Isolation of an Escherichia coli mutant deficient in glutathione synthesis. A mutant of Escherichia coli that contains essentially no detectable glutathione has been isolated. The mutant contains a very low level of the enzyme glutathione synthetase and accumulates lambda-glutamyl cysteine at a concentration approximately equal to the level of glutathione found in its parent. No significant differences in growth were observed between the mutant and its parent. However, the activity of at least one enzyme was found to be affected by the absence of glutathione; the specific activity of the B1 subunit of ribonucleoside diphosphate reductase was greatly reduced. The possibility that the decreased B1 activity is due to a mutation in the structural gene coding for B1 or its regulatory gene could be eliminated. This suggests that one role of glutathione in the cell is to maintain at least this one protein in an active state. We propose the designation gshB for the gene coding for glutathione synthetase.


Escherichia coli deoxyribonucleic acid synthesis mutants: their effect upon bacteriophage P2 and satellite bacteriophage P4 deoxyribonucleic acid synthesis. Escherichia coli C strains containing different deoxyribonucleic acid (DNA) synthesis mutations have been tested for their support of the DNA synthesis of bacteriophage P2 and its satellite phage P4. Bacteriophage P2 requires functional dnaB, dnaE, and dnaG E. coli gene products for DNA synthesis, whereas it does not require the products of the dnaA, dnaC, or dnaH genes. In contrast, the satellite virus P4 requires functional dnaE and dnaH gene products for DNA synthesis and does not need the products of the dnaA, dnaB, dnaC, and dnaG genes. Thus the P2 and P4 genomes are replicated differently, even though they are packaged in heads made of the same protein.


Indirect selection of bacterial plasmids lacking identifiable phenotypic properties. A procedure is described that uses an indicator plasmid (pSC201) to identify cells in a bacterial population that have been co-transformed with a second plasmid lacking detectable phenotypic properties. Under appropriate conditions of indirect selection, between 50 and 85% of transformants carrying the indicator plasmid also contain the nonselected plasmid. A temperature-sensitive mutation in the replication functions of the indicator plasmid enables its elimination from doubly transformed bacteria. Using this procedure, we have isolated bacteria that carry only the small cryptic plasmid. P15A, of the Escherichia coli strain 15. This genetic element, which contains only 2,300 nucleotide pairs, is thus capable of functioning as a replicon independently of the two larger plasmids normally associated with it in E. coli 15 strains (Ikeda, Inuzuka, and Tomizawa, 1970).


Growth rate of Enterobacteriaceae at elevated temperatures: limitation by methionine. The effect of elevated temperatures on growth rate was studied in five strains of Enterobacteriaceae. In all the strains tested a shift to the elevated temperature resulted in an immediate decrease in growth rate which was due to limitation in the availability of endogenous methionine. The first biosynthetic enzyme of the methionine pathway-homoserine transsuccinylase-was studied in extracts of Aerobacter aerogenes, Salmonella typhimurium, and Escherichia coli and was shown to be temperature sensitive in all of them.


Role of methionine in the regulation of serine hydroxymethyltransferase in Eschericia coli. Significant derepression of serine hydroxymethyltransferase is observed when metE or metF mutants of Escherichia coli K-12 are grown on D-methionine sulfoxide instead of L-methionine. The derepression is not prevented by addition of glycine, adenosine, guanosine, guanosine, and thymidine to the growth medium of methionine-limited metF cells showing that the effect is not due to a secondary deficiency of these nutrients. On the other hand, methionine-limited growth of a metA mutant leads to derepression of met regulon enzymes, but only a marginal increase in serine hydroxymethyltransferase activity. A prototrophic metJ strain grown on minimal medium has about the same serine hydroxymethyltransferase as the wild type. The enzyme activity of the metJ strain is not influenced by methionine, but it is partially repressed by glycine, adenosine, and thymidine. metK strains have about twice as much serine hydroxymethyltransferase activity as wild-type cells when grown on minimal medium; but when both types of cells are grown on medium supplemented with glycine, adenosine, guanosine, and thymidine, their enzyme activities are about the same. The results show that methionine limitation can lead to depression of serine hydroxymethyltransferase, but that the regulatory system is different from the one which controls the methionine regulon.


Mutations affecting transport of the hexitols D-mannitol, D-glucitol, and galactitol in Escherichia coli K-12: isolation and mapping. Mutants of Escherichia coli K-12 unable to grow on any of the three naturally occurring hexitols D-manitol, D-glucitol, and galactitol and, among these specifically, mutants with altered transport and phosphorylating activity have been isolated. Different isolation procedures have been utilized, including suicide by D-[3H]mannitol, chemotaxis, and resistance to the toxic hexitol analogue 2-deoxy-arabino-hexitol. Mutations thus obtained have been mapped in four distinct operons. (i) Mutations affecting an enzyme II-complexmt1 activity of the phosphoenolpyruvate-dependent phosphotransferase system all map in gene mtlA. This gene has previously been shown (Solomon and Lin, 1972) to be part of an operon, mtl, located at 71 min on the E. coli linkage map containing, in addition to mtlA, the cis-dominant regulatory gene mtlC and mtlD, the structural gene for the enzyme D-mannitol-1-phosphate dehydrogenase. The gene order in this operon, induced by D-mannitol, is mtlC A D. (ii) Mutations in gene gutA affecting a second enzyme II-complexgut of the phosphotransferase system map at 51 min, clustered in operon gutC A D together with the cis-dominant regulatory gene gutC and the structural gene gutD for the enzyme D-glucitol-6-phosphate dehydrogenase. The gut operon, previously called sbl or srl, is induced by D-glucitol. (iii) Mutations affecting the transport and catabolism of galactitol are clustered in a third operon, gatC A D, located at 40.5 min. This operon again contains a cis-dominant regulatory gene, gatC, the structural gene gatD for galactitol-1-phosphate dehydrogenase, and gene gatA coding for a thrid hexitol-specific enzyme II-complexgat. Other genes coding for two additional enzymes involved in galactitol catabolism apparently are not linked to gatC A D. (iv) A fourth class of mutants pleiotropically negative for hexitol growth and transport maps in the pts operon. Triple-negative mutants (mtlA gutA gatA) do not have further transport or phosphorylating activity for any of the three hexitols.


"Killer" character does not influence the transmission of mitochondrial genes in Saccharomyces cerevisiae. Three cytoplasmic genetic elements have been shown to be separate from mitochondrial deoxyribonucleic acid (DNA), [rho], in Saccharomyces cerevisiae: the killer character [k], omicron-DNA, and psi [psi]. Griffiths has suggested genetic interactions between [VENR] and [TETR] mutants possibly located on omicron-DNA and mitochondrial genetic markers, but possible interactions between the best characterized of the three, the killer character, and mitochondrial DNA have not been investigated. To test this we isolated cycloheximide-induced nonkiller segregants (NKS) of killer cells with suitable genetic markers and mated them in [k] x [k], [k] x [k], and (NKS) x (NKS) combinations. No differences in quantitative mitochondrial marker transmission between these groups were found in crosses illustrating the mitochondrial phenomena of bias, polarity, and suppressiveness. Our studies show that no intercellular interactions between [k] and (NKS) cells influence mitochondrial transmission genetics. Intracellular interactions between the smaller double-stranded ribonucleic acid of [k] and mitochondrial DNA also were not detected.


