180
ANALYTICAL CHEMISTRY
(105) Oberhelman, G. O., Am. J. Sci., 39, 530 (1915). Estimation of pentavalent vanadium by sodium thiosulfate. (106) Oudemans, A. C., Jr., Z . anal. Chem., 6 , 129 (1867). Procedure for direct titration of iron by means of hyposulfates of sodium. (107) Paal, C., and Friederici, L., Ber., 64B, 1766 (1931). Action of sodium hypophosphite on aqueous nickel salt solutions. (108) Ibid., 6SB, 19 (1932). Action of hydrazine on aqueous solutions of nickel salts. (109) Pinkus, A., and Aronsfrau, Ch., Bull. soc. chim. Belges, 41, 549 (1932). Titration of manganese by Proctor Smith process. (110) Pinkus, A., and Ramakers, L., Ibid., 41, 529 (1932). Titration of manganese by Proctor Smith process. (111) Quartaroli, A., IS Congr. intern. q u h pura aplicada, Madrid, 3, 223 (1934). Change (darkening) of cupric hydroxide in relation to tautomers of hydrogen peroxide. (112) Rao, G. G., Ramanjaneyulu, J. V. S., and Rao, V. M., Current Sci., 13, 319 (1944). Catalysis in volumetric analysis. (1 13) Hao, G. G., Ranian,janeyulu, J. V. S., and Rao, V. XI.,Proc. ,Yatl. I n s l . Sczence, I n d i a , 11, 331 (1945). Catalysis in volumetric analysis. (114) Rossler. H., Chcm.-Ztn., 24, 733 (1900). Behavior of rhodium in alloys with noble~metals. (115) Ruff,O., Z . anorg. t i . ( i l l n e w ~Chem., . 185, 387 (1930). Studies in fractional precipitation. Influences of foreign substances in crystal lattice. (116) Ruff, O., and Hiruch, 13.. Ihid., 151,81(1926). Fractional precipitation. Carrying down. .ipparent contradictions of theoretical presumptions. Feigl’s hypothesis on sulfide formation. (117) Sandell, E. H.. ~ S A I . . CHEM.,20, 253 (1948). Colorimetric determinations of traces of gold. (118) Sandell, E. B., and Kolthoff, I. M.,J . A m . Chent. Soc., 56, 1426 (1934). Chronometric catalytic method for determination of micro quantities of iodine. (119) Sastri, B. N., and Yreenivasaya, hZ., Mikrochemie, 14, 159 (1934). Detection of enzymes by spot tests. (120) Schaer, Edward, Anu.,323, 32’(1902). Intensifying (“Activirende”) action of reducing agents, colloidal noble metals, alkaloids, and other basic substances for oxidizing agents. (121) Schmidt, E., and Tornow, E., Chem.-Ztg., 56, 187 (1932). Electrochemical detection of minute quantities of mercury. (122) Schonbein, C. F., J . p m k t . Chem., 89, 1(863). Influence of sulfuric acid on lead salts. (123) Smith, G. McP.. J. An!. Chem. Soc., 39, 1152 (1917). Contamination of precipitates in gravimetric analysis. Solid solution and adsorption us. higher order compounds. (124) Storch, L., Bel... 16, 2015 (1883). Solubility of metallic sulfides in thio acids.
