Preparing Biological Samples for Column Chemistry
Filtering Cultures 1.1 Oxalate Wash Preparation Before filtration , make an oxalate wash that will be used in order to remove extracellular iron from diatoms .
Mix consecutively from a to f in 600 ml of ultra pure .
Take up to 1 L ultra pure once Ph 8 is reached .
15.3g , EDTA ; 14.7g , Sodium Citrate ; 0.74g , Potassium Chloride ; 5g , Sodium Chloride ;
Several drops of Sodium Hydroxide until a Ph of 6 - 712.6g , Oxalic Acid is 1.2 .
Harvest Diatoms Once They Have Been Filtered :
Once cultures have been grown to their optimal density ( approximately 3500 RFU ) , they need to be harvested .
In order to do so , the following items are needed : a pair of tweezers that have been cleaned in 10 % HCl overnight , polycarbonate filters ( 0.1um ) that have been washed in 10 % HCl overnight and rinsed in UPW , 3 bottles : one to collect waste , one that contains ASW and one that contains UPW , oxalate wash , a 60ml syringe , and syringe filter tip .
Label several 15ml tubes with the designated solution numbers on them .
One tube should be labeled for unwashed solutions and a separate tube labeled for washed solutions .
For example , the tubes may say , “solution 1 # 1 unwashed” , “solution 1 # 2 washed” .
Once tubes are labeled , assemble the filtration apparatus .
Use polyethylene gloves to handle the tweezers , remove one polycarbonate filter from its container and place on the filter rig .
Screw the reservoir onto the filter rig and rinse the filter with UPW , allowing the water to be filtered through .
Pour 250ml of culture onto the filter and allow it to drain .
Rinse the filter three times with 5ml of ASW .
Once filter has completely drained , unscrew the reservoir with polyethylene gloves , remove the filter with tweezers , and place it in the corresponding 15ml tube that is labeled “unwashed” .
Syringe filter 60ml of oxalate wash into the remaining 750ml of culture .
Invert bottle once and allow to sit for 30min .
Invert bottle a second time and place a new polycarbonate filter onto the filter rig , rinse with UPW and pour the remaining culture onto filter .
Rinse filter three times with 5ml ASW , drain completely , remove filter , and place in corresponding tube labeled “washed” .
Repeat these steps until all of the 1L bottles have been filtered .
Rinse tweezers with UPW as well as the filter rig , and put everything back in its place .
Rinse the bottles out with UPW and take them to the clean lab to be cleaned .
All cultures , once filtered , need to be run through columns in order to examine iron limitation .
Preparation for Isotopic Analysis Transfer filter to a clean 15 mL tube for storage .
Transfer the filter into a small teflon ( PFA ) vial .
Add 1 mL conc . TE clean HCl and 1 mL conc . TE clean HNO3 to each vial .
CAREFUL : This reaction will produce a corrosive and foul - smelling orange gas .
Place caps loosely on vials and place vials in a fume hood .
IF YOU DECIDE TO PUT THEM ON A HOT PLATE , DO NOT TURN ON HEAT .
( If hotplate is not available , find another good place to store them overnight in a fume hood . )
After the samples have reacted overnight , remove the filters from the vials with a pair of teflon tweezers .
Try to pick up the filter in a way that most of the remaining diatom shells remain on the filter .
The filter will also have a lot of the acid still in it , and this acid contains valuable amounts of Fe .
Rinse off this acid back into the PFA vial by twice pipetting 1 mL of TE H2O over each filter .
Evaporate samples to dryness on a hot plate .
Reconstitute samples in 200 uL of 10N HCl + 0.001 % H2O2 .
Collect a subsample before samples are run through columns .
Run samples through ‘mini’ columns ( 135 uL ) according to established procedures .
While running columns , clean each of your PFA vials by blasting then with TE water from the tap and putting them on the hot plate with 0.1 N HNO3 for an hour or two as columns run .
Elute from the columns into the same PFA vials your samples came out of .
Dry down overnight ( or for a few hours ) .
Re dissolve in 100ul of conc . HNO3 .
Put on hot plate in bio lab , capped , for 30min at 350 .
Take off hot plate in clean lab and put on hot plate in biological lab to dry down .
Reconstitute in 1100ul 0.1N HNO3 Take off 100ul of sample and dilute to 1ml with . 1N HNO3 , put in 15 ml vial .
Give the sample from the step above to Tim to run on element .
( This gives the concentration that allows you to calculate step 22 ) .
When you have calculated the amount of Fe in each sample , add enough double - spike to get a 1 : 2 sample : spike ratio .
The sample is ready for isotopic analysis .
