Showing papers in "Journal of Bacteriology in 1936"

The Use of Semi-solid Agar for the Detection of Bacterial Motility.

Abstract: Although the hanging-drop method for the detection of bacterial motility can be employed successfully, the procedure has several distinct disadvantages. It is so tedious that the determination of this bacterial characteristic is frequently neglected in the routine laboratory. Furthermore, the results are often uncertain because it is difficult to observe motility when only a few of the cells in a culture are motile. Finally, it is necessary to provide relatively young cultures for the examination. The use of semi-solid media for the determination of bacterial motility, on the other hand, eliminates the shortcomings of a hanging-drop technique. The results are macroscopic. They are cumulative, thereby particularly qualifying the method for use in the routine laboratory, where examinations cannot always be carried out at a specific time. Moreover, this method practically eliminates the possibility of overlooking motility when only a small proportion of motile cells are present, because the localized out-growths, which occur wherever motile cells are deposited along the stab, can hardly escape notice. Semi-solid media have been employed for many years in the study of bacterial motility. Rosenthal (1895) reported marked differences in the size and shape of bacterial colonies which had developed in semi-solid nutrient gelatin. Klie (1896) called attention to the spreading and thread-like appearance of colonies

Semisolid Media for Cultivation and Identification of Sporulating Anaerobes

Abstract: This paper amplifies the studies presented serially during the past four years at the annual meetings of our Society, on the use of semisolid media as applied to the routine isolation and identification of the sporulating anaerobic bacilli. These media have been applied tentatively to a variety of aerobes as well, with indications of practical utility here also. This development, however, is a subject for later presentation. The sporulating anaerobes are commonly cultivated in deep solid agar. Formerly such cultures were usually protected by a vaseline or other oily cap, which appears to be an unessential complication, providing the depth of medium is sufficient. Fluid media are also routinely used for certain procedures. For these a vaseline or mechanical seal is requisite, unless incubated in an oxygen-free environment. However, it appears that the two, principles may be combined, as was probably first shown by Pringsheim (1910), by adding only sufficient agar to the fluid base to check convection currents, thus inhibiting aeration. This observation was substantiated and amplified by Jackson and Muer (1911), Lignieres (1919), Hitchens (1921, 1922), Murray and Headlee (1931), among others. When this principle is applied to a liver-peptone broth all of the common anaerobes grow abundantly in the open and un-

A Suggested Standard Method for the Investigation of Electrophoresis.

Abstract: Although the number of papers on electrophoresis is rapidly increasing, the errors in technique to be found in many of them would seem to make a discussion of the necessary safeguards in using such methods desirable. Numerous techniques are available in the literature but the one described in this paper has been used with such uniform results by several investigators, over a period of ten years, that it seems advisable to advance it as a possible standard procedure and to summarize in one place the method in some detail. Many of the developments involved have not been previously described.

An Interesting New Species of Luminous Bacteria

Abstract: The purpose of this paper is to give a full description of an interesting new species of luminous bacteria. Its characteristics make it promising material for investigations dealing with the metabolism of such organisms. At the same time it has been thought desirable to give a more complete account than hitherto recorded of another species, extensively studied in the physiological laboratory of Princeton University (Harvey, 1925; Hill, 1932; Shoup, 1929; Taylor, 1934; and others), and identified as Bacillus Fischeri (Beij.) Migula (Migula, 1900; Hill and Shoup, 1929), or more recently, according to newer nomenclature, Achromobacter Fischeri (Beij.) Bergey et al. (Bergey, 1934; Shoup, 1934; Korr, 1935 a, b; Johnson, 1935, a, b). The cultures of A. Fischeri, utilized in the present study were originally isolated by Dr. Korr from a dead squid at Woods Hole, Massachusetts, in the summer of 1933. The new species was isolated by the senior author from a dead amphipod (Talorchestia, sp.) at Woods Hole, during the summer of 1935. It is morphologically very similar to A. Fischeri, but may be readily distinguished from it, as well as from many other species of luminous bacteria, by virtue of the fact that neither its growth nor luminescence is favored by glycerol, and it produces no acid from this substrate in culture. Other definite physiological and cultural characteristics separate it from all adequately described species that have been reported in the literature.' Because of the numerous fundamental con-

Changes in the Electrokinetic Potential of Bacteria at Various Phases of the Culture Cycle.