(125) Straumanis, AI., and Ence, E., 2. anorg. u. allgem. Chem.. 228, 334 (1936). System zinc mercuric thiocyanate-copper mercuric thiocyanate. (126) Tammann, G., 2. anorg. u. ollgem. Chem., 107, 1 (1919). Chemical and galvanic properties of mixed crystals and their atomic structure. (127) Tananaev, N.A., and Budkevich, A . A , , Z . anal. Chem., 103, 353 (1935). Detection of oxalate ion. (128) Tananaev, N.A., and Panchenko, G. A,, J . Russ. Phys.-Chem. SOC.,61, 1051 (1929). Detection of vanadium and tungsten. and Gessner, H., Z . anorg. u. allgem. Cheni., 86, 1 (129) Thiel, 9., (1913). Nickel sulfide and cobalt sulfide. Apparent anomalies in behavior of nickel sulfide with acid. (130) Thiel, A., and Ohl, H., Ibid., 61, 396 (1909). Precipitation of nickel sulfide from aqueous solutions. (131) Traube, W.,and Lange, W., Ber., 58B, 2773 (1925). Contribution to knowledge of reduction-oxidation and autoxidation processes. (132) Treadwell, W.D., and Guitermann, K. S..2. anal. Chem., 52, 459 (1913). Separation of cadmium from zinc. (133) Usler, G., Ibid., 34, 391 (1895). Separation of mercury from metals of arsenic and copper groups. (134) Wicke, C., Z . Chemie, 8, 89 (1865). Xew degree of oxidation of nickel and its volumetric determination. (135) Ibid., p. 685. Catalytic conversion of sulfur dioxide into sulfuric acid. (136) Wieland. H., Ber., 45, 679 (1912). Combustion of carbon monoxide. (137) Wieland, H., and Franke, W.,Arm., 464, 101 (1928). Mechanism of oxidation processes. Activation of oxygen by iron. (138) Willard, H. H., and Young, Philena, J . A m . Chem. Sac., 50, 1322, 1368 (1928). Ceric sulfate as a volumetric oxidizing agent. (139) Wilm, T., J. Russ. Phus. Chem. Gesell., 1, 60 (1887). Qualitative separation of tin and mercury. (140) Wohler, L., and Metz, L., 2. anoru. u. allgem. Chem., 149, 297 (1925). Separation of platinum metals. (141) Wohler, L., and Spengel, A,, Z . a n a l . Cheni., 50, 165 (1911). Separation of platinum and tin. (142) Wohlers, H. E., Z . anorg. Chem., 59, 203 (1908). Adsorption phenomena of inorganic salts. (143) Woker, G., Ber., 47, 1024 (1914). Theory of oxidation enzymes, peroxides and catalase reactions of formaldehyde and acetaldehyde. (144) Voker, G., “Chemical Analysis,” Stuttgart, William Bottger, 1911. Catalvsis. Role of catalvsis in analvtical chemistry. (145) Zenghelis, C., Z ; anal. Chem., 49, 729 (1910). Sensitive rea;tion for hydrogen. RECEIVED June 28, 1950.
Medium for Assay of Vitamins with lactic Acid Bacteria LAURA 31. FLYKN, VICTOR B. WILLI.441S, BOYD L. O’DELL, AND ALBERT G. HOGAN University of Missouri, Columbia, Mo. The purpose of this work was to devise an easily prepared and flexible medium suitable for use in microbiological assays of several vitamins. This medium supports excellent growth of four bacterial species commonly used in assays (Lactobacillus casei, Streptococcus faecalis, Lactobacillus arabinosus, and Lactobacillus leichmannii). Growth responses can be measured either turbidimetrically or acidimetrically. The cultures containing only the crystalline vitamins grow in this medium at a rate comparable to that obtained in cultures containing extracts of natural materials. The curves for standard cultures and for unknowns are parallel straight lines when responses are plotted against doses on a log-log grid. One basal medium, with minor changes, can thus be used in assays of folic acid, riboflavin, nicotinic acid, and B I Z activity. The medium is easily assembled, because the constituents are available commercially, and only one adsorption, to purify the casein hydrolyzate, is required. Taken together
these advantages permit a marked saving of time, especially in a small laboratory that may be called upon to assay several vitamins more or less simultaneously.
A
RECEXT review by Snell ( 1 3 ) calls attention to the many
excellent methods which utilize microorganisms for vitamin assays. The multiplicity of methods, hoxever, complicates the management of a small laboratory which may make occasional assays of many different vitamins. The popular methods of assaying these vitamins prescribe different species of bacteria for each vitamin and each method specifies its own medium. Thus, technicians may spend an inordinate amount of time preparing supplements and basal media of limited use. A medium capable of supporting optimum growth for various bacterial species and flexible enough t o be used for several vitamins would be markedly advantageous. Preferably, most of the constituents should be available commercially in dry form to simplify storage, and adsorptions, digestions, and purifications should be kept to a mini-
181
V O L U M E 23, N O . 1, J A N U A R Y 1 9 5 1 Table I. Basal Medium Stock Solution (Medium I) for Assay of Folic Acid with L. casei Double Strength Medium (Su5cient for 100 Tubes) &oms
Acid-hydrolyzed casein (Darco treated) Sodium acetate Glucose
5.0 20.0 20.0
M Q. Asparagine Tryptophan-(L)O Cysteine Guanine hydrochloride Adenine sulfate Uracil Xanthine Glutathione Tween 8 O b
300
Thiamine hydrochloride Riboflavin Calcium pantothenate Nicotinic acid Pyridoxine hydrochloride Aminobenzoic acid %tin Folic acid (pteroylglutamic acid) C
200 500
100 250 5 5 5
10 2.5 50.0
-r 400 400 2000 500 10
...