Genetic mapping of a mutation that causes ribonucleases III deficiency in Escherichia coli. the mutation that causes ribonuclease III (RNase III) deficiency in strain AB301-105 of Kindler et al. (1973) has been mapped by use of F' merodiploids, Hfr matings, and P1 transduction. This mutation, rnc-105, lies close to nadB, near 49 min on the genetic map of Escherichia coli. The rnc-105 mutation has been transferred from its original genetic background by transduction and conjugation, and these new strains have the same defects in ribonucleic acid processing reported previously for AB301-105. Strains that carry rnc-105 grow more slowly than parental rnc+ strains, but the difference in growth rate seems to depend on the genetic background of each strain. Bacteriophage T7 grows about equally well in RNase III+ and III- female strains of E. coli, even though the specific cuts that RNase III makes in T7 ribonucleic acid are not made in the RNase III- strains. A low-phosphate defined medium in which most E. coli strains seem to grow well was developed. This medium is equally useful for labeling ribonucleic acids with 32PO4 and as a selective medium for genetic manipulations. It was used to determine the growth requirements of strain AB301-105, which are biotin and succinate in addition to the methionine and histidine requirements of the parental strain. The biotin mutation lies near the position expected from known mutations of E. coli, but the succinate mutation apparently does not. The possibility that the succinate requirement could be due to the RNase III deficiency is discussed. A uraP mutation was isolated for use in transferring rnc-105 between strains by conjugation. It lies near 47 min, somewhat removed from the commonly accepted position for uraP.


Mapping and characterization of a mutation in Escherichia coli that reduces the level of ribonuclease III specific for double-stranded ribonucleic acid. Localization of a mutation affecting ribonuclease III activity (an enzyme specific for double-stranded ribonucleic acid) in Escherichia coli was attempted. By a series of matings and transduction experiments, the mutation rnc-105 was mapped near the nadB gene. In strains carrying this mutation, another mutation (ranA2074) was also found. Based on available data, their order on the E. coli chromosome appears to be tyrA, ranA, nadB, rnc, purI. Strains carrying either the ranA2074 or the rnc-105 mutation fail to grow at 45 C in enriched medium, whereas strains carrying only the rnc-105 mutation are defective in ribonuclease III activity. Strains carrying either of these mutations grow more slowly than corresponding wild-type strains in all media tested at all temperatures; the rnc-105 mutation reduces the growth rate more than the ranA2074 mutation. T4 and T7 bacteriophages form plaques with a lower efficiency on strains carrying the rnc-105 mutation than on other strains. Thus we suggest that ribonuclease III is beneficial for normal growth of E. Coli and that at higher temperatures it becomes indispensable.


Transfer ribonucleic acid methylase deficiency found in UGA supressor strains. Extracts of recessive UGA suppressor strains, designated supK, are deficient in transfer ribonucleic acid (tRNA)-methylating activity when compared to wild-type extracts. Moreover, the tRNA from suppressor strains is methyl deficient when compared to wild-type tRNA. This deficiency is due to the lack of a single tRNA methylase activity in suppressor strains. UGA suppressor activity may be caused by the miscoding of one or more methyl-deficient tRNA's in supK strains.


Nature and properties of hexitol transport systems in Escherichia coli. In Escherichia coli K-12 the naturally occurring hexitols D-mannitol, D-glucitol, and galactitol are taken up and phosphorylated via three distinct transport systems by a mechanism called either group translocation or vectorial phosphorylation. For every system, a membrane-bound enzyme II-complex of the phosphoenolpyruvate-dependent phosphotransferase system has been found, each requiring phosphoenolpyruvate, enzyme I, and HPr or alternatively P-HPr as the phosphate donor. Cells with a constitutive synthesis of all hexitol transport systems but with low P-HPr levels have very low transport and phosphorylating activities in vivo, although 40 to 90% of the enzyme II-complex activities are detected in cell extracts of such mutants. No indications for additional hexitol transport systems, especially for systems able to transport and accumulate free hexitols as in Klebsiella aerogenes, have been found. Substrate Km, and Vmax of the three transport systems for several hexitols and hexitol analogues have been determined by growth rates, transport activities, and in vitro phosphorylating activities. Each system was found to take up several hexitols, but only one hexitol serves as the inducer. This inducer invariably is the substrate with the highest affinity. Since bacterial transport systems, as a general rule, seem to have a relatively broad substrate specificity, in contrast to a more restricted inducer specificity, we propose to name the system inducible by D-mannitol and coded by the gene mtlA the D-mannitol transport system, the system inducible by D-glucitol and coded by gutA the D-glucitol transport system, and the system inducible by galactitol and coded by gatA the galactitol transport system.


Effects on specific antibodies on the catalytic activity of L-asparaginase from Serratia marcescens and Escherichia coli. Rabbit antisera against highly purified L-asparaginase from Serratia marcescens and from Escherichia coli showed up to 60% inhibition of the catalytic amidohydrolysis of L-asparagine when combined with the homologous enzyme. This inhibition was diminished somewhat against the heterologous enzyme. Kinetic studies in the presence of these antisera showed an increased Kmapp for both homologous and heterologous enzymes using L-asparagine as substrate. In contrast, kinetic studies employing the poor substrate, L-glutamine, showed activation attributable to specific antibodies. This was seen in lower Kmapp values and up to twofold increases in the Vmax over the normal rabbit serum controls. The high degree of cross-inhibition (approximately 80%) and the low degree of cross-reactivity in the quantitative precipitin test (approximately 34%) suggest that these two enzymes possess structural similarities located mainly in the regions of the catalytic sites.


Lag in adaptation to lactose as a probe to the timing of permease incorporation into the cell membrane. If bacteria are incapable of forming and incorporating proteins into the cytoplasmic membranes in all phases of the cell cycle, then not all cells from an asynchronous culture should be capable of growth when switched to a new carbon and energy source whose metabolism requires new membrane function. The transfer of an inducible culture to low lactose provides such a situation since the cells cannot grow unless galactoside permease can function to concentrate the lactose internally. From such experiments, it was concluded that the Y gene product of the lac operon is synthesized, incorporated, and can start functioning in active transport, at any time throughout the bulk of the cell cycle. Not only were the lags before growth re-ensued much shorter than would be expected if the membrane transport capability could only be developed in  a small portion of the cycle, but brief pulses of a gratuitous inducer shortened the lags much further. Three types of Escherichia coli ML 30 culture were studied: cells that had exhausted the limiting glucose; cells taken directly from glucose-limited chemostats; and a washed suspension of highly catabolite repressed cells from cultures grown in high levels of glucose and gluconate. The growth studies reported here were performed on-line with a minicomputer. They represent at least an order of magnitude increase in accuracy in estimating growth parameters over previous instrumentation.


Isolation of plasmid deoxyribonucleic acid from two strains of Bacteroides. Two clinical isolates of Bacteroides contained covalently closed circular deoxyribonucleic acid (DNA) as shown by sedimentation in an alkaline sucrose gradient, CsCl ethidium bromide equilibrium centrifugation, and electron microscopy. Bacteriodes fragilis N1175 contained a homogeneous species of plasmid DNA with a molecular weight of 25 x 10(6). Bacteroides ochraceus 2228 contained two distinct, covalently closed circular DNA elements. The larger cosedimented with the covalently closed circular DNA form of the R plasmid, R100, corresponding to a molecular weight of 70 x 10(6); the smaller sedimented as a 58S molecule with a calculated molecular weight of 25 x 10(6). The roles of these plasmids are unknown. Neither strain transferred antibiotic resistance to plasmid-negative Bacteroides or Escherichia coli, and neither produced bacteriocins active against other Bacteroides or sensitive indicator strains of E. coli.


Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae. The interdependence of spindle plaque with other aspects of cell division and conjugation in Saccharomyces cerevisiae has been investigated. Three forms of the spindle plaque appear sequentially before the formation of the complete, intranuclear spindle. The single plaque is present initially in the mitotic cycle; it becomes transformed into a satellite-bearing single plaque during the latter part of G1. Subsequently, plaque duplication yields the double plaque characteristic of the early phase of budding, which coincides with the period of chromosome replication (S). The eventual separation of these plaques to form a complete spindle, with a single plaque at each pole, is nearly coincident with the completion of S. The form of the plaque differs in two independent cases of G1 arrest: the single plaque is found in a cell in stationary arrest of growth, whereas a cell arrested by mating factors in preparation for conjugation contains a satellite-bearing single plaque. The latter form is retained during zygote formation, where it serves as the initial site of fusion of each prezygotic nuceus with the other. This fusion results in the formation of  a single zygotic nucleus with a satellite-bearing single plaque, which is subsequently transformed into a double plaque as the zygote buds. The double plaque is situated adjacent to the site of bud emergence in both vegetative cells and zygotes.


Sizing of bdellovibrio during growth.

Sizing of bdellovibrio during growth. A technique for the counting and relative sizing of host-independent bdellovibrio during growth, with the aid of a modified Coulter counter, is described.


Recipient ability of bacteriophage-resistant mutants of Escherichia coli K-12. The ability of a wide range of bacteriophage-resistant mutants to act as recipients in conjugation with F'lac pro and R100-1 donors has been studied. A number of mutant types defective in recipient ability with F'lac pro, as well as mutants which were hyperrecptive with R100-1, have been detected.


Bacteriophage Mu-1-induced permeability mutants in Escherichia coli K-12. Apparent permeability mutations were produced in Escherichia coli K-12 by bacteriophage mu-1 mutagenesis. They are pleiotropic mutations showing sensitivity to a number of detergents and unrelated antibiotics, and presumably they affect cell wall or membrane biosynthesis. One of the mutations was genetically mapped at a site in or near the acrA and mtc loci at approximately 10.5 min on the Taylor and Trotter map (1972).


Characterization of V-prime factors in Escherichia coli K-12. An episome derived from an Hfr(v) (Hfr isolated from a V colicinogenic parent) strain of Escherichia coli K-12 was isolated and characterized. The direction of gene transfer was inverted from that in the original parental strain.


Transductional mapping of gene trmA responsible for the production of 5-methyluridine in transfer ribonucleic acid of Escherichia coli. The gene trmA, responsible for the production of 5-methyluridine (ribothymidine) in transfer ribonucleic acid, has been located at 79 min on the chromosomal map of Escherichia coli K-12. In five-factor crosses the gene order was shown to be argH-trmA-rif-thiA-metA. The co-transduction frequency between argH and trmA was 65%. Furthermore, the trmA5 mutation was shown to be recessive, in agreement with the notion that the trmA gene is the structural gene for the transfer tibonucleic acid (5-methyluridine) methyltransferase.


Physiological and biochemical studies on the function of 5-methyluridine in the transfer ribonucleic acid of Escherichia coli. Matched pairs of transductant strains differing by the presence of absence of 5-methyluridine (ribothymidine) (m5U) in their transfer ribonucleic acid (tRNA) were used to study the function of this modified nucleoside in Escherichia coli. Ordinary measurements of growth rate in different media revealed no effect of the loss of m5U in tRNA. A gene located close to trmA (the structural cistron for the methyltransferase that produces m5U in tRNA), however, was found to reduce the growth rates significantly, depending on the medium and the temperature of cultivation. Measurement of codon recognition, macromolecular composition, tRNA binding to the ribosome, and the rate of protein chain elongation in vivo indicated no disadvantage caused by the lack of m5U. The regulation of ilv and his operons seemed also to be unaffected by the absence of m5U in the tRNA. In a mixed population experiment, however, cells possessing m5U in their tRNA seemed to have a distinct advantage over cells lacking this modified nucleoside. This experiment provides the first indication of the overall value of m5U in tRNA.


Uridine diphosphate galactose-4-epimerase. Uridine monophosphate-dependent reduction by alpha- and beta-D-glucose. Rates of UMP-dependent reduction of the DPN+ associated with Escherichia coli UDP-galactose-4-epimerase at 27 degrees and 0.2 M ionic strength in 0.1 M Tris-HCl buffer, pH 8.5, are reported. The reaction exhibits excellent pseudo-first order behavior when D-glucose is at anomeric equilibrium. The effects of [UMP] and [glucose] on the observed first order rate constants are consistent with the following equation. The symbols phi are empirical parameters. (See article). The data indicate that the pathway involves random equilibrium binding of UMP and glucose followed by rate-limiting decomposition of the ternary complex to epimerase-DNPH. The binding parameters indicate that the principal activating effect of UMP is not simply to increase the affinity of the enzyme for glucose. UMP appears to increase the reactivity or availability of enzyme-bound DPN+. The kinetic isotope effect for the reaction of D-]1-2H]glucose (kH/kD) is 4.2, which confirms that C-1 is oxidized and that hydride transfer is rate limiting. Both of the purified anomers, alpha- and beta-D-glucose, reduce the enzyme-bound DPN+. As indicated by the deviations from pseudo-first order kinetics because of concurrent mutarotation, the beta anomer is the more reactive, reacting about 4 to 5 times faster than the alpha anomer at concentrations well below saturation. Is is suggested that the lack of stereo-specificity in this reaction may be attributed to the two anomers being productively bound with their opposite faces projecting toward C-4 of bound DPN+. Nonstereospecific oxidation of alpha- and beta-D-glucose may be a model for the mechanism of UDP-hexose epimerization, which also involves nonstereospecific hydride transfer.


Reactivation of the lipid-depleted pyruvate oxidase system from Escherichia coli with cell envelope neutral lipids. The pyruvate oxidase system of Escherichia coli is composed of a soluble flavoprotein, pyruvate oxidase (EC 1.2.2.2, pyruvate:ferricytochrome b1 oxidoreductase), and an electron transport system associated with the cell envelope-membrane fraction. The membrane particles contain 15% lipid by weight. Fractionation of the lipids revealed that abut one-third are neutral lipids and two-thirds are phospholipids. The relative ratio of ubiquinone to menaquinone within the neutral lipid fraction is 15:1 on a molar basis. Removal of the lipids from the membrane particles by extraction with aqueous acetone or hydrolysis of the phospholipids by treatment with Bacillus cereus phospholipase C results in a complete loss of electron transport activity. Analysis of the particles extracted with aqueous acetone revealed that practically all the neutral lipids and 65% of the phospholipids are removed by this treatment. Phospholipase treatment results in a loss of 75% of the membrane phospholipid phosphorus; however, the diglycerides and the neutral lipids produced by phospholipase hydrolysis remain associated with the particles. Addition of neutral lipid and a detergent, hepta-DL-alanyl dodecylamide to the acetone-extracted material results in a restoration of 37% of the original particle activity. Addition of neutral lipid and hepta-DL-alanyl dodecylamide to phospholipase-treated particles completely restores the original electron transport activity. Furthermore, addition of ubiquinone from either yeast (UQ6) or E. coli (UQ8) will restore pyruvate oxidase activity when the quinones are supplemented with photoinactivated neutral lipid. No restoration of activity to phospholipase-treated particles is noted upon the addition of either menaquinone 6 or menaquinone 8 to the reconstitution system. In fact, these compounds appear to suppress restoration of activity when they are added to reaction mixtures containing neutral lipid and phospholipase-treated particles.


