Topic
Introduction

##### Activity 1.1

Activity 1.2

Activity 1.3

Particle Counting Form

Assessment

Developer Bio

User Evaluation Form

Counting and Calculating Procedures
(Using Worksheets)

Activity 1.3

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:

1. Choose the scanning lens first.
2. Place the stage micrometer on the stage and focus.
3. Through the right ocular lens, make sure the micrometer and the ocular scales are aligned.
4. Adjust the ocular lens until its scale (0-100) is aligned with the micrometer.
After this activity, students will be able to:
1. Calibrate the ocular lens.
2. Use a microscope to count the particles in different size ranges.
3. Use a formula and worksheet to calculate the total count for all sizes.
4. Figure out the overall level of cleanliness for their liquid.

Procedures

Counting the particles

1. 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.
2. 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.
3. 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.
4. 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)(# particles counted).
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.

Assessment Tools

1. What is SCF, and how is it used to get the total count?
2. When counting the particles on the borders of the filter, which ones should not be counted?
3. When counting particles using the 4x objective lens, how many squares do you count? What about under 10x and 20x?
Instructor Learning Strategies

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.

 Equipment Stage micrometer Dual counter Side light source Binocular microscope with 4x, 10x, and 20x objective lenses Microscope scale (ocular) Materials

Particle Counting Worksheet

Troubleshooting Tips

1. 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 count.
2. The calibration of the oculars needs to be done each time a new microscope is used.
3. It is also beneficial to take frequent breaks between counts to ease the stress on the eyes.

Answers to Assessment Tools
1. 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 counted).
2. The ones on the lower or right hand borders should not be counted.
3. Under 4x objective, all squares should be counted; under 10x and 20x objectives, 10 squares may be counted if SCF is used.