Hi, I'm trying to estimate carbon fixation of cyanobacteria by adding C13-labelled NaHCO3 and then measure C13 incorporation by IRMS.
My question is how to prepare my samples. As I don't have appropriate filters for my samples now, can I just centrifuge it and wash the pellet with Milli-Q water or sterile seawater (to remove inorganic C13 outside the cells)? But after I get the washed pellet, how can I dry my samples? Is it similar to drying other solid materials like tissues or soils (105 celsius degree for >24h)?
Thanks in advance.
Dear Xingqin,
Attached please find a publication that covers the answer to your question.
Hoping this will be helpful,
Rafik
Assuming you are planning on analysing your samples by EA-IRMS you should freeze dry the isolated organic pellets after washing them with deionized water. Given intra-cellular water might still contain some bicarbonate it might be necessary to acid wash freeze dried pellets (5% HOAc or 5% HCl), wash them again with DI water until acid free and freeze dry again. Please, note sample preparation protocols suitable for MS or nanoSIMS may not be (and typically are not) suitable for IRMS.
Dear Wolfram,
Thanks a lot! This is very helpful. It is EA-IRMS that I'm going to use. I have already dried my samples with a freeze-dryer. And you're right about washing pellets with deionized water, because washing with seawater may cause overestimation of dry weight.
I have an additional question about the duration of acid washing. I've seen paper talking about washing with HCl (2N) for 24h and then wash twice with DI water, 24h each. Does it necessary to wash it that long? Also, one disadvantage of washing with diluted acid is the loss of water-soluble organic carbon. Harris et al. (2001) overcame this problem by exposing soil samples to HCl vapor (HCl-fumigation method). Do you think this method would also work for removing intra-cellular bicarbonate?
Before acid washing your samples you either need to freeze them with liquid N2 or to ultra-sonicate the pellet to disrupt (open up) the cell membranes. Given the realtively small amount of total intra-cellular water, acid washing to remove any (bi)carbonate still present should not take longer than a few minutes. Using dilute acid over a short period of time should not attack the organic material. BTW, one should not and cannot read across sample preparation methods if sample matrices are significantly different (e.g. soil v. organic pellet) especially when carbonate may be present as solid mineral as opposed to being dissolved in aqueous solution.
Dear Wolfram,
These are valuable tips and suggestions. Thank you very much!!
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