Development of an accurate and convenient method by which detection of cellulolytic organisms as well as the types of their cellulase i.e. extra- or intra-cellular, was attempted.
A selective agar medium on which only cellulolytic organisms can form colonies was formulated. The media composed of $NaNO_3$, 0.1%; $K_2HPO_4$, 0.1%, KCl, 0.05% $MgSO_4 \cdot 7H_2O$, 0.05%, yeast extract, 0.005%, CMC, 0.5%, and agar, 0.1%.
Nine different microorganisms were tried to grow on the newly developed medium. They include $\underline{E}. \underline{coli}$ HB101, $\underline{Cellulomonas}$ CSI-1, $\underline{B}. \underline{cereus}$, $\underline{B}. \underline{megaterium}$, $\underline{B}. \underline{amyloliquefaciens}$ HI, $\underline{B}. \underline{stearothermophilus}$, and three strains of $\underline{B}. \underline{subtilis}$, RM125, BD170, and SB202. Out of these nine species and strains, seven could form colonies on the medium remaining two species, i.e. $\underline{E}. \underline{coli}$ HB101 $\underline{B}. \underline{megaterium}$, not grown. The seven organism which could grow on this selective medium were thus qualified as cellulolytic bacteria.
When these seven organism were reexamined by Congo Red tests, however, only five of them showed halos around their colonies and the remaining two species, $\underline{Cellulomonas}$ CSI-1 and $\underline{B}. \underline{cereus}$, showed no halos. The Congo Red test was, therefore, not qualified for testing overall cellulolytic activity of microorganisms. It was concluded from the results that the overall cellulolytic activity of an organism can be detected by growing on the newly developed selective medium. Further tests with Congo Red dye may distinguish extracellular cellulase from intracellular one.
섬유소 분해능이 있는 bacteria 를 분리 동정하거나 유전자 재조합에 의하여 형질 전환주 중에서 이 분해능을 갖는 재조합주를 신속하고도 확실하게 분리해 내는 일은, 섬유소 분해능을 가진 유전자를 복제 및 발현 시키는데 일차적인 조건이 된다.
한정된 양 (0.005%) 의 yeast extract 를 함유한 기본 배지에 0.5% 의 CMC 를 첨가 시킴으로써, 오직 섬유소 분해능이 있는 bacteria 만을 분리할수 있었고, 뒤이은 Congo Red dye 처리에 의하여 세포벽 밖으로 분리 가능한 섬유소 분해 효소를 생산하는 bacteria 를 다시 구별해 낼수 있었다.
한편 개발된 CMC 기본 배지를 CMC 대신 다른 탄소원 중합체를 대체 시킴으로써, 다른 종류의 탄수 화물 분해능을 갖는 bacteria도 쉽게 분리해 낼수 있음을 알았다.