method of Brown (1) on beads, in evacuated tubes in the refrigerator. Bacteria from beads were subcultured a t least twice, in the appropriate “liquid subculturing medium” (Table 11), before use in an assay or before storage on agar stabs. The incubation temperature used for all the bacteria was 33” C. Inoculated agar stabs were incubated 24 hours and stored in the refrigerator. Fresh stab cultures were made each week. Preparation of Inocula. Cultures for inocula were incubated for 16 to 18 hours at 33” C., and refrigerated until used. The cultures were then centrifuged, and the packed cells were “washed” twice, diluted to small volume, and filtered aseptically through a thin pad of cotton. Sterile “vitamin-free medium” (the basal medium without the vitamins, tryptophan, cysteine, glutathione, and Tween) was used in all washings and dilutions. The filtered suspension was diluted, its turbidity was measured with an electrophotometer, and it was then diluted further to give the desired concentration of bacteria. Each tube in the assays was inoculated with 1 ml. of the final dilute suspension. (An estimate of the number of bacteria added as inoculum in each assay is shown in Table 111.) Preparation of Sample Solutions. Each sample for folic acid assay was blended with 150 ml. of 0.05 M hosphate buffer, pH 7.2, per gram of sample (dry basis). 8aprylic alcohol wm added to prevent foaming and the mixture was autoclaved 15 minutes a t 15 pounds. I t was then cooled, and incubated with
&om 0.5 0.5 0.2 0.01 0.01
KzHPOk KHzPOd MgS04.7HaO NaCl FeS04.7HzO hInS0r.HnOd
Table 11. .ilterations of Medium I for Various Uses
0.1
Vse
Glucose, cysteine, and. glutathione added as 6olids. Other ingredients added in form of solutions. Combine ingredients, adjust to pH 6.8, and make up to 400 ml. with HtO. L-tryptophan may be replaced by 200 mg. of DL-tryptophan. Obtained from Atlas Powder Co., Wilmin ton, Del. For complete medium add 0.5 7 folip aciffor 100 tubes. d Add MnSO4,HzO to medium after adlusting pH. a
b c
mum -4 commercially available, standardized, dehydrated medium would be useful. The media now available in this form have not been subjected, however, to tests rigorous enough to make possible definite conclusions concerning their value in research. Studies made several years ago in the laboratory of the authors, of factors affecting the early growth of L. casei, resulted in the development of a semisynthetic medium in which the organism grew well. Tests revealed its adaptability to assays of folic acid. It has since been tried in the determination of other vitamins with other bacteria. This paper reports the results of these investigntions. EXPERIMENTAL
Organism
Medium Modified
Modifications and Xew Numbern
I1 Add 0.57 folic acid, 0.5
Liquid subculturing medium
L. casei
Agar storage medium
L. casei
.4gar storage medium
S.faeco2is L. leichmannii
IV
Folic acid assay
L. cosei
I
Folic acid assay
8.faecclie
I
VI Omit sodium acetate and soKH~POI. Add 2&.5 dium citrate ihy rate) and change amt. of KIHPOi to 3.10 g.
Riboflavin assay
L . casei
I
VI1
Nicotinic acid assay
L. orobinosus
I
VI11 Omit nirotinic acid and Tween 80, add 0.57 folic acid
BIZactivity assay
L. leichmannii
I
I X Add 0.57 folic acid, 1.5 g. choline chloride, 1.5 g.
I
g. Wilson’s liver fraction
L . arabinOsU8 L . arobinosus
L, and H10 to 1000 ml. Bacto &%o:se agar
I11 Add 6 g. sodium acetate and 1000 ml. water to 27.5 g. dry agar mixture
V Add 12 g. agar
g.
Omit
riboflavin.
0.57 folic acid
add
inositol, 50 mg. pyridoxComposition of Media for Varied Uses. Table I shows the amine, and 50 mg. Tween 80 composition of the foundation medium, as used for the assay of folic acid with L. c u s ~ i . I t differs from a Sufficient for 100 tubes. other media commonly used for this organism in the absence of peptone, the Table 111. Ranges of Response in Standard Cultures addition of glutathione and Tween 80, Growth Response the substitution of the more easily Estimated No. ,of Limiting Vitamin Turbidimetric Acidimetric, soluble cysteine for cystine, and an inTest Bacteria in rnxation 0.1 N acid proOrganism Inoculuma period L-values duced in 72 hr crease in the amount of manganese sulHours M1. fate used. Tests have shown that each L. casei 1.67 X lo4 Folic 2.0 x 10-3 40 0.1427 2.50 of these changes improves the medium. acid 40 0.9390 18.80 Alterations in Medium I to make it suitS.faecalis 5 X 106 Folic 18 0,0505 3.40 acid 7.0 X 10-3 18 0.5530 10.80 able for other purposes are shown in L . casei 3.3 X 106 Ribo24 0.0200 2.00 Table 11. flavin 2.5 X 10-1 24 0,7000 20.00
a-:~t~~~S~;ft~$’n
Maintenance of Stock Cultures. The bacterial strains used- L. casei Yo. 7469, S. faecalis No. 8043, L. arabinosus No. 8014, and L. leichmannii No. 4797-were obtained from the American Type Culture Collection, Georgetown University, Washington, D. C. Desiccated stock cultures were stored according to the
X 105
L . arabinoaua
2.5
L. leichmannii
3.0 X 100
Sicotinic acid Vitamin Biz
6.0 X 10-1 6.4 X 10-4
24 24 24 24
0.1079 1.0110 0,0494 0.3210
2.60 15.80 1,60
9.80
a Estimates based on assumptions that barely visible suspension (optical density -0.0315) containa 10’ bacteria, and, that inoculum ciiltures contained 2.5 X 108 bacteria after 16-18 hours’ incubation in media prescribed.