Abstract: The investigations of Schultz and Ritz (1910), Reichenbach (1911), Sherman and Albus (1923, 1924), Heiberg (1932), and Sherman and Cameron (1934) have demonstrated that bacteria from freshly made cultures are killed by various agents and temperatures which are innocuous to older cells. Bayne-Jones and Rhees (1929), and Schmidt and Bayne-Jones (1933) have shown an increased rate of heat production per cell during this stage of \"physiological youth.\" Martin (1932), Walker, Winslow, Huntington and Mooney (1934), and Mooney and Winslow (1935) have reported an increased 02 consumption and CO2 and NH3 yield at the same period. In the light of these results, it is obviously a problem of considerable importance to investigate the changes which the bacterial surface may undergo during this phase of the culture cycle. Any alterations in surface properties might be reflected by changes in the electrokinetic potential unless some peculiar set of circumstances were to keep the surface charge density constant. Although the literature of bacterial electrophoresis is voluminous (cf. Abramson, 1934a), no systematic investigation of the influence of the age of the culture on the electrokinetic potential of its cells has been carried out under standard conditions. In 1924, Shibley found that young cells of Pneumococcus Type I and

The Spread of Tularemia through Water, as a New Factor in Its Epidemiology.

Abstract: Rodents are the chief sources of infection of tularemia and the reservoir of its virus in the external world. The r6le of the waterrat, which, in the U. S. S. R., is the principal source of tularemic infection for man is well known. In the United States the same part is played by the wild rabbit. In the U. S. S. R. the r6le of the hare, rabbit, and mouse,-in Norway, that of the wild rathas been proved. Other rodents may serve as reservoirs of virus, for instance: Apidemus silvaticu8, Ondatra zibethica, Didelphi8 virginiana, Marmota flaviventer, Marmota bobac, Microtm ilaeu8, gerbitu8 tamarcnu8, Citelu8 beecheyi Rich., Citellu8 pygmeu Pall, Microtu8 call/ornwcu8-aestuarinus, etc. Animals that do not belong to the rodent-class, such as oppossums, foxes, and lemmings, have also been recorded as sources of infection. The modes by which tularemia spreads from these sources of infection are also being carefully investigated. Contact with water-rats while hunting or flaying them has been well studied in the U. S. S. R. (Zarhy, Hatanever, Golov, Volfertz, Suvorov, Voronkova, Gauzner, Belitzer, Kniazevsky, Berdnikov, Tiflov and others). In the United States contact with wild rabbits during skinning and especially during the preparation of their carcasses is the cause of infection in 61 per cent of cases (Francis). The spread of tularemia through contact with the domestic rabbit was proved for the first time in the U. S. S. R. byMillerincollaboration with Kreutzer, Kvashnina and Grzebina. Its spread through contact with mice has also been proved by Miller con243

Effect of the Chemical Constitution of Soaps upon their Germicidal Properties.

