Isol­a­tion and cul­tiv­a­tion of Mi­croor­gan­isms

Isolation and cultivation of microorganisms

The di­versity of mi­cro­bial spe­cies is nearly end­less. Cur­rently the use of stand­ard me­dia in a high through­put ap­proach will al­ways yield strains rep­res­ent­ing novel spe­cies and gen­era. This is not the fo­cus of our cul­tiv­a­tion pro­gram.

The cul­tiv­a­tion-in­de­pend­ent ge­n­omic ana­lyses of hab­it­ats provide us with a real­istic view on the abund­ant mi­croor­gan­isms in the sea. For can­did­ates of a global im­port­ance in the car­bon cycle, we use mo­lecu­lar tools – strain-spe­cific PCRs and in situ hy­brid­iz­a­tions – to quantify the pres­ence in en­rich­ment cul­tures. Nov­elty arises of­ten from a change in meth­ods. Mi­cro­bi­o­lo­gists have used nu­tri­ent-rich single-fed batch cul­tures to isol­ate bac­teria for over hun­dred years, tar­get­ing the fast­est grow­ing or­gan­isms. This hap­pens very rarely in nature. And the abund­ant bac­teria in the sea ap­par­ently do not like these rich me­dia. We di­lute the cells with sterile sea­wa­ter dir­ectly after sampling to isol­ate in­di­vidual cells and in­cub­ate for long times to get slowly grow­ing bac­teria. The com­bin­a­tion of phys­ical isol­a­tion into hun­dreds of cul­tures, suf­fi­cient time to grow and mo­lecu­lar tools to ana­lyze en­rich­ment cul­tures still needs the per­fect me­dium for the mi­croor­gan­ism of in­terest. A per­fect me­dium provides all nu­tri­ents at non-toxic con­cen­tra­tions, ideally in a per­man­ent con­tinu­ous-fed mode at mi­cro­molar con­cen­tra­tion, like in nature.

A con­tinu­ous-fed mode for hun­dreds of cul­tures can only be real­ized in ex­cep­tional cases. One solu­tion to this prob­lem is the down­scal­ing of our ex­pect­a­tions. Clear wa­ter can con­tain one mil­lion of cells per ml, and ex­actly this growth, to the na­ked eye in­vis­ible, is the best strategy to ob­tain and cul­tiv­ate many of the abund­ant bac­teria from the sea. Once the strains have grown in me­dia with en­vir­on­mental nu­tri­ent con­cen­tra­tions, se­lect­ive pres­sure to­wards growth at higher cell con­cen­tra­tions can be ap­plied and vis­ibly tur­bid cul­tures can even­tu­ally be ob­tained.    

The ocean is not a broth, the nu­tri­ent con­tent is low. But in the ocean there are in the wa­ter column many local, dy­namic and of­ten nu­tri­ent-rich hab­it­ats for mi­croor­gan­isms: the sur­face of al­gae and zo­oplank­ton, the di­gest­ive sys­tems of zo­oplank­ton and fishes, the particles formed by the sea and zo­oplank­tonic fecal pel­lets, cell ag­greg­ates and dif­fus­ive bound­ary lay­ers around nu­tri­ent re­leas­ing al­gae and other particles. The isol­a­tion of mi­croor­gan­isms from these hab­it­ats is one of the great chal­lenges in mi­cro­bial ocean­o­graphy.