ANALYTICAL CHEMISTRY
182
desiccated chicken pancreas as suggested by Burkholder ( 2 ) , using 20 mg. of dry pancreas per gram dry Reight of sample, under toluene for 24 hours at 37" C. After incubation it was autoclaved briefly ( 5 minutes at 15 pounds), cooled, and filtered. Samples assayed for riboflavin and niacin were subjected t o a preliminary acid hydrolvsis (autoclaving 15 minutes at 15 pounds in 0.1 N sulfuric acidj, neutralized with sodium acetate, and enzyme-hydrolyzed overnight at p H 4.5 with a mixture of takadiastase, papain, and Pectinol A. The Wilson's liver fraction L was not hydrolyzed, but was assayed in a suspension in water.
>
I 10
2
LOGARITHMIC SCALE 1020 I 2 5 1020
5
IO 08, lot
5 05 z 02 8 01 v
09g
L 5 02
05L
8
07,
a
(L
I
6 2 51
03 g
2
FA-3 L
.
9
6 .
s - 4
I
,
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3
15 0 I 1
>
9
6 I
,
,
12 15" ,
8 1 2 0 4 8 ARITHMETIC SCALE WFlGHT OF SAMPI F PER TUBE
,
e
,
Figure 1. Comparison of Arithmetic and Log-Log Grids Turbidity of L. casei cultures measured after 16 and 40 hours' incubation. Concentration of folic acid instandard cultures (1 and 3) is IO-' microgram times number shown in abscissa (F.A.). Weight of 8pinach in test cultures (2 and 4) is 10-6 gram times number shown on abscissa (S)
Sterilization. Inoculation. and Incubation. The ematv tubes and metal caps were sterilized immediately before 6aEh test The basal medium stock solutions were added in 4-ml. volume, rather than s-rnl., t o permit adding the inocula in a I-ml. volume. An aliquot of standard solution, or diluted sample extract, and water t o bring the volume t o 9 ml. were added to each tube. Sterilization times found suitable under existing laboratory conditions were 10 minutes a t 15 pounds for Medium VI for 8. faecalzs, and 13 minutes at 15 pounds for the media for the other organisms. Each tube was inoculated with 1 ml. of bacterial suspension, and all cultures were incubated a t 33' C. Responses of the bacteria t o the vitamins were evaluated in two ways: in terms of photometric density, and in terms of 0.1 N acid formed during 72 hours' incubation. The time of reading turbidimetric growth responses was adjusted to the type and speed of grov th of the cultures under the experimental conditions. Lengths of incubation times in the studies reported here are shown in Table 111. Plotting and Calculation. The average response in triplicate cultures at each dosage wvas calculated, and dose-response curves were plotted on a log-log basis as suggested by Wood ( I d ) , using graph paper with a log-log grid. Doses of vitamins in the standard cultures were expressed as micrograms of vitamin per tube. Doses of unknowns were given in terms of Keight of sample per tube. IVhen curves from the assays met the criterion of parallelism, all points representing the same response indicated the same level of vitamin, making the calculations quirk and easy. Useful Details of Technique. Xatural sources of groLvth stimulants, such as liver fractions, tomato juice, and tryptone are essential in media used for subculturing and storing exacting bacteria. Ihidentified factors in these natural materials ensure the maintenance of vigorous giowth and sensitivity in cultures of bacteria to be used in assays. Vigorously growing cultures which have been incubated only 16 to 18 hours are bettcr foi inocula than cultures incubated for 24 hours Adding the inoculum in a volume of 1 ml , aseptically through a Brewer pipet, makes inocula in diffeient tubes more uniform. This enables good turbidity readings and reproducibility in replicate cultures. It is imperative that the amino acid preparations used in basal media shall be free from the vitamins t o be assayed. This is
especially true in the case of folic acid, because cultures of S. fuecalis show maximum growth when the medium contains only 8 millimicrograms of folic acid per 10 ml. of culture. Naximum growth of L. casei is attained at a still ]offer concentration, approximately 3 millimicrograms of the vitamin per 10 ml. Many commercial casein hydrolyzates and some preparations of 1tryptophan require special treatmtnt before use in media for assays of folic acid, Solutions of hydrolyzates or tryptophan ( 9 ) (approximately 10% concentration) may he made suitable for assays of this vitamin, even with L. cnsei, by adsorption with Darco 60 at pK 3.0 to 3.5,using 10 t o 15 grams of Darco for each 100 grams of amino acid mixture. Heating the Darco for 2 hours a t 100" C. before use, and stirring the Darco suspension for an hour a t 80" C. as recommended by Couch and Richardson ( 3 )improve the procedure. Solutions of hydrolyzate or amino acids should be tested after adsorption, in a short assay, and the adsorption should be repeated if the "blank" is high. RESULTS AND DISCUSSION
