Low-Tech Protozoa Procedure

 A more cost efficient way to count the number of protozoa in a soil sample.

 

 

 

Materials:

(3)          Petri Dishes

              Soil Core

               Mortar and Pestle

               Balance

(1 gal.)    Distilled Water

               Serialogical Pipette

               Pipette Pump

               Uhlig Extractor : two 8 ounce plastic cups                                                     

               Scissors

                Two sheets of 6 x 6" nylon Bridal Vei565r 

                Rubberband

                12.5 Qualitative Filter Paper

                Capillary Tube

                Methyl-Green Stain

                Microscope Slide

                Slipcover

                Disposable Graduated Beral-type Pipette

                Microscope

                Ringstand

                Glass Funnel

                Ring

 (2)          3 oz. Plastic Cups

 

 Preparation:

     Uhlig Extractor

1)       Cut one of the plastic cups at the first line from the bottom of the cup.                                                 

2)       Cut the second cup along the same line in such a way that two strips of the cup are still left below the first line. These two leftover strips will be used as “legs” to stand the cup.

 

 

 

3)       Place the two pieces of 6 x 6 inch nylon bridal veil over the bottom opening of the first cup. Layer these   pieces so the lines do not cross. 

4)       Tightly hold the bridal veil in position as the second cup is placed over it in a parallel fashion.

5)       Stand it on its legs.

 
 

3rd Filter

1)       Secure the ring on the ringstand 8 inches from the stand's base.

2)       Place a glass funnel in the ring.

3)       Fold a 12.5 cm qualitative filter paper in half. Fold it in half one more time.

4)       Open the folder filter paper in such a way that there are three folds on one side and there is one fold on the other side.

 

Protozoa Extraction

    1.Place 15 cm sample of soil into the bottom of a clean, empty Petri dish and allow

to dry for 24 hours or until completely dry.

    2. Grind the sample with a mortar and pestle until it is powder-like.

     3. Pour the ground soil into a three ounce plastic cup.

    4. Cover the cup with a mesh screen and secure it onto the cup using a rubberband.

    5. Place a second clean Petri dish onto the balance. Set the balance to 0.

    6. Sift 9-10 grams of soil into the second Petri dish. Record how much soil was sifted. (It would be easier to just sift between these numbers rather than trying to sift the same amount of soil each time [9.5 grams or something of that nature])

    7. Using a serological pipette that has been secured into a pipette pump, add 20 mL of distilled water to saturate the soil.

    8. Cover the Petri dish with its lid and allow it to sit for 7 hours (if time does not allow, refrigerate sample after 7 hours).

    9. Fill a third clean Petri dish with 30 mL of distilled water using a serological pipette and its pump.

  10. Place a Uhlig extractor in the filled Petri dish with its legs facing down.

   11. Pour/scoop the soil sample into a modified Uhlig extractor. (If after adding the soil sample to the extractor, the water in the Petri dish does not touch the mesh, add more distilled water to the Petri dish in 5 mL increments. Record the amount of water used.)  

   12. Remove the extractor and pour the solution into the funnel made of 12.5 cm qualitative filter paper. *Do not scoop the settled soil from the previous soil solution, just pour the liquid.

   13. As soon as the filter is completely empty of solution, the filtering is complete.

 

Counting Protozoa: 

      1. Using a capillary tube, deposit 7 µl of methyl-green stain on a clean microscope slide. Then using a disposable graduated Beral-type pipette, add 18 µl (The first demarcation on the pipette) of the 2nd filtrate from step 6 to the stain on the microscope slide and cover with an 18 x 18 mm2 cover slip.

        2. Examine under a light microscope at 40X (for quantitative) or 100X (for qualitative) observations of the various protozoa living in the soil.

         3. Use the following equations to determine the population density of protozoa in the soil sample (the 40X is preferred method; us 100X formula only when absolutely necessary):

[(# per field \of view at 40X) · (total ml of 2nd filtrate) · 747 ] ÷ (grams of sifted soil) = # of protozoa per gram of soil ;

[(# per field of view at 100X) · (total ml of 2nd filtrate) · 5102 ] ÷ (grams of sifted soil) \= # of protozoa per gram of soil

 

    

*The original procedure was modified by 2005 T.A.'s  (Leah Miller and Kalyani Ravi) of E.S.S.R.E.