Abstract: Studies on the properties of soaps are of importance because of their common use as detergents, in the preparation of vaccines, in the treatment of disease, and because of their natural occurrence in the animal body where they may have some effect in determining the resistance of the animal organism to infection. Reichenbach (1908) studied the action of soaps on Escherichia coli and found that potassium stearate, pahmitate, and myristate were effective in killing this organism, while the oleate, erucate, and linoleate were inactive. Walker (1924-1926) showed that sodium and potassium soaps of the same acid did not vary greatly in their germicidal action. The lower members of the series such as the butyrate, valerate, caprylate, and caproate, had little or no germicidal effect. He found the pneumococcus to be very susceptible to laurates, oleates linoleates, and linolenates. Streptococci were killed much like the pneumococci but at higher concentrations of the soap. Escherichia coli, Eberthella typhi, Shigella paradysenteriae, Salmonella paratyphi were killed by moderate concentrations (N/20 to N/40) of the saturated soaps (laurates to stearates) but were very resistant to the unsaturated soaps. Eggerth (1926-1931) studied the germicidal action of alpha substituted soaps. He found that the soaps of the alpha bromo fatty acids were usually more germicidal than the unsubstituted. The alpha hydroxy soaps exhibited a high germicidal activity

Adsorption of Bacteria in Salt Lakes.

Abstract: The adsorption of bacteria by porous and powder-like substances has for a long time been the subject of research. It was shown by Kruger (1899) that the number of bacteria present in water may be reduced by addition of coke, sand, clay, brick-dust, magnesium oxide and other powder-like substances. The adsorbent action is much greater in the case of substances with a low specific gravity than with those whose specific gravity is high. The results were confirmed by a number of other investigators. According to Eisenberg (1918), the degree of adsorption of bacteria depends upon their lipoid contents which is higher in the cells of the gram-positive bacteria than in those of gramnegative forms. However, there is no sharp dividing line between these two groups, as regards adsorptive capacity: some gram-positive species (Corynebacterium diphtheriae) are only faintly adsorbed, while some gram-negative species (Serratia marcescens) are well adsorbed. The problem of absorption of bacteria is of particular interest in soil investigation (Chudiakow, 1925-26; Dianowa and Woroshilowa, 1925). The degree of adsorption depends upon the size of the soil particles. The smaller their size the higher is the percentage of adsorbed bacteria. This holds true, however, only up to a certain limit beyond which there is a sharp decline in the degree of adsorption. Soil particles which do not exceed 0.270 do not adsorb bacteria at all. On the contrary, Karpinska (1925) has shown that such particles may be adsorbed by large bacteria, as by the chains of Bacillus mycoides. The same phe-

A Study of the Species Lactobacillus plantarum (Orla-Jensen) Bergey et al.

Abstract: In systematic work it is essential that the characters used to define species shall be of such a nature that at least the majority of the strains can be properly and readily identified. In only relatively few cases have descriptions of species of bacteria been sufficiently comprehensive to include even known variations and, as a result, new strains or variants of species are frequently described as new species. For such reasons it was found difficult to arrange a key for the species of the genus Lactobacillus for Bergey's Manual of Determinative Bacteriology (1934). Many so-called species could not be separated from one another. This situation was particularly evident in those species of non-gasproducing organisms normally found in plant materials or in soil. In a study of over four hundred strains of non-gas-producing organisms isolated from fermenting substances no characters were observed which could satisfactorily be used to separate the cultures into groups that corresponded to described species. It seemed, therefore, that nearly all of the descriptions that have been given are not only too incomplete, but are also too restricted in nature and that more inclusive descriptions should be used. The description of the species Streptobacterium plantarum OrlaJensen (1919) syn. Lactobacillus plantarum Bergey et al., is the

Studies on Hemolytic Streptococci: I. Methods of Classification.

Abstract: That a satisfactory method for the classification of the hemolytic streptococci has not yet come into practical use is illustrated in the last edition (1934) of Bergey's Manual of Determinative Bacteriology, by the description of six species, pyogenes, scarlatinae, puerperalis, erysipelatis, epidemicus and cuniculi, with no distinctive characters other than that they are said to cause different diseases, and that, for some, specific serologic reactions are claimed. Ability to produce a specific disease is a character which may be utilized in bacterial classification, in so far as it may contribute to the differentiation of species. The fact is now well established, however, by the works of many investigators, and especially by the agglutinin absorption studies of Williams and her collaborators, and also those of Griffith, that a given serological type of streptococcus may cause more than one kind of disease. There is also abundant epidemiological evidence that a given strain of streptococcus can cause more than one kind of disease, in the many reports to be found in the literature of more than one streptococcus disease occurring in the same epidemic. For example, among 50 septic sore throat epidemics reported by Williams, cases of scarlet fever occurred in 18, and cases of erysipelas occurred in 11. Hence it is obvious that an attempted differentiation of streptococci into species on the basis of disease source ends in confusion.

