radiocarbon uptake. The method has been described by Carlucci and
Silbernagel (Canadian J. Microbiol., 12: 1079, 1966).METHOD A.
CAPABILITIESRange: 2-35 mg B i /liter (greater quantities after initial dilution)
1. PRECISION AT THE 20 mitG B i/LITER LEVEL
The correct value lies in the range:
Mean of
ndeterminations
±- 1.3/ni mitgB i/liter.2. PRECISION AT THE 5 MG B i/LITER LEVEL
The correct value lies in the range:
Mean of
ndeterminations
±-1.0/ni inggB i/liter.(The precision of the method depends largely on the presence or absence of inhibitors to the algae in the sample. Precision increases if the level of seawater toxicity decreases.)
B.
OUTLINE OF METHODSeawater samples are filter-sterilized and, if necessary, diluted with a vitamin-free sea water. The dispensed sea water is then supplemented with nutrients. The solutions are inoculated with
Brstarvedcells of
Monochrysis lutheriand incubated for
46hr. Carbon-14 labelled sodium carbonate is then added and the uptake of labelled carbon by the cells measured over a
2-hrexposure in a
constant-lightincubator. Rates of carbon-14 uptake are proportional to
B 1concentrations over certain ranges. Internal standardization is used to account for inhibitors present in the sea water.
C. SPECIAL APPARATUS AND EQUIPMENT
See
III.5,C.D.
SAMPLING PROCEDURE AND SAMPLE STORAGESee
III.5,D.E.
SPECIAL REAGENTS 1. VITAMIN-FREE WATERSee
III.5,E.1.2. NUTRIENT SOLUTIONS
See
III.5,E.2a.169
170 A PRACTICAL HANDBOOK OF SEAWATER ANALYSIS
(b) Vitamin solution. Dissolve 11 mg crystalline B12 and 10 mg of biotin in 100 ml of distilled water. Dilute 10 ml of this solution to 100 ml. Sterilize the diluted vitamin solution by passage through a fritted-glass, UF grade filter. Store in
10-ml portions in test tubes at -20 C in a deep-freezer.
3. RADIOACTIVE CARBONATE
See III.5,E.3.
4. ALGAL INOCULUM (B1 FREE)
To each of two sterile 125-ml Erlenmeyer flasks add 50 ml vitamin-free sea water and plug the flask with a sterile cotton plug enclosed in cheesecloth. Add to each flask 0.25 ml of nutrient solution a, and 0.05 ml of solution b. To one flask add 0.05 ml of Bl solution prepared as described later in Section G. This flask contains a complete medium that is used to maintain algal stocks. Transfer 5.5 ml of an actively growing culture of Monochrysis lutheri (culture should be visibly turbid with cells) to this flask. Incubate the transfer at 22 C in the light incubator for 2 days, then transfer 0.5 ml of this culture to the remaining flask which contains Bl-free medium. After 5 days incubation, the culture in this flask is in the log phase, of growth, almost stripped of Bl and is ready to be used as an inoculum.
The preparation of this inoculum is critical and the times of incubation given should be adhered to closely. Some trial experiments may be necessary to obtain a final suitable inoculum which must contain approximately 5.0 X 105 cells/ml, with no excess vitamin Bl and with the cells still physiologically active.
F. EXPERIMENTAL
PROCEDURE
1. Thaw the samples (Note a) and add duplicate aliquots of between 5 and 20 ml (Note b) aseptically to 50-ml micro-Fernbach flasks. Where necessary, bring the volume in each flask to 20 ml by the aseptic addition of vitamin-free sea water.
2. Add to each flask 0.1 ml of nutrient solution a, and 0.02 ml of nutrient solution b (Note c). To one of each duplicate add a 10 mµg/liter addition of vitamin Bl to serve as an internal standard as described in Section G below (Note d).
