Microbiologic Assay for Niacin Effect of Total Volume p e r Tube on Accuracy D. RI. HEGSTED AND C. E. ALVISTUR Department of Nutrition, Ministry of Public Health and Social V’elfare, Lima, Peru, Institute of Inter-American Affairs, Inter-American Regional Ofice, Technical Cooperation Administration, Department of State, Department of Nutrition, Haroard School of Public Health, and Department of Biological Chemistry, Harcard Medical School, Boston, Mass.
HE microbiologic techniques for the determination ot rvarious vitamins and amino acids are well known and widely used. They are particularly valuable in routine work, since neither highly skilled technicians nor expensive or complicated equipment are necessary. Ordinarily, about 20 tubes are required t o yield a standard curve and from 8 t o 10 tubes are used for e ich unknonn. 4 s the assay is usually run, a constant amount of basnl medium is measured in each tube, varying amounts of the standard vitamin solution or unknown are added, and the volume of each tube is equalized by the addition of water. The tubes are then plugged or covered, sterilized, inoculated, and incubated until they are ready for titration. The number of separate steps is considerable and when a hundred to several hundred tubes are set up, omission of any step will result in appreciable saving of time. It occurred to the authors that if the vitamin under investigation is the sole limiting factor in growth, as it should be in an ideal assay, considerable variation in the volume of the solution in each tube might be permissible without appreciable change in
0.90
12-
P
10-
s
i
I 8
0
2 60
8
s
i 8-
5
silicate glass test tubw. In the ordinary “constant volume” assay, 5 ml. of double-strength basal medium was placed in eacl) test tube, appropriate volumes of standard niacin solution or t h e extracts to be assayed were then added, and the total volume was completed to 10 ml. with distilled xater. The tubes \vex then plugged, autoclaved, inoculated, and incubated a t 37” C. in the usual manner. Titrations bvere made after 72 hours with approximately 0.1 S sodium hydroxide. In studying the effect of changes in the final volume, all other factors such as the amount of basal medium per tube, the amount and kind of inoculum, etc., were the same as in the constant volume aPsay which was made a t the same time for comparison. The volume effect was investigated in two ways: by comparing standard curves produced when the total volume was 8, 9, or 10 ml. per tube, and by the so-called variable volume assay, in which the basal medium was added in a volume of 8 ml. to each tube. The standard solution of niacin or the extract to be assayed was then added in volumes ranging from 0.2 to 2 ml. depending upon the amount of the solution used. The solutions for this kind of assay were approximately five times as concentrated as thoce used in the conventional assay, where volumes u p to 5 ml. may be added.
I VARIABLE V O L U M E CONSTANT ”
1
4-
r I
2-
8
0
I
I
I
I
I
I
I
1
I
I
8.75
8.95
9.00
9.30 9.50
8.80 9.40
9.80 9.60
RESULTS
Table 1 demonstrates the slight effect on the standard curve when the total volume is varied from 8 to 10 ml. These curves are not obviously different. Variance analysis (Table 11) Show, however, that the final volume does have a detectable influence on the titrat,ion. The F value of 5.22 approaches significance a t the 1% level of probability. There also appears to be a significant interaction between the level of niacin added and the final volume on the titration obtained. In Table I1 are also shown the results of variance analysis of three other similar experiments. In experiment I11 the effect of volume and the interaction failed to reach a significant level. The
ANALYTICAL CHEMISTRY
1636 Table 11. F Values Obtained from Analyses of Variance o f Data i n Table I (Experiment 1) a n d through Other Similar Experiments Experiment
Niacin added
F
Source of Variation Volumea
F
Interactionb
within 10% of the mean (1). Tubes yielding high or low value at either end of the assay -‘ere discarded. The values obtained by the two assays are not significantly different for any of the materials tested. The standard error of the mean value is also approximately the same in both assays.
F
I I1 111
1002 5.22 3.39 2778 586 3.33 1430 0.14 1.35 2731 5.37 2.65 IV a Significance a t 1% probability level requires F value of 5.78 or larger; at 5% probability level 3.47. b Significance a t 1% ‘probability level requires F value of 3.17 or larger; a t 5% probability level, 3.47.
Table IV.
Niacin Content f Std. Error, Y/G.
Constant Substance volume Dark bread 29 f 1 . 5 Whole wheat 52 k 0 . 4 Peanuts 186 k 3 . 7 Fresh carrots 6.3 I.O.lb Whole liver 50 zt 1.1 16 k 0 . 4 Beans Skim milk powder 10 k 0 . 4 a N o t significant for any material tested. Only five tubes in assay.