The Life Cycle of the Organism Causing Yeast Meningitis

Abstract: In studying the characteristics of several strains of the organism commonly called Torulk histolytica, some rather prominent structures were noted, the significance of which seems to have been overlooked in previous descriptions. The study of the nature of these bodies disclosed a sequence of changes in morphology which necessitates a revision of the classification of at least some of the members of this group. One of the strains studied was isolated on four occasions from the spinal fluid of a patient in the University of Iowa Hospitals. This man had symptoms indicative of lesions in the central nervous system for two years prior to admission in 1934. In January, 1936, his disturbance had neither progressed nor subsided. Another strain was recovered from the spinal fluid of a patient in the Minneapolis General Hospital in 1935. His illness, from onset to death in three months, was characterized by symptoms referable to the central nervous system and at necropsy these tissues exhibited typical lytic lesions. We are indebted to Dr. A. T. Henrici and Dr. N. H. Lufkin for the culture and history of this latter case.

Growth and Fermentation of Bacteria Near Their Minimum Temperature.

Abstract: The object of this paper was to study the minimum temperature of fermentation separately from that for growth, and to explain, if possible, the cause for a minimum temperature. Since both these life functions are chemical reactions, they should continue, though at a greatly reduced speed, until the medium freezes solid. This is certainly not always the case with the growth of bacteria; most of them completely cease to grow at temperatures 5 to 10 or more degrees above the freezing point.

Some Chemical Factors Influencing Growth and Pigmentation of Certain Microörganisms.

Abstract: This is a report on some observations regarding the effects of metals and other substances on the growth and pigmentation of bacteria. A brief explanation of our interest in this field will enable the reader to follow the general trend of our thesis. In a contribution from this laboratory (Kharasch, Legault, Wilder and Gerard, 1936) the factors which govern the oxidation of certain thiol systems to the disulfides have been evaluated. The thiol systems generally are extremely sensitive to traces of heavy metal ion catalysts, particularly those of manganese (Mn++), copper (Cu++), and iron (Fe +++). The effectiveness of these ions as catalysts varies widely. Manganese is catalytically the most effective, while iron is weakest and copper intermediate. It was further demonstrated that the catalytic effects of the metal ions, particularly those of copper and iron, could be inhibited by the addition of various extracts to the system. Thus, beef extract was found to inhibit the catalytic effect of iron, but not that of copper or manganese, in the oxidation of thiols by oxygen. Liver extract on the other hand, was found to inhibit powerfully the catalytic effect of both iron and copper ions, but not that of the manganese ion. The inhibition of copper ion catalysis in thiol systems to which liver extracts had been added was most striking. Furthermore, chicken liver extracts on a

Sonic Energy as a Lethal Agent for Yeast and Bacteria.

Abstract: Crystalline quartz has a certain characteristic which is fundamental to the experiments which we have performed. It is piezo active. Wood and Loomis have described an apparatus which we have utilized with certain modifications and which gives rise to the piezo effect. With this apparatus we have conducted certain experiments dealing with the killing effect of this source of energy upon bacteria and yeasts.' Wood and Loomis (1927) have demonstrated that ultrasonic radiation can disrupt certain higher forms of life. Williams and Gaines (1930) have shown germicidal effect of this wave motion but they used much lower frequency than that which we have applied. Harvey and Loomis (1929) demonstrated the death of luminous bacteria within 1 and 11 hours when treated by this energy. Chambers and Flosdorf (1936) have utilized a low frequency for extraction of certain fractions of the bacterial body. The reader is referred to the description of Wood and Loomis for an understanding of the principles involved and of the circuit utilized. We departed from their type in certain respects, as follows. The generator consisted of a circular cast-bronze housing of approximately 20 cm. diameter at the bottom and curved inward to 10 cm. in diameter at the top. One side was grounded. The bottom of this container was of plate glass and upon it was supported a flat-topped mushroom-shaped electrode upon which

Gram-Negative Bacilli of the Genus Bacteroides.