Logarithmic versus Arithmetic Plotting. Both methods of plotting are in common use and both usually yield excellent re-
Table IV. Objective Analysis of Methods of Plotting Factors Important in Construction and Use of Graphs Arithmetic Method Logarithmic Method 1. Simplicity and Graphs are simple, Graphs may seem hard to understand, withease of underunderstandable, and out explanation, to standing familiar to all technicians without mathematical training 2. hid afforded in Differenres in growth Critical evaluation of data is easy, since critical evaluarates of cultures conp a r a l l e l i s m of tion of experitaining standards straight lines for mental data and test solutions are standard and unnot always evident known cultures is an important indication of validity of assay Occasional e r r a t i c Lack of parallelism and occasional e r r a t i o points are sometimes points are immedinot easily detected ately evident 3. Objectivity, Drawing curves to fit Process of drawing and ease in data with precision straight assay lines drawing curves may become subis ohjective lor data jective task, and is inherently more difficult than drawing straight line 4. Length of por- Usable portion of curve Many assays have is brief if range of wider working range tion of curve o n this basis usable for calassay is narrow culation 5. Avoidance of Lines converge near Lines are not crowded crowding on origin on graph graph Vse of graphs on log6. Possibility of It is often impractical or impossible to log basis may permaking calcumake calculations mit calculation of lations from from a few points results from assays fewpointshigh high on curve for based on poor estion curve mates of potency unknown if estimate of potency of sample was poor 7. Time and Calculations are time- Calculations are preconsuming, and evalcise and quick, since judgment reuation of data reany one point on a quired for calquires judgment line may be used, culations in choosing invalid if lines are parallel points to be discarded
V O L U M E 23, NO. 1, J A N U A R Y 1 9 5 1
183
I
.
FOLIC ACID 10 B E E F a C H I C K E N PANCREAS 0 M I L K SOLIDS m SOYBEAN OILMEAL
0~~~ D-~GM IO-~GM
, 2
I
5
10
20
I
,
.
50
80
WEIGHT OF SAMPLE PER TUBE
Growth Response of L . cusei in Acid
Figure 2.
of Folic
Assay
Turbidity measured after 40 hours’ incubation
pret this break, data were compiled correlating estimated numbers of bacteria with photometric density. A dense suspension of L. casei was diluted stepwise to a “barely visible turbidity,’’ and the photometric densities of the diluted suspensions were determined. The “barely visible” suspension (photometric density 0.0315, transmission 93%) was arbitrarily assigned a value of 1 X lo7 bacteria per ml., as suggested by Hinshelwood (6). Estimates were then made of the numbers of bacteria in the more concentrated suspensions. When the photometric densities shown in Figure 2 were changed to estimated numbers of bacteria by the use of these data, the dose-response curves showed the expected pattern of parallelism over the entire range. The important point is that in each series of assays, when dose-response curvea were plotted on a log-log basis, the curve for the standard waa a straight line parallel to the straight-line curves for the unknowns, providing the incubation time was adequate. Blanks and Ranges of Growth Response. A number of points emphasized in the text are illustrated in Table 111: the size of the inoculum, the amount of vitamin added in the standard assay cultures, the blanks, and the spread of the response. Although graphs on a log-log grid do not show the response to zero-dosage (blank), extrapolation toward zero dosage and observation of the slopes of the standard curves indicate that the blanks are low. Table 111shows the blanks in typical assays and compares them with the marked growth responses a t the highest levels of the vitamins. These blanks may be lowered by cutting down the size of the inocula. However, beyond a certain point a reduction in the inoculum size is a disadvantage in that one must allow a longer incubation time before the growth obtained is proportional to the amount of the vitamin present.