Evidence for the coordinate control of glycogen synthesis, glucose utilization, and glycolysis in Escherichia coli. I. Quantitative covariance of the rate of glucose utilization and the cellular level of fructose 1,6-diphosphate during exponential growth and nutrient limitation. In cultures of Escherichia coli W4597(K) and G34 under various nutritional conditions the rates of glucose utilization and cellular levels of fructose-1,6-P2 are quantitatively related by the Hill equation where the value of the Hill coefficient is approximately equal to 2. This is the first evidence that fructose-P2, or any metabolite which covaries with fructose-P2, modulates glucose utilization in E. coli. In light of previous observations from our laboratory this new observation and those in the succeeding report provide the first evidence that in E. coli glycolysis, glycogen synthesis and glucose utilization are coordinately regulated, thus providing for the coupling of ATP utilization and production under various metabolic circumstances. Alterations in the level of ATP apparently affect the velocity of phosphofructokinase, the rate-limiting enzyme in glycolysis, altering the cellular levels of glucose-6-P or fructose-P2. Changes in the levels of these hexose phosphates are quantitatively related to alterations in the rates of glucose utilization and glycogen synthesis in the intact E. coli cell.


Evidence for the coordinate control of glycogen synthesis, glucose utilization, and glycolysis in Escherichia coli. II. Quantitative correlation of the inhibition of glycogen synthesis and the stimulation of glucose utilization by 2,4-dinitrophenol with the effects on the cellular levels of glucose 6-phosphate, fructose, 1,6-diphosphate, and total adenylates. In cultures of Escherichia coli W4597(K) and G34 under various nutritional conditions the rates of glucose utilization and cellular levels of fructose-1,6-P2 are quantitatively related by the Hill equation where the value of the Hill coefficient is approximately equal to 2. This is the first evidence that fructose-P2, or any metabolite which covaries with fructose-P2, modulates glucose utilization in E. coli. In light of previous observations from our laboratory this new observation and those in the succeeding report provide the first evidence that in E. coli glycolsis, glycogen synthesis and glucose utilization are coordinately regulated, thus providing for the coupling of ATP utilization and production under various metabolic circumstances. Alterations in the level of ATP apparently affect the velocity of phosphofructokinase, the rate-limiting enzyme in glycolsis, altering the cellular levels of glucose-6-P or fructose-P2. Changes in the levels of these hexose phosphates are quantitatively related to alterations in the rates of glucose utilization and glycogen synthesis in the intact E. coli cell.


Studies on the mechanism of inhibition of Salmonella typhimurium by 1,2,4-triazole. The inhibition of Salmonella typhimurium by 1,2,4-triazole appears to be mediated through an effect on L-cysteine biosynthesis. O-Acetylserine sulfhydrylase A, the final enzyme in the L-cysteine biosynthetic pathway, was found to catalyze a reaction (triazolylase) between O-acetyl-L-serine and 1,2,4-triazole, giving 1,2,4-triazole-1-alanine as a product. In wild type S. typhimurium grown on 4 mM 1,2,4-triazole, 97% of the total O-acetyl-L-serine synthesized in vivo is incorporated into 1,2,4-triazole-1-alanine. 1,2,4-triazole also significantly lowers the levels of several of the enzymes necessary for sulfate reduction. This effect is presumably due to the ability of the inhibitor to lower intracellular concentrations of O-acetyl-L-serine, an inducer of these enzymes. Inhibition of growth is probably caused by L-cysteine starvation, arising from the decreased availability of the L-cysteine precursors, sulfide and O-acetyl-L-serine. Two 1,2,4-triazole-resistant strains bearing mutations in cysK, the structural gene for O-acetylserine sulfhydrylase A, incorporate only small quantities of O-acetyl-L-serine into 1,2,4-triazole-1-alanine in vivo. In vitro studies, using purified preparations of O-acetylserine sulfhydrylase A, revealed greater losses of triazolylase activity than sulfhydrylase activity in the enzymes from both cysK mutants. Resistance to 1,2,4-triazole apparently can arise from mutations leading to a preferential loss of triazolylase activity or from mutations which diminish both activities to the extent that high concentrations of O-acetyl-L-serine and sulfide accumulate behind the sulfhydrylase reaction.


Outer membrane as a diffusion barrier in Salmonella typhimurium. Penetration of oligo- and polysaccharides into isolated outer membrane vesicles and cells with degraded peptidoglycan layer. In Escherichia coli and Salmonella typhimurium, the cell wall that contains both the outer membrane layer and the peptidoglycan layer acts as a barrier of the molecular sieve type for the penetration of uncharged saccharides (G. Decad, T. Nakae, and H. Nikaido (1974) Fed. Proc. 33, 1240). Here we examined which of the layers of the cell wall limited the size of the penetrating molecules, by studying the penetration of saccharides into (a) cells whose peptidoglycan layer had been destroyed by lysozyme treatment or growth in the presence of penicillin and (b) isolated outer membrane vesicles. We found that peptidoglycan-defective cells were similar to intact, plasmolyzed cells in that they allowed a partial penetration of stachyose (molecular weight 666), but essentially excluded saccharides with molecular weights higher than 900 to 1000. We also found that the isolated outer membrane acted as a penetration barrier for saccharides. These observations led us to conclude that the outer membrane, rather than peptidoglycan, sets the size limit for the penetration of uncharged, hydrophilic molecules through the E. coli or S. typhimurium cell wall. The isolated outer membrane, however, had an exclusion limit much higher than that found in intact cells. This "leakiness" could be decreased either by the use of mutants producing extremely deficient lipopolysaccharide, or by trypsin treatment of the isolated membrane followed by heating and slow cooling in the presence of Mg2+. We feel that these observations are consistent with the hypothesis that the resealing of the ruptured outer membrane during the isolation procedure is often incomplete, and that cracks and holes thus generated are responsible for the "leakiness" of the isolated membrane vesicles.


Inactivation of normal beta-D-galactosidase by antibodies to defective forms of the enzyme. A counterpart of the antibody-mediated activation of genetically defective enzymes is reported here. Antibodies elicited by certain mutant forms of beta-D-galactosidase (EC 3.2.1.23) of Escherichia coli were found to inactivate the normal form of the enzyme. (Antibodies elicited by normal beta-D-galactosidase do not affect the enzyme's catalytic activity.) We present evidence that the inactivating antibodies are directed against one or a few determinants of the enzyme. The level of inactivation caused by the antibodies was independent of temperature below 25 degrees and increased with temperature above 25 degrees. The inactivation was proportional to the concentration of antiserum until a maximum level of 50% inactivation was reached. Antibodies capable of inactivating up to 87% of the activity were obtained after the antiserum was partially absorbed in an affinity column. This antibody preparation showed a 10-fold enrichment of inactivating antibodies over other antibodies direct against the enzyme. The antibody-mediated inactivation caused a reduction in the Vmax of beta-D-galactosidase without affecting the apparent Km of the enzyme. In contrast to antibodies to normal beta-D-galactosidase, inactivating antibodies changed the response of the enzyme to cations. To explain these results, we present a model in which there is a temperature-dependent equilibrium between two active forms of beta-D-galactosidase. Inactivation results from a conformational change induced by the binding of inactivating antibodies to only one of these two forms.


