under the 10x
and 20x objective lenses. SCF=960/(3.08)(L)(N); In this
formula: 960= the effective filtering area (mm 2), which
is a constant number; 3.08= the width of a grid square (mm),
which is also a constant number; L= the calibrated length
of the micrometer scale (from the ocular calibration performed before
the activity); and N= the number of counted squares, which could
be for example 10 or 20 squares. Make sure that when you choose
a certain number of squares to count, that you will be using the
same number consistently. The significance of this formula
is in its use in the total count calculation: Total # of particles=(SCF)(#
- If SCF is not known or available at the
time, you may use the generic SCF. For 10x objective lens, use 31.16;
and for 20x objective lens, use 62.33. These numbers were calculated
assuming L=100, and N=10. So if you decide to use the generic SCF,
you have to count 10 squares.
- Using the 4x objective lens (total magnification=40x),
view the slide and count the particles in the 2 following size ranges: >4
and 2-4 lines on the scale in the ocular. Under this magnification,
all the squares should be counted, and no SCF is needed. Write the
numbers counted in your table in the corresponding columns in the
worksheet that is provided. For particle size >4, write
in the >100 micron column, and particle size between 2 and 4,
write in the 50-100 micron column.
- Using the 10x objective lens (total magnification
= 100x), view the slide, pick 10 squares and as you go through (up,
down,or sideways), count the particles in the size range 2.5-5 lines
on the ocular scale. Then multiply this number by your SCF or the
generic SCF for the 10x objective (31.16). The result will go in
the 25-50 micron column in the worksheet.
- Using the 20x objective lens (total magnification=200x),
view the slide, pick 10 squares (preferrably the same 10 used above),
and as you go through, count the number of particles in the size
ranges 1-3 and 3-5 lines in the ocular scale. Then multiply these
numbers by your 20x SCF or the generic SCF for the 20x objective(62.33).
The result will go in the 5-15 and 15-20 micron columns respectively.
- Now compare your numbers in each column
with the corresponding columns of the NAS table to find out what
is the specification standard (cleanliness level) of your liquid.
Note: When counting under
4x objective lens, count the particles on the upper or left hand border
of a counting area, but not the ones on the lower or right hand border.
This will give you a 100 complete squares.
Instructor Learning Strategies
- What is SCF, and how is it used to get
the total count?
- When counting the particles on the borders
of the filter, which ones should not be counted?
- When counting particles using the 4x objective
lens, how many squares do you count? What about under 10x and 20x?
Activity Level of Difficulty: Beginner
Suggested Teaching Strategies: Students need a copy of the procedures
along with the worksheet for each sample count. They can work in pairs
or by themselves, but after each count is completed to the cleanliness
level, they should compare their results with each other.
Time Commitment: 10 minutes per size range; overall up to an hour.
- Stage micrometer
- Dual counter
- Side light source
- Binocular microscope with 4x, 10x,
and 20x objective lenses
- Microscope scale (ocular)
Particle Counting Worksheet
- The methods used for counting depends on
the students' preferences; however, it is important to be consistent
every time. Once one method is chosen, it has to be used for each
- The calibration of the oculars needs to
be done each time a new microscope is used.
- It is also beneficial to take frequent
breaks between counts to ease the stress on the eyes.
Answers to Assessment Tools
- SCF is the Statistical Calibration Factor,
from the following formula: 960/(3.08)(L)(N). It is used
to calculate the total count under the 10x and 20x objectives according
to the following: Total number of particles=(SCF) (# particles
- The ones on the lower or right hand borders
should not be counted.
- Under 4x objective, all squares should
be counted; under 10x and 20x objectives, 10 squares may be
counted if SCF is used.