t O
*
k
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@ SPINACH
n I
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@ SOYBEAN M E A L @ CHICK PANCREAS
0.2
Titrations made after 72 hours’ incubation ,
0.1
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W E I G H T OF S A M P L E PER TUBE
sults. Because t.he authors, however, prefer logarithmic plotting, it seems worth while to list objectively the advantages and disadvantages of each method (Table ITT). Figure 1 presents typical data from a folic acid assay witahL. casei,-plotted on both logarithmic and arithmetic bases for comparison. Results calculated from the arithmetic dose-response curves for 16 hours incubation viould not be valid, and it is necessary to wait until the turbidity responses reach the comparative values shown a t the end of 40 hours. Thc,sc facts might not be evident, from the arithmetic graph, to a person unaccustomed to Jvorking with curves. Thc snme data can be evaluated a n d interpreted easily if plotted on a log-log basis. Graphs of photometric densities of cultures incubated for 16 hours do not show parallelism; therefore the assay data are not reliable a t this early stage. ( I t is assumed that the spinach extract contained materials which stimulated the early growth of the cultures). After 40 hours’ incubation the dose-response curves on the log-log grid h:ive become parallel, and valid assay results may be calculated. Parallelism of Dose-Response Curves. The straight-line doseresponse curves on graphs of optical densities from turbidimetric data sometimes exhibit a break, as shown in Figure 2. To inter-
Figure 4. Growth Response of S. faecalis in Folic Acid
Assay
of
Turbidity measured after 18 hours’ incubatioa
As shown in Table 111, the estrc~mc~ sensitivity of L . casei to folic acid makes it necessary to uso i t smallcr inoculum than that used in assays of the same vitamin with S. Jaecalis. The rapid growth of L . casei and L. arabinosus in assays of riboflavin and of nicotinic acid in this medium permits the use of very small inocula in these assays, with low blanks and wide ranges of response. Small inocula are preferable in assays of B12 with L. Zeichr mannii. The authors have found this niediuni snd its variations satisfactory for routinc assays, without the additiou of other growth stimulants. This is not to say that the medium could not be improved by changes suggested sincr the work reported in this paper was complrtrd. Folic Acid Assays with L.casei or S. j a e c d i s . Figures I, 2, and 3 present data from typical assays of folic acid, with L. casei
ANALYTICAL CHEMISTRY
184
crepancy may indicate inadequate extraction of the vitamin. Even greater hIicrobiologica1 Assays deviation is shown between assays for the Turbidimetric Acidimetric dry turnip greens, the microbiological L. casei L. caeei assays indicating only 43% of the Roberts S. faecalis, Roberts S. faecalis, Medium & h e l l Medium Medium & 8nell Medium Chick amount found in the chick assay. The Material Assayed I medium VI I medium VI Assaysa difference between results from microMicrograms per gram biological and chick assays of folic acid Beef, dehydratedanddefatted 0.392 0.315 0,360 0.380 0,340 0.400 0.300 in egg yolk does not necessarily indicate Chicken pancreas, dehydrated 3.40 2.38 3.16 2.22 ... and defatted incomplete extraction of the vitamin, 0.392 0.447 0.’605 2.1 0.538 Egg yolk. dehydrated 0.052 0:044 0.098 0,040 0.57 0.076 0.042 Milk solids fat-free although this may be the case. It was 9.93 8.95 9.93 9.26 9.96 10.38 14.0 Mustard greens, dehydrated expected that the egg yolk would be low 4.27 4.10 4.19 4.93 4.68 4.29 5.0 Soy flour 6.05 7.10 6.69 5.95 6.31 6.17 ... Soy meal in folic acid, so the test material was fed 14.27 14.43 15.92 15.33 16.33 14.92 17.8 Spinach, dehydrated ... ... ... 13.9 5.99 Turnip greens, dehydrated at a high level in the diet. The synthetic 22.93 20.90 ... ... 26.3 20.65 ... Yeast, brewers’ basal diet contained acid-washed casein a Chick assays made with technical assistance of J: E. Savage. Synthetic pteroylglutamic acid and all the crystalline B-comples vita(Folvite 7-7904) used as standard in both rmcrobiological and chick assays. Preparation would be considered 90 or 98% pure, depending upon which physical constants are accepted as oorrect (4). mins commercially available a t the time. It Mas not, however, supplemented with vitamin Bl2 (10, f a ) or a good source of the animal protein factor. The egg yolk Table VI. Results of Assays of Several Vitamins probably contained large amounts of the Microbiological Assays animal protein factor, or other unidentiResults Assays by Material Test TurbidiAcidiOther fied growth stimulants. The growth of A 8 sayed Vitamin orgamsm Medium metric metric Methods the chicks that received egg yolk was Y/Q. Y/Q. 5/Q. Flour, enriched Riboflavin L. case; VI1 0.00256 much better than the growth of those A.O.A.C. .. . 