Action of the highly purified, membrane-bound enzyme phosphatidylserine decarboxylase Escherichia coli toward phosphatidylserine in mixed micelles and erythrocyte ghosts in the presence of surfactant. Phosphatidylserine decarboxylase, Escheichia coli, was purified to near-homogeneity by the procedure of Dowhan, W., Wickner, W. T., and Kennedy, E. P. ((1974) J. Biol. Chem. 249, 3079-3084) and assayed by following the production of CO2 using gas chromatography. The purified enzyme has an absolute requirement for the surfactant Triton X-100. The function of Triton in the assay is evaluated and a kinetic scheme describing the action of this membrane-bound enzyme in the micellar system provided by the surfactant is presented. According to this scheme, the enzyme first binds to a mixed micelle, composed of phosphatidylserine and Triton, where the dissociation constant is KSA. The enzyme, now part of the mixed micelle, then binds the substrate phosphatidylserine in its active site and this binding is related to the Michaelis constant, KMB. KSA, expressed as the sum of the molar concentrations of Triton and phosphatidylserine, is about 0.04 M. KMB, expressed as the mole fraction of phosphatidylserine in the mixed micelles, is about 0.03. Phosphatidylserine decarboxylase activity toward phosphatidylserine in human erythrocyte ghosts was also determined. The amount of phsophatidylserine converted to phosphatidylethanolamine and CO2 was found to be related to the amount of phosphatidylserine solubilized from the membrane by Triton X-100. In the absence of Triton, no significant activity of the enzyme toward the ghosts was detected even after subjecting the ghosts to lyophilization, homogenization, or sonication.


DNA repair synthesis dependent on the uvrA,B gene products in toluene-treated cells. Ultraviolet irradiation of toluene-treated Escherichia coli causes an inhibition of replicative DNA synthesis. This is followed by the appearance of nonconservative DNA repair synthesis which does not require either the polymerase or 5' leads to 3' exonucleolytic activities of DNA polymerase I. The repair synthesis may be catalyzed by DNA polymerase III activity but does not require a functional DNA polymerase II. The ultraviolet-induced synthesis requires ATP and is dependent on a functional uvrA and uvrB gene product. However, other uvr gene products are not required for the synthesis. The recB function is also not required.


The in vitro translation of a terminating signal by a single Escherichia coli ribosome. The fate of the subunits. A complex was isolated containing one 70 S ribosome bound to the RNA of the f2 bacteriophage in the region of the coat gene. Either the f2 RNA and 70 S ribosome or both ribosomal subunits in this complex were differentially labeled with tritiated lysine or phosphorous 32 RNA. Incubation of this complex in a ribosome-free protein-synthesizing system lacking initiating factor and appreciable nuclease activities allowed the synthesis of coat protein and subsequent termination. Using this techique, it was found that termination resulted in the release of ribosome as subunits. Intermediate in this process is a transient 46 S complex composed of the f2 RNA and 30 S ribosomal subunit, presumably the result of the initial release of the 50 S ribosomal subunit. This 46 S complex subsequently breaks down to a free f2 RNA and 30 S ribosomal subunit. The rate of this latter process is faster in the presence of crude initiating factors. These results are discussed in terms of the mechanism of termination and reading of the intercistronic region.


The isolation of RNA homologous to the genetic control elements of the lactose operon. A sequential DNA-RNA hybridization procedure is described whereby RNA homologous to a target DNA region 35 to 140 base pairs in length can be purified up to 6700-fold from a complex in vitro transcript to a homogeneity sufficient for sequence analysis. Requirements for the procedure include: (a) uniform transcription over the target DNA region in vitro; (b) specialized transducing phages which carry genetic deletions defining the target region on either side; and (c) specialized transducing phages which carry the target DNA in opposite orientations. These requirements have been met for the genetic control region (promoter, operator) of the lactose operon of Escherichia coli, to which the method was applied. The procedure is independent of the activity of the genetic control signals under study and can therefore be applied without modification to the study of point mutations introduced into the template.


Purification of Escherichia coli endonuclease specific for apurinic sites in DNA. The endonuclease specific for apurinic sites in DNA has been isolated from Escherichia coli B41 as a pure monomeric protein of 32,000 daltons. The enzyme hydrolyzes a phosphodiester bond near the apurinic sites in double-stranded DNA; it does not hydrolyze untreated DNA and its action on alkylated DNA is restricted to the apurinic sites always present. This enzyme is not endonuclease II which is most probably a mixture of two enzymes, one a glycosidase (Kirtikar, D. M., and Goldthwait, D. A. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 2022-2026), the other an endonuclease for apurinic sites which is the enzyme isolated in this work.


A comparison of iliac marrow and biodegradable ceramic in periodontal defects. Defects were created in the tooth-supporting bone of six beagle dogs. Biodegradable tricalcium phosphate was compared to cancellous marrow as a treatment method. The findings indicated that ankylosis and resorption of the tooth can occur when using marrow. The ceramic-filled defects healed slower; but the material itself was well tolerated by the tissues, initiated new bone formation, filled in the defect, and has good potential for the treatment of advanced periodontitis.


Thermodynamic considerations of the setting reaction in Ag3Sn amalgams. The setting reaction of Ag3Sn amalgams may be thought of as a liquid phase sintering problem in which the reactants seek to form products with the lowest thermodynamic potential. We discuss the thermodynamic and kinetic considerations of this reaction. The lowering of free energy has been given as the reason for the formation of the gamma1, gamma2,and beta1 phases and a qualitative reaction coordinate diagram has been hypothesized. The barrier height for the formation of the beta1 phase has been determined to be 137 kJ/mol.


Use of PMMA in expansion dental implants. The Polymer Expansion Implant System, which is a system for the substitution of missing dental roots in natural or surgical alveolus, has been developed by the author. The polymer used is poly (methyl methacrylate) (PMMA). A material is sought that has three of the basic physical and mechanical qualities of human dentine: low modulus of elasticity, thermal and electrical passiveness, and ideal porosity. In addition it should be resistant, easily worked, biocompatible, and of low cost. Of the biomedical polymers, PMMA has these qualities, and it can be used as a substitute for dental roots. The design of bodies to be implanted as substitutes for dental roots requires: anatomical measurements of the maxilla and sockets; methods of retention by the bone tissue; and methods of insertion. The formation of lamina dura around an implanted body is a reply of the organism to the forces acting on it and a demonstration of the good interaction of the porosity of the material, the peri-implant collagenous fibers and bone. X-rays that demonstrate the behavior of bone to a PMMA implant are shown.


A failed vitallium/stainless steel total hip replacement: a case report with histological and metallurgical examination. A case of a failed total hip replacement consisting of a Vitallium hip socket and a stainless steel femoral head prosthesis is presented. The mixed-metal combination and inadequate design resulted in severe abrasive wear and fretting corrosion of the bearing surface, and the accumulation of stainless steel wear debris and corrosion products in the periarticular tissues was extensive. The tissue response was primarily fibrotic scarring with severe histiocytic and mild foreign body giant cell reaction. Despite the massive accumulation of wear debris and corrosion products, however, there was minimal chronic inflammation and no acute inflammation.


[Renal preservation: comparative effectiveness of 2 simple perfusion technics]. The authors compare the efficacy of two perfusates, Collins' solution and Perfudex, for conservation of kidneys for short periods by perfusion and hypothermic immersion. 22 dog kidneys, removed by lombotomy, were preserved for 24 hours with one or other of these solutions and then re-implanted into their host. Radio-isotopic renography, the uptake of Hg 197 Cl3 and a hippuran renogram, used to assess the function of the transplants, suggested the functional superiority of kidneys preserved with Collins solutions. Histological controls carried out beyond the 10 th post-operative week, confirmed these results. The correlations observed between these various findings also assessed the reliability of radio-isotope examinations in the exploration of renal function.


