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Culture Clean Up

Culture Clean Up

Reisolating a strain so that there are no other prokaryotes or eukaryotes growing with it is a laborious, multistep process that is not the same for all algae.  Here we outline the NCMA approach. 

First, depending upon the alga and the contaminant,  reisolation may be achieved by serial dilution, streaking on an agar plate and picking colonies, manual single cell picks from liquid media or single cell isolation and sorting using flow cytometry, all with or without the additional use of antibiotics or antifungals. 

Second, after a reisolate has been made, we confirm its success using one of the following methods.

  1. If the contaminant was a bacterium, the newly reisolated strain is tested with our bacterial test media.  For an additional fee, 16s ribosomal RNA can be analyzed in eukaryotic strains to determine if there is any bacterial ribosome present.
  2. If the contaminant was a protozoan, the newly reisolated strain is monitored microscopically for the continued presence of the protozoan.

What appears as a simple process can take two months to a year to complete. So why does this process take so long?  First, it takes time to grow up reisolates from a small amount of biomass. Second, we put the reisolate through the testing phase twice, each of which takes three weeks.  Third, if the contaminant was a protozoan, a culture of the reisolate usually needs to be quite dense to be able to visualize a protozoan contaminant. This is because protozoans can exist as a small percentage of total cells within a culture of a strain, and they may have many different appearances in their growth cycle. They are most easily visualized when swimming. They can be quite difficult to visualize when they are not.

In spite of everything above, sometimes this process fails because:

  1. Many algal strains grow better with their bacteria, and some have an obligate requirement for compounds produced by their co-occurring bacteria.
  2. Some bacteria attach to the outside of some species, making it incredibly difficult to separate the algae from their bacteria.
  3. Some algae are not good candidates for antibiotic treatment as all eukaryotic algae have organelles that are of bacterial origin (mitochondria and chloroplasts).  Prokaryotic algae (i.e., Cyanobacteria) may or may not survive antibiotic treatment. , They are bacteria after all.
  4. Bacterial contaminants may not be sensitive to the antibiotics chosen for treatment, as available antibiotics were developed for use in mammalian systems, not bacteria from the marine environment.
  5. Fungicides are particularly toxic to algae, and it seems that the fungi, at least in our experience, recover more quickly from such treatment than do algae.

 

Which algae are the best candidates?

Algae that are small and grow on agar plates are the best candidates for reisolation. These include, but, not exclusively, the green algae (e.g., Chlorella, Nannochloropsis, Tetraselmis, etc. , pennate diatoms, some centric diatoms.

 

Costs

  1. Plating, dilution and/or antibiotic techniques charged at $150/hour plus materials. At least three hours is estimated, success or failure, and down payment of this charge is required.  The actual time spent will be itemized and charged, if in excess of the down payment, and the balance is expected before reisolated strains are returned.
  2. Flow cytometry sorting techniques start at $800/culture plus materials, success or failure, and down payment of this charge is required. The actual time spent will be itemized and charged, and payment is expected before reisolated strains are returned.

 

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