Strategies for Reducing Bacterial Load

From ChoanoWiki

1. Filtration
   • This is probably the best way (very good efficiency, and gentle on the choanos) but it's very slow. We use differential size filtration with two different cell culture inserts 'BD Falcon Cell Culture Inserts 8.0micrometer Falcon No.:353093' and 'BD Falcon* Cell Culture Inserts 3.0micrometer Falcon No.:353092' The 8um filter blocks bacterial clumps but allows the choanos and bacteria to pass through, then the 3um filter traps the choanos while allowing the bacteria to pass.
2. Differential Centrifugation
   • This is the quickest and easiest way to eliminate most bacteria, but it does physically traumatize the choanos. Bacterial clumps will spin down at relatively low g (~10xg), the choanos a bit higher (~500xg), and individual bacteria much higher (~5000xg). These values are approximate, and keeping fast swimming choanos pelleted can be challenging (though cold helps, and azide helps even more, if you don't need them alive).
3. Shaking
   • Growing cells on a rotational shaker at ~100rpm helps somewhat. Also, the remaining bacteria tend to form larger clumps that are easier to centrifuge out.
4. antibiotic treatment
   • Works, but may be some side effects---there seems to be an increase in blebbing of choano cells.