Effect of prolonged LH-releasing hormone administration on gonadotropin response in patients with hypothalamic and pituitary tumors. The effect of prolonged administration of synthetic LH-RH (400 mug daily im for 5 days) on gonadotropin response to LH-RH (100 mug single injection iv) was compared in 13 patients with pituitary tumors or suprasellar tumors. No patients with pituitary tumors exhibited an augmented response on serum FSH and LH after prolonged LH-RH administration. A paradoxical fall in response to LH-RH test occurred in 3 patients with pituitary tumors and in a patient with hypothalamic tumor in whom gonadotropin responses were normal on the first LH-RH test. On the other hand, 2 patients with suprasellar tumors with a low response to the initial LH-RH test showed significant increase on a second LH-RHtest after consecutive LH-RH administration. Consecutive administration of LH-RH may help to distinguish between the hypogonadism of pituitary origin and that of hypothalamic origin when the initial LH response to a single dose of LH-RH is subnormal.


Pituitary response to synthetic luteinizing hormone-releasing hormone in patients with Turner's syndrone. Synthetic luteinizing hormone-releasing hormone (LHRH) was administered intravenously in a dose of 25 mug to 9 patients with Turner's syndrome of 45,XO karyotypes, 5 normal menstruating women, and 5 postmenopausal women. In patients with Turner's syndrome, the basal serum LH level was higher than that of the normal subjects and lower than that of postmenopausal women. The LH response to LHRH, when expressed as a percent of control, was comparable to that of the normal luteal phase and significantly higher than that of postmenopausal women. The basal FSH level was more elevated than the basal LH level. The FSH response to LHRH, when expressed as a percent of control, was similar to that of postmenopausal women and lower than that of normal subjects. The results suggest that pituitary function is preserved in patients with Turner's syndrome. I interpret the pituitary responsiveness and/or reserve of patients with Turner's syndrome as larger than that of postmenopausal women. A greater response of LH than of FSH to LHRH was observed in this study and is contrary to findings reported by others on teenage patients with Turner's syndrome. This suggests that the sensitivity of gonadotropins changes with age in Turner's syndrome.


Detection of bacteriuria by luciferase assay of adenosine triphosphate. A selective method for distinguishing bacterial and nonbacterial adenosine triphosphate (ATP) in clinical bacteriological specimens was studied. The method involved incubation of samples with the detergent Triton X-100 and the ATP-hydrolyzing enzyme apyrase. The incubation selectively destroyed ATP in suspensions of various human cells while not affecting the ATP content in microbial cells. ATP remaining in the sample after incubation was extracted in boiling buffer and assayed by the firefly luciferase assay. Application of the method to 469 clinical urine specimens showed that the ATP level after treatment with Triton/apyrase was correlated to bacterial counts and that the sensitivity of the assay was sufficient for the detection of 10(5) bacteria/ml. The ATP levels per bacterial cell remaining in the urine specimen after treatment with Triton/apyrase were close to values observed in laboratory-grown cultures. The specificity and sensitivity of the luciferase assay for the detection of urinary bacteria and its possible use as a bacteriuria screening method are discussed.


Unusual Enterobacteriaceae: a Salmonella cubana that is urease positive. This is the first report of a naturally occurring Salmonella that is urea positive. The strain was identified as Salmonella cubana and it was typical in all biochemical, serological, and bacteriophage reactions, except that is produced urease strongly.


[Comparative experimental study of inverting and everting sutures in the colon]. The authors carried out 88 sutures of the ascending colon in Wistar rats, 44 in inversion and the remainder in eversion. The suture material used was monofilament 9/0 nylon. The intestinal diameter was measured using an instrument and the healing process studied by means of the following parameters: granulocytes, oedema, fibrin, fibroblasts and re-epithelialisation of the mucosa, on histology. The results prove that inverted sutures heal within 10 days, whereas everted sutures are not healed after 17 days. Furthermore, we demonstrated the fact that, after 72 hours, the intestinal caliber above the suture showed no appreciable differences compared with that of the normal colon.


Evaluation of the Bactrol Disks, a set of quality control cultures. A set of commercially available quality control cultures was evaluated. These cultures were found to be an acceptable alternate to lyophilized or continuously subcultured cultures for a quality control program.


Rapid hippurate hydrolysis method for presumptive identification of group B streptococci. A rapid test to detect the hydrolysis of sodium hippurate by beta-hemolytic streptococci within 2 h was developed. All group B streptococci tested were positive using this method and all other groups were negative.


Comparison of four methods for determining nitrate utilization by cryptococci. This study evaluated the following methods for determining nitrate utilization: Wickerham broth, a special nitrate broth, Delft plate, and nitrate strip. With 236 isolates of cryptococci as test organisms, the special nitrate broth method gave 99% correct results and the Wickerham broth method gave 98%. The nitrate strip and Delft plate methods gave correct results in 94 and 86% of tests, respectively. The special nitrate broth method is judged superior because it provides accurate results within 48 h, compared to 14 days with the Wickerham broth method.


Effect of acetone fixation on infectivity and antigenicity of respiratory syncytial virus and adenovirus in the fluorescent antibody test. An investigation was made on the effect of acetone fixation on infectivity and immunofluorescent antigenicity of respiratory syncytial virus and adenovirus type 5, lipid- and nonlipid-containing viruses, respectively. Viruses were allowed to replicate in HEp-2 cells, and the cells were then fixed in acetone at 5 C for periods ranging from 30 s to 7 days. Treatment for 10 min was sufficient to inactivate respiratory syncytial virus, whereas infectious adenovirus type 5 could be isolated from cells immersed in acetone for 7 days. There was a gradual reduction, to 50% of that observed at 30 s, in the intensity of fluorescent antibody staining of both viruses with increasing fixation time, but no significant decreases in fluorescent antibody end-point titers of antisera to either virus were observed.


Standardization and evaluation of the CAMP reaction for the prompt, presumptive identification of Streptococcus agalactiae (Lancefield group B) in clinical material. Primary cultures of clinical material were screened for the presence of colonies suspected of being Streptococcus agalactiae (Lancefield group B). Sixty-three such cultures and 108 other isolates of beta-hemolytic streptococci (groups A, C, and G), encountered during the first 3 months of the investigation, were studied by Lancefield grouping, sodium hippurate hydrolysis, and a standardized CAMP test. All streptococci were inoculated perpendicularly to streaks of a beta-toxin-producing staphylococcus on sheep blood agar plates and incubated aerobically in a candle jar and anaerobically at 37 C. Plates were examined after 5 to 6 and 18 h of incubation. The production of a distinct "arrowhead" of hemolysis was indicative of a positive CAMP reaction. All group B streptococci produced a positive CAMP reaction in the candle jar or anaerobically, usually within 5 to 6 h, and aerobically after 18 h of incubation. All group A streptococci produced a positive reaction only under anaerobic conditions. Groups C and G streptococci were negative under all atmospheres. The CAMP reaction is a prompt and reliable procedure for the presumptive identification of group B streptococci when a candle jar atmosphere is used during incubation.


