VOL.

84, 1962

NOTES

869

TABLE 1. Equivalence zone data from lysozyme-antilysozyme flocculation tests*

Tube no. Total protein

Lysozyme added
jAg

Lysozymertt and wash

fluids

supernatants with

body present antibody/ boypeetantibody rabbit serumanie

antilysozyme
pg

Total anti-

Molecular ratio

antibocty/antigen

(Ppt)y

activity in
pg

Lysozyme ppt

Ag

pg

10 9 8 7 6

341.6 424.8 539.8 513.3

398.2

29 38.8 58 77.5 116.5

2.09 1.56 0.94 1.14 1.85

++ ++

314.7 387.5 482.7 436.9 283.6

11.6 10.3 8.4 5.7 2.4

1.05/1 1/1.03 1/1.2 1/1.8 1/4.2

0.007 0.10 0.245 0.31 0.39

* Employing 0.5 ml of a 1:2 dilution of antiserum; these data represent a typical result from eight replicate experiments.

= 160,000), the molecular ratios in which the antibody and antigen molecules were combined at equivalence were calculated. These results are presented in Table 1. It was noted that at equivalence and at slight antibody excess the antibody:antigen ratio is approximately 1:1. Assuming that the valence of the antibody is 2,

the valence of lysozyme must also be 2 to permit polymer formation and a precipitate in this system. This investigation was carried out during the tenure of a Predoctoral Fellowship from the Division of General Medical Sciences, U. S. Public Health Service.

MINOR ELEMENT COMPOSITION OF YEAST EXTRACT' C. L. GRANT2 AND DAVID PRAMER of Agricultural Microbiology and Soil Science, Rutgers, The State University, New Brunswick, Departments
New Jersey

Received for publication May 14, 1962

Yeast Extract is employed commonly as a of exogenous factors essential for growth of fastidious microorganisms, but frequently it is necessary or desirable to replace the crude extract with known growth factors and to cultivate microorganisms in chemically defined, rather than complex, media. Recently, the ash of Yeast Extract was demonstrated to be required for growth of the nematode-trapping fungus, Arthrobotrys conoides, in a medium that contained glucose, biotin, thiamine, and various inorganic salts (Coscarelli and Pramer, J. Bacteriol. 84:60, 1962). To provide an experimental basis for the formulation of a mineral solution that was suitable for identification of the required element, Yeast Extract was ashed and the residue analyzed by quantitative spectrochemical methods. The
source

equipment employed included a Jarrell-Ash 3.4-imi grating spectrograph and microphotometer, and a National Spectrographic power source and plate processor. Yeast Extract (Difco) was dried to constant weight at 70 C and 5-g samples were transferred to acid-cleaned Vycor evaporating dishes. These were placed in a muffle furnace, and the temperature was increased gradually to 450 C. The samples were ashed overnight, weighed, and dissolved in 5 ml of redistilled 6 N HCl and 5 ml of deionized water. The solutions were quantitatively transferred to volumetric flasks and made to 50 ml with deionized water. Samples, equivalent to 1.0 g of oven-dry sample, were analyzed for Al, Ba, Cu, Fe, Mg, Mn, and Sr, using the spectrographic procedures described by Shimp et al. (Soil Sci. 83:51, 1957). The I Paper of the Journal Series, New Jersey New Brunswick. elements Cd, Co, Cr, Ga, Mo, Ni, Pb, Sn, Ti, Agricultural Experiment Station, V, and Zn were determined in the remaining 2 Present address: Engineering Experiment of New Hampshire, Durham. solutions (equivalent to 4 g of oven-dry sample) Station, University

870

NOTES
TABLE 1. Minor element composition of Yeast Extract
Element (pg/g dry wt.)

J. BACTERIOL.

number

Ash

Al

Ba

Cd

Co

Cr

Cu

Fe

Ga

Mg

Mn

Mo

Ni

Pb

Sn

Sr

Ti

Zn

385062 406870 416490 419285 421072

12.25 12.71 12.84 14.24 12.33

3.0 2.1 3.0 3.8 3.5

1.1 1.0 1.3 1.7 1.3

1.2 1.3 2.0 1.8 1.3

6.1 10.7 91.7 121 1.0 9.4 53.5 156 4.0 17.4 68.7 155 4.3 11.0 41.6 135 2.0 11.6 101.0185

0.01 1160 0.13 1580 0.20 1540 0.02 980 0.07 1100

3.2 2.0 2.3 1.4 2.4

3.7 6.310.5 2.6 30.8 4.6 8.0 32.9 4.2 6.3 9.0 2.6 9.1 11.9 12.0

0.18 1.0 0.03 1.0 0.14 1.4 0.05 1.2 0.04 0.84

3.2 36.3 104.0 1.4 31.2 58.6 2.9 66.1 104.0 2.9 45.8 57.3 4.8 39.1 46.2

Mean.... 12.87 3.1 1.3 1.5 3.5 12.0 71.3 150 0.09 1270 2.3 5.9 18.2 6.8 0.09 1.1 3.0 43.7 74.0 C(%)*... 6.2 21 21 23 58 26 35 16|93 21|29 47 70 62 77 20 40 31 38
*

Coefficient of variation of individual values.

using the preconcentration spectrographic method originally outlined by Mitchell and Scott [J. Soc. Chem. Ind. (London) 66:330, 1947] as modified by Shimp et al. (Soil Sci. 83:51, 1957). All spectrographic determinations were made in duplicate. The precision, based on agreement between duplicates for all elements, was i1 11 %, expressed as the average coefficient of variation. The results of analyses of samples representing five different batches of Yeast Extract are listed in Table 1. Individual values, means, and coefficients of variation for each of 18 elements are presented. The information is of general

interest and of particular importance when the ash of Yeast Extract is demonstrated to be essential for growth. It facilitated identification of zinc as the element required by A. conoides, and is offered as an aid in deciding on reasonable levels and judicious combinations of minor elements to be employed in experimental nutrition.
The authors are grateful for the capable technical assistance of lrista Uudna. This investigation was supported in part by National Science Foundation research grants G5949 and G19211.

A SIMPLE PHENYLALANINE PAPER STRIP METHOD FOR IDENTIFICATION OF PROTEUS STRAINS

M. GOLDIN AND ARLEEN GLENN Division of Bacteriology, Department of Pathology, Mount Sinai Hospital and Research Foundation, Chicago, Illinois

Received for publication May 17, 1962

The ability of an organism of the Enterobacteriaceae to deaminate phenylalanine to form phenylpyruvic acid is limited to the ProteusProvidence (Proteus inconstans) group and is claimed to be more reliable and specific than the commonly used urease reaction (Henriksen, J. Bacteriol. 60:225, 1950; Shaw and Clarke, J. Gen. Microbiol. 13:155, 1955; Buttiaux et al., Ann. inst. Pasteur 90:133, 1956). Smith and Free (Am. J. Med. Technol. 28:24, 1962) recently reported the use of Phenistix for the differentiation of Proteus. This test, however, requires suspend-

ing cells from a young culture in an aqueous solution of phenylalanine. We have attempted to modify the phenylalanine reaction so that it could be done with a minimum of time and difficulty, by impregnating filter paper strips with the amino acid. Bachrach (J. Clin. Pathol. 13:526, 1960) reported that the identification of Proteus-Providence strains was facilitated by the use of filter paper strips impregnated with tryptophan and urea. We have confirmed the value of this test and have attempted to modify the phenylalanine test so that