3. To each bioassay flask add 0.2 ml of the inoculum of Monochrysis lutheri prepared as described in Section E above, making the initial concentration of cells in each bioassay flask about 104 cells/ml. Should the inoculum cell concentration be notably lower or higher than 5 X 105 cells/ml add to each flask volumes pro-portionally greater or less than 0.5 ml. The addition should have a volume in the range 0.3-0.7 ml.
4. Allow all flasks to incubate in the light incubator at 22 C for at least 46 hr and not more than 50 hr.
5. Add 1.0 ml of 14C-labelled bicarbonate solution, containing 1 µc of activity, at 2 min intervals, to each flask; mix the contents well and replace the flask in the incubator for exactly 2 more hr (Note e).
6. Filter the contents of each flask through a 25-mm diameter HA Millipore filter, wash the sides of the flask with a policeman to detach any cells, and rinse with
111.7. THIAMIN (VITAMIN B1) 171 filtered sea water. Continue the determination as described in the method for photo-synthetic rates, Section V.3, counting the activity with a suitable gas-flow geiger counter. Arrange the times for the addition of radioactive carbonate and filtration so that each sample receives exactly 2 hr (-!-5 min) incubation with the isotope.
7. Using the radioactive count obtained from each sample, read the apparent concentration of vitamin B, from a calibration curve prepared with each batch of samples as described in Section G below. Let this concentration be A mµg B,/liter.
Read the apparent concentration of vitamin Bl in the flask (containing sample plus internal standard) which was taken through the analysis with each sample. Let this concentration be B mµg B1/liter. Calculate the concentration of vitamin Bl in the sample from the expression:
mµg B1/liter = A xB20 ^ x v
where v is the number of ml of sample originally taken for the analysis. If B - A is less than 6.5 repeat the assay using a smaller sample (Note d).
NOTES
(a) The samples should be sterile. This can be achieved if solutions are filtered at the time of collection aseptically through PH Millipore filters. Otherwise solutions obtained as described in Section D above should be sterilized by refiltering immediately prior to the bioassay.
(b) It is necessary to dilute the sample if its Bi content is greater than about 25 mµg Bl/liter or if it has greater than 35% inhibition as determined by the recovery of internal standards (see Note d below).
(c) To avoid adding nutrients by separate aliquots, suitable proportions of the various nutrients may be mixed together just prior to use and supplementation can be made with one aliquot. The final concentration of nutrients in all flasks should be the same as obtained by adding the volumes separately as indicated in F.2 above, i.e., add 0.12 ml of the mixture.
(d) Internal standardization is important because unknown substances in natural sea waters can cause considerable inhibition to the growth of the alga. If inhibition exceeds 25-35%, the calculation described in F.7 becomes unreliable, warranting a repeat determination with a greater dilution of the sample with the vitamin-free sea water.
(e) This time is critical and should be controlled to within 5 min.
G. CALIBRATION
1. STANDARD THIAMINE (VITAMIN BY) SOLUTION
Dry thiamine hydrochloride for 4 hr at 100 C. Dissolve 10.0 mg of the dried thiamine hydrochloride in 100 ml of distilled water. Sterilize the solution by passing it through a fritted-glass filter, UF grade and store in 10-ml portions at -20 C.
2. DILUTION OF Bl SOLUTION
Dilute 1.0 ml of the concentrated standard Bl solution prepared above to 100 ml with distilled water; dilute 1.0 ml of this solution to 100 ml.
1 ml - 10.0 mµg
This solution will be referred to as solution A. Make further dilutions using vitamin-free sea water as follows:
7 ml of solution A to 10 ml (solution B) 5 ml of solution A to 10 ml (solution C)
172 A PRACTICAL HANDBOOK OF SEAWATER ANALYSIS 4 ml of solution A to 10 ml (solution D)
3 ml of solution A to 10 ml (solution E) 2 ml of solution A to 10 ml (solution F) 1 ml of solution A to 10 ml (solution G)
3. PROCEDURE