Rapid identification of group B streptococci by counterimmunoelectrophoresis. Beta-hemolytic streptococcal isolates have been examined by counterimmunoelectrophoresis (CIE) with group B antiserum to determine whether this techinque is of value in the rapid identification of group B strains. Ninety stock cultures and 100 clinical isolates of beta-hemolytic streptococci including representatives of groups A, D, C, G, and B were inoculated into Todd-Hewitt broth; after incubation at 37 C for 1, 2, 3, and 4 h, aliquots of the whole broth cultures were removed and tested by CIE. Antigen was not regularly detected in the 1-, 2-, and 3-h samples, but after 4 h all 126 group B streptococcal strains identified by the capillary precipitin reaction gave CIE precipitin bands with group B antiserum. None of the 58 non-group B strains gave precipitin reactions with this antiserum. Cerebrospinal fluid from an infant with group B streptococcal meningitis and peritoneal fluid from a patient with group B streptococcal peritonitis had free group B antigen detected by the CIE technique. CIE of broth cultures and direct body fluids appears to be a rapid and sensitive method for the identification of group B streptococcal strains.


Pagano-Levin Candida test medium: evaluation using vaginal samples. Since 1958 pagano-Levin medium (PLM) has been used as an aid in the identification of Candida albicans. However, no statistical analysis of its effectiveness based on large number of random human specimens has been reported. The present study compared PLM (now called Candida test) with Sabouraud's plus antimicrobials and Littman's ox-gall agar without antimicrobials. Of 500 random vaginal samples 24.8 were true positives, 71.9% were true negatives, 2.2% were false positives, and 1.2% were false negatives on PLM. If only samples identified as C. albicans were considered, 95.4% were true positives and 4.6% were false negatives. PLM did not inhibit C. albicans from the vagina. No one medium was found superior to the others for the purpose of isolating and identifying C. albicans. Of the five strains of C. parapsilosis, the only other Candida species isolated, all the samples grew on PLM but most were inhibited on Sabouraud's medium plus antimicrobials.


Serotyping and biotyping of 160 Escherichia coli strains: comparative study. One hundred and sixty Escherichia coli strains were serotyped and biotyped. Serotyping revealed 68 different types, with 25 strains not typable. Biotyping was possible in all strains but revealed 55 different types. One biotype could be subdivided into 35 different serotypes, indicating that for this biotype the series of biochemical and fermentation reactions could be extended for further differentiation.


Sensitivity of bhk-21 cells supplemented with diethylaminoethyl-dextran for detection of street rabies virus in saliva samples. A tissue culture system for detecting rabies virus from saliva samples of suspected animals was developed and compared to suckling mouse inoculation. Swab samples were obtained from the mouth of the animal heads received for rabies diagnosis; these swabs were submerged in maintenance medium. The maintenance medium was inoculated intracerebrally into suckling mice and onto BHK-21 cells with diethylaminoethyl (DEAE)-dextran (BHK/DEAE) and without (BHK). Rabies immunofluorescence was performed on the brain of the mice dying during the observation period and also on both tissue culture systems every day after infection. The BHK-DEAE system detected 28 positive samples obtained from 48 rabid animals and the BHK system detected 18. By suckling mouse inoculation only 11 of the same positive samples were detected. A total of 90 samples was studied by the three methods. Rabies virus was detected by the tissue culture methods earlier than by suckling mouse inoculation. The BHK-DEAE method was an economic and fast method for rabies virus detection in saliva samples, which could be used for ecological and pathogenesis studies, as well for rabies diagnosis before the death of the suspected animal.


Suitability of peracetic acid for sterilization of media for mycoplasma cultures. The utility of peracetic acid for sterilization of serum and yeast extract additions to mycoplasma medium was studied by culturing six Mycoplasma species. Culture media containing additions that had been sterilized with peracetic acid proved to be as good as filtered components. The use of 0.05 to 0.1% peracetic acid is recommended to sterilize the serum and yeast extract additions since savings in time and equipment can be accomplished.


Simplified microimmunofluorescence test with trachoma-lymphogranuloma venereum (Chlamydia trachomatis) antigens for use as a screening test for antibody. A simplified microimmunofluorescence test with trachoma-lymphogranuloma venereum (Chlamydia trachomatis) antigens has been devised as a screening test for antibody in human sera. The test differs from our standard procedure by amalgamating 15 different immunotypes into nine antigen pools, by using only three serum dilutions, and by dropping use of duplicate slides. The screening test could be performed on at least six times as many sera as the standard test for a given unit of effort. It was shown to have a sensitivity equal to the standard test and an ability to determine specific immunotype on the basis of antibody pattern (72%) only slightly reduced from the standard test (84%). The screening test was carried out on 876 patients from different population groups in the Seattle area. The tests showed that antibody was present frequently in persons attending venereal disease clinics (60%) and commonly (25%) in a group of adults without venereal disease. Nine percent of children under 15 years of age tested had antibody; the significance of this finding is unknown.


Identification of streptococci: serogrouping by immunofluorescence. This paper deals with the fluorescent antibody (FA) method for identifying six commonly occuring and two rare groups of streptococci by using commercially prepared (Difco) conjugates. We have shown that group-specific FA produced frequent cross-reactions with heterologous groups of organisms. These reactions varied with different strains of the same serogroup. Nonetheless, there was distinct overall patterns in the intensity and appearance of the homologous and heterologous reactions. When monitored by the precipitin test with Rantz and Randall extracts, these patterns led to the correct identification of 90 to 100% of specimens of serogroup A, B, C, and G streptococci. Many members of groups D and F also showed distinctive reaction patterns. However, there was a significant number of strains of both groups D and F that either failed to strain or stained poorly with the homologous conjugate. As a result, the identification of these serogroups by FA was less reliable.


Antibody response of patients with malignancies to bacteremia with gram-negative bacteria. Antibody response, determined by means of the indirect bacterial hemagglutination tests, was studied in 58 consecutive patients with various malignancies whose blood culture yielded growth of Enterobacteriaceae or Pseudomonas and from whom serum specimens were obtained. Of these patients 59% had a significant antibody response. The invading microorganisms were Escherichia coli in 33 and Klebsiella in 19 subjects, an antibody response being documented with essentially equal frequency (60 and 57% of the subjects, respectively). Two patients had positive blood cultures for both E. coli and Klebsiella, one of whom had a significant response to one isolate only. A specific antibody resonse was documented in 67% of the subjects from whom blood for antibody titration was obtained at least 5 days after the blood culture, but from only 21% of patients whose serum was procured during the first 5 days after the blood culture. Similarly, such an antibody response was identified in 73% of subjects with two consecutive serum specimens, but in only 28% of the patients with a single serum specimen for antibody titration. Documentation of the immune response may be of diagnostic aid in differentiating between infection and contamination even in patients with underlying malignancy and under potentially immunosuppressive therapy.


Laboratory experience with a radiometric method for detecting bacteremia. Two bacteriologic systems for detecting bacteria in blood were compared; the automated radiometric BACTEC and the conventional method used in our laboratory for many years. BACTEC consisted of two bottles with 30 ml and the conventional method with 50 ml of media for aerobes and anaerobes. The BACTEC bottles were inoculated with 2 to 3 ml and the conventional with 4 to 5 ml of blood at the patient's bedside. Out of the 3,045 blood specimens cultured (804 patients), 262 (117 patients) were positive by one or both methods. The conventional system detected 5more cultures. The explanation of the differences is discussed. Positive blood cultures were detected by the BACTEC procedure as early as 6 h after the blood collection. In the first 24 h, on the average, 77% of aerobic organisms were detected by the BACTEC as compared to 48% by the conventional system. All anaerobic BACTEC cultures were positive within 4 days, whereas the conventional system detected at that time 74%. At day 4, 67% of fungi were detected by the BACTEC and only 27% by the conventional system. Of the 3,045 blood cultures examined by the BACTEC, 208 were recorded as false positive with growth index readings ranging from 30 to 59.


