The simplest way is to fix the sample by adding an equal volume of 50% formalin (18.75% formaldehyde).  Add two drops of brilliant Green dye to a 10 ml subsample and allow to stand at least 4 hours. Place a measured drop of the sample onto a glass slide and count all cells at 100x magnification. Generally 0.05 to 0.1 ml  is the volume counted. If the number of cells is too high to count, dilute the sample as needed. Brilliant Green Dye is prepared as follows:

2.0 g Brilliant Green; 2.0 ml glacial acetic acid; Dilute to 100 ml with distilled water.

If Brilliant Green is not available, methylene blue, methyl green or Lugol's iodine can be used. Lugol's Iodine fades rapidly, so the sample should be counted immediately after it is added.

Good Luck!. 

Hi

This is the protocol used in my lab. It is long (15 minutes per sample) but it is very reliable.

Good luck

Ruminal content (approximately 1 L) was strained through 4 layers of cheesecloth and a 5-mL portion of the strained ruminal fluid was preserved using 5 mL of methyl green formalin-saline solution for protozoa enumeration (Ogimoto and Imai, 1981). Protozoa samples were stored at room temperature in darkness until counting. Protozoa were microscopically enumerated using a counting chamber (Neubauer Improved Bright-Line counting cell, 0.1 mm depth; Hausser Scientific, Horshamm, PA) and genera were identified as outlined by Dehority (1993).

References

Dehority, B. A. 1993. Laboratory manual for classification and morphology of rumen ciliate protozoa. CRC Press Inc. . Boca Raton, FL.

Dehority, B. A. 1993. Laboratory manual for classification and morphology of rumen ciliate protozoa. CRC Press Inc. Boca Raton, FL.

Dear Dr.Durk Dehority, Thank you for the protocol and encouragement. I follow.

Dear Dr.Fadi Hassanat, Thanks for helping me with reliable protocol.