Table 111. F Values Obtained from Analyses of Variance of Constant Volume Versus Variable Volume Standard Curves Experiment
I
I1
Kiacin added
F
2599 997
Source of Variation Volume F 2 . 4Sa 0.40’
Niacin Content of Various Samples Determined by Two Assay Methods
Interaction F 5 . 16b 4.24
o t significant a t 5% probability level. * NSignificant a t 1% probability level.
Variable volume 30 k 1 . 2 52k0.5 191 I. 4 5.7 I.O.1 49 h O . 8 17 k0.6 11 k O . 8
ta
0.18 0.1 1.19 0 84 0.14 1.36 1.50
5
it is allowed to vary from 8 to 10 ml. Analysis of variance of these results and another similar experiment (Table 111) indicates that the volume is not a significant variable. A significant interaction between volume and niacin content is, however, apparent. The principal point of interest is whether this will have any significant effect upon the accuracy of the assay. Assuming the unknown material is of the correct concentration, so that the acceptable values fall over the center of the standard curve (this is always necessary for an acceptable assay by any method), this interaction should not appreciably affect the assay. This appears t o be true. In Table IV are presented the results obtained with a variety of materials when assayed by either the constant volume or variable volume assay. An acceptable assay was considered to be one in which at least 6 tubes agree
CONCLUSION
In the usual microbiological assay variations in the total volume of 2 ml. or less have relatively small effects on the results obtained. An acceptable procedure for routine assays is to add 8 ml. of basal media t o each tube and the standards or unknown in volumes not to exceed 2 ml. It is unnecessary to equalize the volume of the tubes over this range. LITERATURE CITED
(1) Snell. E. E.. “hficrobioloeical Methods in Vitamin Research.” \-,
in “Vitamin Methods,”-Val. I, edited by Paul Gyorgy, Kew York, ilcademic Press, 1950. (2) Snell, E. E., and Wright, L. D., J . E d . Chem., 139, 675 (1941) RECEIVEDfor review April 1, 1952. Accepted June 13, 1952. Study supported in part b y grants-in-aid from t h e Abbott Laboratories, North Chicago, Ill., National Biscuit Co., Kew York, N. Y., and Williams and Waterman Fund, New York, N. Y.
Gravimetric Determination of Thorium and Rare Earth Elements in Magnesium Alloys G. E. WENGERT, R. C. WALKER, M. F. LOUCKS, AND V. A. STENGER The Dow Chemical Co., Midland, Mich. T H E creep resistance of magnesium-base alloys a t elevated temperatures is increased by incorporation of certain rare earth elements and thorium in the alloy composition (8, 4). Utilization of this fact in the production of magnesium parts for high temperature service has required the development of analytical methods for alloys containing various proportions of these elements. Common procedures for precipitating rare earths offer difficulties of one sort or another when high concentrations of magnesium are present. The oxalates, for example, are appreciably soluble in acidified magnesium salt solutions. If a great excess of oxalic acid is added, or not enough mineral acid, magnesium oxalate may be coprecipitated. The more strongly basic rare earth elements such as lanthanum cannot be precipitated completely as hydroxides unless the alkalinity is so high that magnesium precipitates also. I n the development of a scheme for determining thorium and the rare earths, provision should be made for the probable presence of zirconium and zinc as alloying agents, along with traces of iron, copper, lead, manganese, and aluminum as impurities. Since the methods may be needed in setting up standards to be used in spectrographic control or for other referee purposes, they should be capable of good accuracy and precision. It is also desirable to obtain the rare earths as oxides in order that they may be
redissolved and tested spectrophotometrically for neodymium and praseodymium ( S , 5 ) or volumetrically for cerium. The use of benzoic acid for precipitating thorium in the presence of rare earth elements has been recommended by Venkataramaniah, Rao, and Rao ( 6 ) . The authors have found that zirconium is completely precipitated under the same conditions and that the procedure can be applied to magnesium alloys. Hopkins ( 1 )has pointed out the suitability of ammonium sebacate for separating rare earth elements from those of the alkaline earth group. In the proposed procedure, these reagents are used for bringing about preliminary separations; final determinations are by the oxalate method with ignition to the oxides. Previous experience has shown that oxalate precipitation in acid solution gives a good Eeparation from manganese and the other trace impurities. REAGENTS
Bromophenol Blue Indicator. Place 0.40 gram of bromophenol blue in a mortar, add 8.25 ml. of 0.1 N sodium hvdroxide and mix until solution is complete. Dilute to 100 ml. f i t h distilled water. Ammonium Chloride. A.C.S. reagent. Sodium Sulfite Solution. Dissolve 2 grams of reagent grade sodium sulfite in 98 ml. of distilled water, and adjust to a pH of 2.4. Prepare fresh as needed.
