Hi Yoana,

Thank you very much for those steps it certainly helps.  I am a bit unsure what you mean by pelletizing? When I think of pelletizing I envision homogenizing a liquid sample to get a concentrated pellet. Are you crushing the filters with a motor and pestle or grinding them down to a fine silt?  I have worked on preparation of fish muscle tissue for mass spectrometry in which we dried the tissue and then ground it down to a fine material.  But the fish samples were being processed by weight where as my algae samples will be processed by cell density.  I will be using a CHN to process the algae samples not a MS.  Do you treat your filters (after they have collected algae sample) with any acid to remove any inorganic carbon or is that accomplished in the pre-bake step??   

Hi Amy,

We use a similar procedure in our lab with pre-ashed GFF filters. Over the years we have found that the extra filter material can occasionally be a problem (incomplete combustion and/or jamming the autosampler) when packing the entire sample into even the large tin cups. 

- If filtered fairly evenly, we have used a paper punch to collect a quantitative sub-sample of the residue. This requires some testing ahead of time to make sure one has enough mass (enough C and N) to measure.  This makes preparing the "burrito" of the tin+sample easier. It also affords use of analytical replicates, if desired.

- If densities are very high (we do this routinely with benthic samples), one can actually pipet cell suspensions directly into the larger tin cups (9 x 10 mm) and dry them before folding the cup for the analyzer. They hold about 900 uL; we often add this amount twice (add-dry-add-dry).

Good luck,

John Wehr

Hi Amy,

I would recommend filtering different volumes to make sure there is no problem with organic matter adsorption and/or cell breakage during filtration. Isochrysis galbana is robust (see Table 5 of Collos et al. 2014. Particulate carbon and nitrogen determinations in tracer studies: the neglected variables. Appl. Rad. Isot. 94: 14-22) but the other one's resistance to filtration is unknown to my knowledge. Otherwise, 13 mm filters are most practical for cultures work and avoid jamming the sampler as mentioned above.

I agree with all of the above mentioned steps.  It is very important to pre combust the filters.  Fisher sells a 'peletizer' which makes it very easy to insert the burrito filters and allows you to squish them.. The smaller the better.  Also you want to make sure that you have enough cell densities since PON is much lower than POC (remember your Redfield ratio of 106:16 C:N).  I would also make sure that you pipette only off the top of your culture or use a relatively actively growing and fresh culture as you may otherwise suck up and analyse dead cells and detritus which will throw off your value.  Important also to get an accurate cell count (i.e. Coulter counter).  Good luck!

Thanks to all for the helpful advice :)