Abstract: This study was undertaken as an extension of two previous reports on the problem of non-sporulating, anaerobic bacilli; namely, that of Thompson and Beaver, (1931, 1932) and that of Beaver, Henthorne, and Macy (1934). The object of this research was the investigation of the morphologic, biologic, and agglutinative properties of several Gram-negative species of nonsporulating anaerobic bacilli; and a comparison of these species with each other and with the other anaerobes encountered, to demonstrate, if possible, the generic relationships of any or all strains.

The Pure Culture Isolation of Ammonia-oxidizing Bacteria.

Abstract: The autotrophic nature and agricultural importance of the ammonia-oxidizing bacteria has long stimulated the interest of bacteriologists. Studies of these organisms have been neglected, however, because of the difficulties in obtaining pure cultures. Such cultures were first described by Winogradsky (1890) and have since been obtained by several other investigators (Bonazzi, 1919; Heubtilt, 1928; Rubentschik, 1929; Nelson, 1931; Boltjes, 1935). Nevertheless, generally useful isolation methods have not been developed, as illustrated by the fact that no pure cultures were available to the writers in 1933. Since that time, Boltjes (1935) has reported an admirable study of the nitrifying bacteria and has isolated pure cultures of Nitrosomonasl and Nitrobacter. Inasmuch as the pertinent literature on the isolation of nitrifying bacteria has been reviewed recently by Winogradsky and Winogradsky (1933) and by Boltjes, it is necessary to point out only that Nelson's method of picking single cells is impractical, that Winogradsky (1935) in his recent work has neither sought nor possessed pure cultures and that Boltjes employed a micromanipulator for picking his colonies. It was the purpose of the present investigation to develop a method of isolation which would require no special apparatus and in which the colonies could be picked by hand, so that the isolation could be made in bacteriological laboratories in general. The

Attempts to Increase the Heat Resistance of Bacterial Spores.

Abstract: Bacteria are conspicuous among spore-forming plants for the great resistance of their spores to drying, to dyes, to germicidal chemicals and particularly to heat. Heat resistance is at times almost unbelievable and it is this property of bacterial spores which makes their destruction by heat sterilization, as in the processing of canned food, so great a problem. A great deal of work on heat resistance has therefore been done, and it has been found that the resistance of any particular species is not fixed but varies with several factors. The conditions under which the spores are produced as well as their age and previous treatment apparently are important. The concentration of spores, pH of the substrate in which they are heated, and the presence or absence of protective colloids or salts are also factors. These points are discussed in the reports of Burke (1919), Bigelow and Esty (1920), Weiss (1921), Esty and Meyer (1922), Magoon (1926a), Curran (1935), and others. Esty and Meyer have also noted that spores of different strains of the same organism vary widely in their heat resistance and that all of the spores in a particular suspension do not have the same resistance, a small percentage apparently being capable of surviving heating for much longer periods than the majority. A recent confirmation of this fact of individual variation is found in the report of Yesair and Cameron (1936) in which they showed difference in specific gravity between the more resistant and the less resistant spores of a population. This variability has suggested to several 1 Part III of a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The University of Wisconsin, 1936. 589

The Estimation of Bacterial Populations with the Aid of a Photoelectric Densitometer.

