Enumeration of Colony forming Units of Trichoderma in formulations-Precautions to be Taken to Avoid Errors during Serial Dilution

Traditionally serial dilution method is used for the enumeration of viable propagules in a microbial formulation. Conidia of some fungal agents like Trichoderma spp. are hydrophobic in nature. They repel water and are not dispersed uniformly in water. To enhance uniform distribution, dispersing agents like Tween 20 have to be used. In many articles in the published literature, high CFUs (>10¹¹ CFUs g^-1) have been either been claimed or reported to be present in formulations. Further considering the volume (4/3 × 3.14 × r³) occupied by each conidium (minimum 2 micron diameter), realistically we cannot expect high Colony Forming Units (CFUs) reported or claimed in many research papers. In the present report an attempt was made to estimate the possible 'number of conidia per cc' and 'number of conidia per g' of Trichoderma harzianum spores (NBAII Th10-MTCC 5584). The results showed that even at higher dilutions (>10^-18 to 10^-20) colonies of Trichoderma appeared on plates which is not theoretically possible. It is proposed that while enumerating the viable propagules in a formulation the serial dilution has to be done with Tween 20 and the tips should be changed for each dilution without fail. The dilution should be restricted to 10⁹ or 10¹⁰. The limitations of the serial dilution beyond 10¹⁰ and precautions to be taken are discussed.