0.60251 0.00245 that received the diet with an optimum (.4,0.A.C . flooroamount of crystalline pteroylglutamic metric) acid, so one cannot assume that folic hlustard greens, de- Riboflavin L. casei VI1 1.62 ... hydrated 1.55 acid was the only limiting factor in the basal diet. Riboflavin Assays with L.casei. Data obtained in assays of riboflavin, using Medium VI1 (Table 11) with L. casei, Av. 8 0 . 8 demonstrate the applicability of the Wilson’s liver frac- Vitamin BIP L . leichmannii IX 0.350d . . . 0.389/ medium to assays of this vitamin. Retion L activity 0.357 jmicrobiolog0.335‘ ical) sults from a typical assay are included in Table VI. Usually only turbidity measa After 24 hours’ incubation. b After 40 hours‘ incubation. urements are made in riboflavin assays, c From data in Figure 5. d From data in Figure 6. because the results calculated from ture Average from 8 testa. bidities (read after 24 or 40 hours’ in/ Data from Peeler et al. (8). cubation) check those obtained by titration. Dehydrated mustard greens, re cently adopted as a check sample in the in hledium I. Folic acid assays with this organism frequently laboratory of the authors, are routinely run in each assay. Riboflavin values for this material, as determined from microrequire a 40-hour incubation period to ensure parallelism in graphs of optical densities. If the speed of an assay is of greater biological assays in Medium VII, have been found to check conimportance than a high degree of precision, turbidity readings sistently within less than loyo. The results agree with those obtained fluorometrically. made after 24 hours’ incubation give fair approximations of the folic acid content of cultures. Data shown in Figure 4, from a A sample of enriched flour was assayed for riboflavin according representative folic acid assay with S. faecalis, demonstrate the to the A.O.A.C. directions as given by Loy ( 7 ) . The riboflavin suitability of the modified medium (Medium VI, Table 11) for content assayed fluorometrically by the official method was 2.45 assays with this organism. With each organism, calculations from acidimetric measurements give the same results as those from turbidimetric measurements. Table V- shows the good correlation between results of microI biological assays in Medium I with L. casei, in Medium VI with 8. faecalis, and in the Roberts and Snell medium ( 1 1 ) with L. cusei. The good agreement between results of assays in these 0.5 lo[ media and results of assays with either L. casei or 8. fueculis in other commonly used media wa8 confirmed in recent studies under 2 MUSTARD GREENS IU’GMS. the auspices of the Association of Official Agricultural Chemists a ENZWE BLANK IO” GMS. ( 4 , 5 ) . The data from chick assays in Table V are of interest because the literature contains few data comparing results of assays t , , I I , ,111 I l l of folic acid, where the same foodstuff has been subjected to both 0.5 IO 2.0 5.0 10.0 20.0 50.0 100.0 microbiological assay and bioassay. There is fair agreement beWEIGHT OF SAMPLE PER TUBE tween microbiological assays and chick assays of soy flour and of Figure 5. Growth Response of L. orabinosus in dehydrated spinach. Folic acid in dried mustard greens, as deAssay of Nicotinic Acid termined by the microbiological method, was only about i O % of Turbidity measured after 24 hours’ incubation the amount in the greens as found in the bioassay. This disTable V.
Comparison of Results of Assays of Folic Acid
2or
8
V O L U M E 23, N O . 1, J A N U A R Y 1 9 5 1
185
milliniicrograms per gram, determined microbiologically by the A.O.A.C. procedure it was 2.51 millimicrograms per gram, and using the medium suggested in this paper the flour was found t o contain 2.56 millimicrograms per gram.
I .o L
I C
a 051
the test organism have been carried out with Medium I X another modification of Medium I. Data from a typical assay are shown in Figure 6. From these data Wilson’s liver fraction L w w calculated t o contain 0.350 microgram of BI? activity per gram. The average result from eight successive assays of this fraction was 0.335 microgram of BIZactivity per gram. These results are comparable t o the value reported by Peeler et nl. (8) (0.389 microgram per gram) from microbiological assays. In the authors limited experience with L. leichmannii periods of unexplained failures have been more frequent than in assays with other organisms. These difficulties may have been due to minor changes in procedures, the importance of which was not appreciated. 4CKYOWLEDGMENTS
I
1
2
.5
IO
I
20
1
I
I I I I I
50
100
WEIGHT OF SAMPLE PER TUBE
Figure 6.
