Overview

Background/
Definitions

Topic
Introduction

Activity 1.1

Activity 1.2

Activity 1.3

Particle Counting Form

Assessment

Developer Bio

Preparing a Sample for Counting
Activity 1.2

Student Learning Strategies

This process is very important, since it involves the filtration of the liquid to capture the particles to be counted on a sterile gridded membrane filter. If performed incorrectly, the membrane filter will capture more or fewer number of particles of various sizes, and the cleanliness level recorded as a result will be erroneous.

After this activity, students will be able to:

  1. Perform a blank analysis preparation used as a baseline for the filtration process.
  2. Use sterile techniques to avoid contamination of the sample.
  3. Use the vacuum/filtration apparatus properly.
  4. Perform a fluid sample filtration for the counting procedure.

Procedure

A. Blank Analysis Preparation

This procedure is used as a baseline for the filtration process.

  1. With a forceps, grab a membrane filter and rinse with either filtered alcohol or distilled water.
  2. Place the membrane filter (grid side up) on the filter holder base and lock the funnel to the filter with a clamp.
  3. Pour 100 ml filtered alcohol or distilled water from a dispenser into a prepared sample bottle and shake.
  4. Remove the cap and the filter cover, and pour the sample (alcohol or distilled water) into the funnel.
  5. Pour 50 ml alcohol or distilled water from a dispenser into the sample bottle.
  6. Pour this sample ( alcohol or distilled water) into the funnel. Cover the funnel at this time.
  7. Attach the vacuum to the filter flask and turn it on. The sample should be filtered until half of the fluid ( 50 ml) remains in the funnel.
  8. Uncover the funnel, and rinse the funnel walls ( sides) with the filtered alcohol ( using a dispenser). Do not dispense the alcohol onto the filter.
  9. Cover the funnel again, and turn on the vacuum, to filter the rest of the sample. The filter should appear dry at this time.
  10. Remove the clamp, release the vacuum, and lift the funnel.
  11. Using a forceps, remove the filter membrane and place it between 2 glass slides. Do not forget to label your slide with the specimen's name.

B. Fluid Sample Filtration

Same as above, with some modifications:

  1. With a forceps, grab a membrane filter and rinse with filtered alcohol or distilled water.
  2. Place the membrane filter (grid side up) on the filter holder base, and lock the funnel to the filter with a clamp.
  3. Shake the sample bottle gently and remove its cap without rotating the film.
  4. Uncover the funnel and pour the contents of the sample bottle into the funnel.
  5. Rinse the sample bottle with 50 ml alcohol using a dispenser, and pour it into the funnel. Cover the funnel.
  6. Attach the vacuum to the filter flask and turn it on. The sample should be filtered until 50 ml of the fluid remains in the funnel.
  7. Uncover the funnel, and rinse the funnel walls with the filtered alcohol (using a dispenser). Do not dispense the alcohol onto the filter.
  8. Cover the funnel again, and turn on the vacuum to filter the rest of the sample untill the filter appears dry.
  9. Remove the clamp, release the vacuum, and lift the funnel.
  10. Using a forceps, remove the filter membrane and place it between 2 slides. Label your slides with the name of the sample.

Note: Perform a particle count with the Blank Analysis Preparation as well as the Fluid Sample Preparation. If the Blank Analysis count is above 10% of the total acceptable sample count, then the filtration apparatus should be cleaned and the procedure should be repeated.

Assessment Tools

  1. What is the purpose for a Blank Analysis Preparation?
  2. What is used to rinse the membrane filter?
  3. Why is the membrane filter placed grid side up on the filter holder base?
  4. Why do you need to cover the funnel while in use?
  5. Why do you need to rinse the funnel walls with filtered alcohol after only half of the fluid is filtered?
  6. What should be performed if the Blank Analysis count is above 10% of the total acceptable sample count?
Instructor Learning Strategies

Activity level of difficulty: Beginner

Suggested teaching strategies: Students need a copy of the procedures, and can work in pairs; however, each student should attempt to operate the vacuum/filtration apparatus once for familiarization.

Time commitment: 10 to 15 minutes per procedure; 20 to 30 minutes overall.

Equipment/Materials





  1. glass filter holder
  2. filter flask, side arm,
    1 liter
  3. vacuum hose, 1/4"
  4. solvent filtering dispenser
  5. solvent filters
  6. gridded sterile membrane filters
  7. filter forceps
  8. vacuum pump
  9. slides
  10. glass filter flask cover
  11. the sample drawn (from the last activity)
Troubleshooting Tips
  1. The funnel should be covered at all times to prevent contamination from any possible sources.
  2. The membrane filter should be positioned properly on the glass slide so that its sides are parallel to the sides of the slide. Otherwise, under the microscope, the lines of the grids will not be straight, which makes the counting very difficult.
Answers to Assessment Tools
  1. It is used as a baseline for the filtration process.
  2. Filtered alcohol or distilled water.
  3. So that the particles are captured on the gridded side which is placed on the slide. The grids help in the counting procedure.
  4. To reduce contamination from other sources.
  5. To ensure all the particles go through the membrane filter including those particles that tend to attach to the funnel walls as the liquid is vacuumed.
  6. The filtration apparatus should be cleaned and the procedure should be repeated.