Abstract: In a study of the amounts of acid formed by cultures of Lactobacillus acidophilus when grown at constant pH (Longsworth and MacInnes, 1935, 1936) it was considered desirable to compare the acid production with the bacterial population of the cultures. Two different methods for estimating the latter were employed. These were the determination of the concentration of viable bacteria by the plating method and the measurement of the optical densities of the bacterial suspensions with the aid of a photoelectric densitometer. It is the purpose of this paper (a) to describe the densitometer, (b) to outline its manipulation and (c) to interpret the results in terms of the existing theory of the phenomenon. Comparisons will also be given between the densitometer readings and the plate counts. The densitometer. The densitometer used in this research utilizes two vacuum phototubes in the bridge circuit shown in figure 1. It is the same in principle but differs widely in constructional details from the instrument described by Stier, Arnold and Stannard (1933-34). A diagram, drawn to scale, of a view of the side of the instrument in cross-section is shown in figure 2. The phototube P is enclosed and electrically shielded by a lighttight housing constructed from brass tubing. The support for the heat-absorbing water cell at W, the cuvette at C and the diaphragms d is made from square brass tubing. One end of this tubing is soldered to the photoelectric cell housing while the other end passes through a square opening in the side of the lamp house H. A section (aa' in length), of the top side of this tubing is cut 307

On the Detection of Nitrate Reduction.

Abstract: For 30 or 40 years one of the features regarded as most important in the characterization of bacteria has been ability to reduce nitrates. This is so true that in nearly any paper in which some species is described, a statement is given as to whether or not it reduces nitrate and in most manuals of determinative bacteriology this characteristic is given under the descriptions of nearly all species. The majority of such books and scientific papers, however, merely make a categorical statement \"nitrates reduced\" or \"nitrates not reduced,\" as if it were so simple a determination as not to require specification of the methods. Unfortunately, this is not the case. It has been pointed out frequently that there is no simple procedure by which the ability of an organism to reduce nitrate can be categorically determined. There are, to be sure, simple tests which show, in the case of a positive reaction, that nitrate reduction has occurred; but to show that reduction does not occur is a complicated undertaking which is rarely attempted in a routine way. As a matter of fact, in a medium containing nitrate, there are many possible ways in which action on this substance may occur, to wit: (1) Reduction to nitrite, ammonia, and free nitrogen, with or without accumulation of the ammonia and nitrite; (2) reduction to nitrite, then to ammonia as a final end product, with or without accumulation of the nitrite; (3) reduction to nitrite, followed by assimilation of the nitrite, with or without its accumulation in

Micrococcus tetragenus Infection: II. Description of Variant Forms.

Abstract: About 40 years ago a controversy existed as to whether numerous strains of Micrococcus tetragenus existed or if the various kinds then studied were merely variant forms of a single strain. Those favoring the first point of view denoted separate strains by the terms, subflavus (Von Besser), aureus, albus (Boutron), citreus (Vincenzi) and ruber (Bujwid, Trdmolieres). Others believed these to be merely variant forms of a single strain. It appears now, as will be shown in this report, that both views were correct; numerous strains exist and each has variant forms. Among the early investigators were Boschi and Bellei (1897) who noted that a yellow variety lost its chromogenic ability and became white on subsequent transfer and believed that one was a variant form of the other. Similar views were held by Jacobelli (1899) and Vincenzi (1897). Teissier (1896) in 1896 noted that the typical tetrad formation which occurred in vivo became less and less marked on subculture in vitro. He also observed the growth of pinpoint daughter colonies in the original large colonies and the development of a collarette around the mother colony. In 1914 Eisenberg noted the appearance of two distinct colony forms, a large form composed of capsulated virulent tetrads and a small form composed of noncapsulated cocci. Baerthlein in 1918 also noted the appearance of small brownish-yellow, delicate colonies among the typical large yellow ones. The large colonies were composed of typical tetrads, the small of small cocci. Wreschner in 1921 isolated a large gray-white virulent capsulated

Absorption of Staphylococcus Bacteriophages.