G r o w t h Response of L. Zeichnzannii in Assay of V i t a m i n BIZActivity
Turbidity measured after 24 hours’ incubation
Tween 80, a polyosvethylene derivative of sorbitol mono-oleate suitable for use in microbiological cultures, was generously s u p plied for this work by the Atlas Powder Co., Wilmington, Del. The dehydrated mustard greens and dehydrated spinach were gifts from California Vegetable Concentrates, Inc.. Huntington Park, Calif., and the s o y flour was a gift from the A. E . Staley Manufacturing Co., Dccatur, Ill. LITER4TURE CITED
Nicotinic Acid Assays with L. arabinosus. Figure 5 , which presrnts data from a typical assay of nicotinic acid, demonstrates the suitability of Medium T’III for the assay of this vitamin v i t h L. arabinosus. Results from the turbidimetric data (Figure 5) indicate that the mustard greens contain 79.1 micrograms of nicotinic acid per gram. From titration data on the same cultures the mustard greens were estimated to contain 78.5 micrograms per gram. This medium has been used successfully for nicotinic acid assays for several months, and turbidity readings of 24-hour cultures have given consistently good results. Table VI s h o w the uniformity of results obtained. All values are for the same batch of dried mustard greens, but they include any errors in sampling and extraction, as a different sample was extracted for each of the three assays. Comparison of the results from turbidimetric and acidimetric data reveals that the values differ from each other by no more than 10% and all fall within less than 6% of the over-all average. B12Activity with L. leichmannii. Assays of Bl2 activity using L Zezchrnannii (American Type Culture Collection No. 4797) as
Brown, J. H., Science, 64,429 (1926). Burkholder, P. R., XcT’eigh. I., and %’ilson, K., A r c h . Biochem., 7,287 (1945). Couch, J. R., and Richardson, L. R., A . and M. College of Texas, College Station, Tex., personal communication. Flynn, L. M., J . Assoc. Ofic.Agr. Chemists, 32,484 (1949). Ibid., 33,633 (1950). Hinshelwood, C. N.,“Chemical Kinetics of the Bacterial Cell,” p. 30, London, Oxford University Press, 1946. Loy, H. W., Jr., J . Assoc. Ofic. Agr. Chemists, 32,461 (1949). Peeler, H. T., Yacowita, H., and Norris, L. C., Proc. Soe. Esptl. B i d . Med., 72,515 (1949). Popper, Rm., Jr.. Research Dept., California Packing Corp., 4204 Hollis St.,Emeryville 8 , Calif., personal communication. Rickes, E. L., Brink, S . M.,Koniusay, P. R., Wood, T. R., and Folkers, K., Science, 107,396 (1948). Roberts, E.C.,and Snell, E. E., J . Bid. Chem , 163,499 (1946). Shorb, M. S., Science, 107,397 (1948). Snell. E. E..Phusiol. Rev.. 28.255 (1948). RECEIVED November 27, 1948.
Contribution from the Department of Agricultural Chemistry, Missouri Agricultural Experiment Station, Journal Series, No. 1117.
Determination of Parathion in Air Samples by UItraviolet Absorption Spectroscopy ROBERT C. HIRT, Stamford Research Laboratories, .4merican Cyanamid Co., Stamford, Conn., AND J. B. GISCLARD, Central Medical Department, .4merican Cyanamid Co., Xew York, N . Y .
A
li ACCURATE determination of the concentration of aro-
matic vapors in the atmosphere is of considerable importance in industrial hygiene investigations. In view of its toxicity, one of the most important aspects of the manufacture of parathion and its powder formulations has been the necessity of maintaining a workroom atmosphere free of this material Part of the program of ensuring safe and satisfactory working conditions has been the routine collection of atmospheric samples, for nhirh a rapid and sensitive ultraviolet spectrophotometric method of anal? -is has been applied. This method was developed for the deterniination of nitro-
twizene, chlorobenzene, and aniline vapors in the atmosphere, and u a s extended to the determination of parathion present as vapor, mist, or air-borne impregnated dust. This method complements the procedure of Averell and Norris (1) for determining small amounts of parathion residues, but the two methods are not interchangeable for most applications. Parathion is also kno\\ n by its chemical name, 0,O-diethyl 0-p-nitrophenyl thiophosphate. I t s properties ( 4 ) , applications (S),and toxicity ( 2 )have been described by various authors. The presenre of the strongly chromophoric pnitrophenyl grouping i n the molecule gives rise to an intense, broad absorption band in
