111.6. DETERMINATION OF BIOTIN INTRODUCTION

The following method uses a biotin-requiring dinoilagellate and measures response to various concentrations of the vitamin by rate of radiocarbon uptake.

The method has been described by Carlucci and Silbernagel (Canadian J. Microbiol., 13: 975, 1967).

METHOD

A. CAPABILITIES

Range: 0.5-4 mlig biotin/liter (greater quantities after dilution) 1. PRECISION AT THE 3Mite BIOTIN/LITER LEVEL

The correct value lies in the range:

Mean of n determinations -±0.28/ni mg biotin/liter.

2. PRECISION AT THE 1 Mi^t20 BIOTIN/LITER LEVEL

The correct value lies in the range:

Mean of n determinations ±0.09/ni mg biotin/liter.

(The precision of the method depends largely on the presence or absence of inhibitors in the seawater sample. The above precision is the worst to be expected with moderate inhibition. In samples where there are no inhibitory properties, the precision is greatly improved.)

B. OUTLINE OF METHOD

Seawater samples are filter-sterilized, and, if necessary, diluted with vitamin-free sea water. The sea water is supplemented with sterile nutrients, inoculated with biotin-starved cells of the dinoflagellate, Amphidinium carteri, and allowed to incubate for 94 hr in a constant-light incubator. Carbon-14 labelled sodium carbon-ate is then added and the uptake of labelled carbon measured over a 2-hr exposure.

Rates of carbon-14 uptake are proportional to biotin concentrations over certain ranges. Internal standardization is used to account for seawater inhibition to alga growth.

C. SPECIAL APPARATUS AND EQUIPMENT See III.5,C.

D. SAMPLING PROCEDURE AND SAMPLE STORAGE See 111.5,D.

E. SPECIAL REAGENTS 1. VITAMIN-FREE WATER See III.5,E.1.

165

166 A PRACTICAL HANDBOOK OF SEAWATER ANALYSIS 2.

NUTRIENT SOLUTIONS

(a) Chelated metals, nitrate and phosphate.

See

III.5,E.2a.

(b) Vitamin solution.

Dissolve

11

mg crystalline

B„

and

10

mg thiamine hydrochloride 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.

ALGA

INOCULUM

(BIOTIN-FREE)

To each of three sterile

125-ml

Erlenmeyer flasks add

50 ml

vitamin-free sea water. Plug the flask with a sterile cotton plug enclosed in cheesecloth. To each flask add

0.25 ml

of nutrient solution

2a,

and

0.05 ml

of solution

2b.

To one flask add

0.05 ml

of biotin solution as described later in Section G. This flask contains the complete medium. Transfer

1 ml

of an actively growing culture of

Amphidinium carteri

(culture should be visibly turbid with cells) to this flask. Incubate the trans-fer in the light incubator at

22 C

for 6± days, then transfer

0.5 ml

of the resulting culture to one of the remaining two flasks containing biotin-free medium. Incubate this culture again for

1

week and then add

5.5 ml

of this second transfer to the remaining flask of biotin-free medium. After

6

days further growth, the cells will be in the log phase of growth, stripped of biotin, and ready to be used as an

inoculum.

The correct preparation of this

inoculum

is essential, and the times of incu-bation given should be adhered to closely. Some trial experiments may be necessary to obtain a final

inoculum

which must contain approximately

5.0 x 10 5 cells/ml,

with no excess biotin and 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

vitarnin-free

sea water.

2. Add to each flask

0.1 ml

of nutrient solution

2a,

and

0.02 ml

of nutrient solution

2b

(Note

c).

To one of each duplicate add a

1.0 nIug/liter

addition of biotin to serve as an internal standard as described in Section G below (Note

d).

3. To each bioassay flask add

0.5 ml

of the

inoculum

of

Amphidinium carteri

prepared as described in Section E. The concentration of cells in the

^inoculum

should be approximately

5.0 X 10 5 cells/ml;

thus, the initial concentration of cells in each bioassay flask is about

104 cells/ml.

Should the

inoculum

cell concentration be notably lower or higher than this, add volumes proportionally greater or less than

0.5 ml

to each flask. 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

94 hr

and not more than

100

hr.

5. Add

1.0 ml

'4C-labelled bicarbonate solution, containing

^{1 pc}

of activity,

111.6 . BIOTIN 16 7 to each flask, at 2-min intervals, 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 diam HA Millipore filter, washing the sides of the flask with a policeman to detach any cells and rinsing with 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.

7 . Read the apparent concentration of biotin from the radioactive count obtained from each sample from a calibration curve prepared with each batch of samples as described in Section G below . Let this concentration be A mµg biotin/liter . Read the apparent concentration of biotin in the flask (containing sample plus internal standard) which was taken through the analysis with each sample. Let this concentration be B . mµg biotin/liter. Calculate the concentration of biotin in the sample from the expression :

mµg biotin/liter = A XB 1 A X v0

where v is the number of ml of sample originally taken for the analysis. If B - A is less than 0 .65 mµg biotin/liter repeat the assay using a smaller sample volume (Note d) .

NOTES

(a) The sample should be sterile . This can be achieved if solutions are filtered at the tim e 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 biotin content is greater than 4 mµg biotin/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 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 ., 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 the inhibition exceeds about 25-35%, calculations presented in E .7 above become unreliable, warranting a repeat determina-tion with a greater diludetermina-tion of the sample with vitamin-free sea water .

(e) This time is critical and should be controlled to within 5 min.

G . CALIBRATIO N

1 . STANDARD BIOTIN SOLUTIO N

Dissolve 10 .0 mg of pure crystalline biotin 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 BIOTIN SOLUTIO N

Dilute 1 .0 ml of the concentrated standard solution prepared above to 100 ml with distilled water ; dilute 1 .0 ml of this solution to 100 ml ; dilute 5 .0 ml of this second dilution to 25 ml with distilled water .

168 A PRACTICAL HANDBOOK OF SEAWATER ANALYSIS 1 ml 2.0 mpg

This solution is to be referred to as solution A. Make further dilution using vitamin-free water as follows:

5 ml of solution A to 10 ml (solution B) 4 ml of solution A to 10 ml (solution C) 3 ml of solution A to 10 ml (solution D) 2 ml of solution A to 10 ml (solution E) 1 ml of solution A to 10 ml (solution F) 0.5 ml of solution A to 10 ml (solution G) 3. PROCEDURE

Add to one of each duplicate sample (Section F.2 above) 0.1 ml of solution F This addition is equivalent to 1.0 mg biotin/liter. The sample with the internal standard is analyzed with the duplicate containing no added biotin and used for the calculation described in Section F.7.

With each series of samples being bioassayed prepare 7 flasks containing 20 ml of vitamin-free sea water enriched with the nutrient solutions as described in F.2.

To one flask make no addition and to the other six flasks add 0.1 ml of solution 13—G, respectively. The concentrations of added biotin in the sea water of the external series will be 5, 4, 3, 2, 1, and 0.5 mg biotin/liter, respectively. Inoculate and incubate these standards as described in Section F.3-6 along with the samples being bioassayed. Prepare a calibration curve by plotting the counts/min against the concentration of biotin in that standard. The biotin concentrations in the samples and the internals are read from the calibration curve.

Note: Cell counts rather than radioactivity may be used to measure vitamin concentrations.

See note in III.5,G.

111.7. DETERMINATION OF THIAMIN (VITAMIN B 1) INTRODUCTION

The following method uses a thiamin-requiring

chrysomonad

as assay organism

Im Dokument A PRACTICAL HANDBOOK OF SEAWATER ANALYSIS (Seite 177-181)