Abstract: In a series of papers beginning in 1933 Levine and Frisch have affirmed that extracts derived from susceptible cultures of Salmonella block the lytic activity of bacteriophages active against that group of bacteria. These investigations have been confirmed by Burnet (1934), utilizing organisms and phages belonging to the same group as well as several strains of Shigella. These extracts apparently reflect quite accurately those surface antigenic components responsible for bacteriophage absorption on living and heat-killed susceptible cultures. In the course of our studies with staphylococcus bacteriophages we have been struck by the fact that these phages are exceedingly difficult to isolate from sources rich in phages for members of the enteric group. It may very well be that one of the factors contributing to this difficulty is the absorbing quality of such substances as molds, yeasts, phage-resistant staphylococci, and body exudates (Applebaum and MacNeal, 1931; Rakieten, 1932). Other bacteriophages, especially those attacking organisms of the enteric group are not affected at all by the same agents that have a capacity for what may be termed \"non-specific absorption\" of staphylococcus phages. The absorption of staphylococcus phages by organisms other than staphylococci (to be reported later) has revealed evidence of heterogenetic antigenic relations between staphylococci and otherwise totally unrelated organisms. This report deals with (1) the absorption and inactivation of staphylococcus bacteriophages by extracts prepared from susceptible and resistant strains

The Preparation of Silicic Acid Jellies for Bacteriological Media.

Abstract: Although silicic acid jellies are inferior to agar, gelatin, blood serum or egg white in the preparation of solid media for the majority of bacteriological requirements, there are certain specialized purposes, such as the isolation of autotrophic bacteria, for which an inorganic jelly is necessary. Theoretical advantages from the utilization of silica gel as a specialized substitute for organic gels have not been realized in practice, largely as a result of the erroneous assumption that inorganic gels are inert in the medium. The present study deals with the influence of time, temperature, dialysis and autoclaving on the pH and the rigidity of silica jellies prepared for bacteriological purposes. Many modifications of two classical methods for the preparation of silicic acid jellies have been described by bacteriologists. The \"sol\" method, first applied in bacteriology by Kuhne (1890) and later used by Winogradsky (1891) and by Omeliansky (1899), was adequately reviewed by Bojanovsky (1925) and recently employed by Boltjes (1935) in obtaining pure cultures of nitrifying bacteria. This procedure has a distinct advantage in that the dialyzed and sterilized sol is gelled after addition of the bacterial suspensions to the plates. However, it was not considered of general utility because of the special apparatus required for the preparation of the dialyzed sol and because the liquid of syneresis exuded on the surface of the plates permits a film of contaminants to cover the pure colonies which may develop in the depths of the gel. Boltjes overcame this difficulty with the aid of a micromanipulator for colony picking.

Bacteriophage as Related to the Root Nodule Bacteria of Alfalfa.

Abstract: Bacteriophages virulent for different strains of Rhizobia of leguminous plants have been isolated by various investigators but not much has been learned regarding their significance in the nodules. Laird (1932) established the heterogeneous nature of the Rhizobia with respect to bacteriophage, but his attempt, as well as that of Almon and Wilson (1933), to find a correlation between the effectiveness of different strains in modulation and sensitiveness to the lytic principle was not successful. Among the theories relative to the means by which atmospheric nitrogen fixed by Rhizobia is made available to the plant, Fred, et al., (1932) have listed the action of bacteriophage, but little or no work has been done in this connection. Demolon and Dunez (1935) isolated a bacteriophage from the soil surrounding the roots of old alfalfa plants and concluded that it interferes with symbiosis between the Rhizobia and the host plant. Within recent years a decrease in yield of alfalfa, Medicago sativa, three to five years after seeding has been noted on irrigated soils in the Yakima and Ellensburg districts in Washington. Marked responses from additions of nitrogen have been obtained in experiments in these districts, pointing to the possibility of the presence of some substance which interferes with normal symbiosis between the nodule bacteria and the alfalfa plant. The object of the work presented here was to determine if Rhizobia bacteri-