Assessment of virus infectivity by the immunofluorescent and immunoperoxidase techniques. The immunofluorescent and immunoperoxidase cell-counting techniques were comparable in precision and reproducibility for the quantitative assessment of influenza virus infectivity. The dose-response function was linear with each procedure, and comparable results were obtained for estimating neutralizing antibodies in antiviral serum.


A simple test system for the separation of staphylococci from micrococci. A simple test system for the separation of staphylococci from micrococci is described, which is based on the ability of staphylococci to produce acid aerobically from glycerol in the presence of 0.4 mug of erythromycin per ml and on their sensitivity to lysostaphin.


Fluorescent antibody studies in chlamydial infections. Irradiated McCoy cells infected with genital strains of Chlamydia trachomatis were grown in wells on slides coated with polytetrafluoroethylene. The inclusions produced in this system formed the antigen in an indirect immunofluorescence test, which detected group-specific chlamydial antibodies in sera from patients attending veneral disease clinics. Chlamydial antibodies were found more frequently and in higher titer in sera from women attending veneral disease clinics then in sera from a less promiscuous population attending a Family Planning Association clinic. Paired sera from 13 patients with nongonococcal urethritis from whom chylamydiae had been isolated were tested against the homologous isolates; seroconversion was demonstrated in only one instance, and antibody was present in the first serum specimens of all the other patients. Chlamydia-specific immunoglobulin M was found in four of eight patients with psittacosis and in a proportion of sera from patients attending veneral disease and Family Planning Association clinics. The antigen for this immunofluorescence test can easily be prepared in laboratories with cell culture facilities for the isolation of C. trachomatis, and the test should be useful for laboratories which cannot undertake the micro-immunofluorescence test.


A more complete evaluation of the color-coded antigen for the automated reagin test. A comparative study, on 500 samples, of the toluidine red antigen, one of the low-cost, color-coded antigens for the Automated Reagin Test, shows a greater sensitivity than the rapid plasma reagin card antigen.


Recovery of Cryptococcus neoformans from modified Dubos liquid medium utilized for isolation of mycobacteria. Cryptococcus neoformans was isolated from nine pathological specimens cultured for mycobacteria. Five of these were recovered only in liquid TBC medium in the absence of growth on conventional substrates.


Serological diagnosis of California (La Crosse) encephalitis by immunofluorescence. A method of indirect immunofluorescence was developed and examined retrospectively as a serological test for the laboratory diagnosis of California encephalitis (CE). LaCrosse virus immunofluorescence immunoglobulin (Ig) G and IgM studies were done on paired sera from 50 patients with acute central nervous system infections. CE had been documented in 25 patients by hemagglutination inhibition, neutralizing, complement fixing, and/or precipitin tests. Five (20%) of the acute and 16 (64%) of the convalescent sera from CE patients had La Crosse IgM antibodies. Seven (28%) of the acute and all of the convalescent CE specimens had La Crosse IgG antibodies. Titers ranged from less than 4 to 256. IgG antibodies were present in all 11 sera collected 1 to 2 years after CE, but IgM antibodies were absent. The 25 serum pairs from patients who did not have CE were negative for IgM and IgG antibodies. This study indicated that La Crosse immunofluorescence antibody tests were as sensitive and specific for CE as conventional hemagglutination-inhibition tests, and would detect at least 20% of patients during their acute illness.


Rapid identification of swarming Proteus using the PathoTec ornithine decarboxylase test strip. The PathoTec ornithine decarboxylase test strip was evaluated for use in identification of swarming Proteus. All 181 strains tested were accurately identified within 4 h.


Growth potential of cottonseed culture media for various clinically significant aerobic bacteria. Enzymatic hydrolysates of various cottonseed flours were prepared with the proteolytic enzymes bromelain, HT-200, Pronase, and trypsin. The growth of various aerobic bacteria of clinical significance in these hydrolysates was compared to that obtained with a standard casein-soybean peptone culture medium, Trypticase soy. The generation times of the majority of bacteria grown in the bromelain cottonseed flour hydrolysate were shorter than that obtained with the standard control broth. A bromelain cottonseed flour hydrolysate agar preparation supported the growth of the bacteria comparably to that of the casein-soybean agar substrate. All the bacterial colonies were larger on the bromelain cottonseed flour hydrolysate blood agar medium than those grown on the control agar. The peptones derived from the enzymatic hydrolysis of cottonseed flour are sufficient to promote the rapid and luxuriant growth of a wide spectrum of aerobic bacteria without the addition of peptone from other sources. It is suggested that cottonseed flour peptones be utilized as a nutrient source in general-purpose media for the clinical microbiology laboratory.


Pigment production by Cryptococcus neoformans from para- and ortho-Diphenols: effect of the nitrogen source. Cryptococcus neoformans produced pigments when p-diphenols were substrates in a glucose-amino acid-salts medium. The best substrates were 2.5-dihydroxybenzoic acid and 2,5-dihydroxybenzenesulfonic acid. In contrast to the cellular pigment production from o-diphenols (hydroxyl groups in the 2,3- or 3,4-position of phenyl ring), the p-diphenols (1,4- or 2,5-positions for the hydroxyl groups) produced large amounts of soluble pigments that diffused into the medium. When an optimal source of nitrogen (glutamine, glycine, and asparagine) was used, 89% of the C. neoformans strains produced pigments from p-diphenols. In contrast, 0 to 67% of the strains produced pigments when a suboptimal nitrogen source (proline, ammonium sulfate, ornithine, and methionine) was used. When glutamine-glycine-asparagine was the nitrogen source, 100% of the C. neoformans strains produced pigments from o0diphenols, whereas 77 to 100% of the strains produced pigment when proline-ammonium sulfate-ornithine-methionine was the nitrogen source. Cryptococcus species other than C. neoformans and all tested Candida species failed to produce pigments from any of the substrates except when hydroquinone was used. A combination of glutamine-glycine-asparagine and 3,4-dihydroxyphenylalanine allowed differentiation of colonies of C. neoformans from C. albicans in 3 to 6 days. These data showed that pigment production from o- and p-diphenols served as an excellent biochemical test for the identification of C. neoformans.


Construction of an interpretive pattern directory for the API 10 S kit and analysis of its diagnostic accuracy. A directory of test patterns and their interpretations has been prepared for identification of Enterobacteriaceae by using the 11-test API 10 S kit. The diagnostic accuracy of the directory and kit were evaluated by using records of test results for 37,476 isolates studied with the 21-test API 20 Enteric kit. Analysis indicates that 96.9% of the isolates would have been correctly identified at the genus level and 95.9% at the species level by using only the subset of tests included in the API 10 S.


Anaerobic bag culture method.

Anaerobic bag culture method. In a new method of anaerobic culture, a transparent, gas-impermeable bag is used and the anaerobic environment is established with copper sulfate-saturated steel wool. An Alka-Seltzer tablet generates carbon dioxide. The agar plate surface can be inspected through the bag at any time without interrupting the anaerobic atmosphere or disturbing other specimens. Methylene blue indicator strips are completely reduced by 4 h after the bag is set up and have remained reduced for as long as 3 weeks. Growth of 16 different stock culture anaerobes was generally equivalent by the bag and GasPak jar methods. Yield and growth of anaerobic isolates also were equivalent with 7 of 10 clinical specimens; from the other 3 specimens, 13 isolates were recovered, 5 by both the bag and jar methods and the rest by one method or the other. No consistent differences were found between the anaerobic bag and GasPak jar methods in the yield of anaerobes from clinical specimens. Early growth (24 h of incubation) of anaerobes from one specimen was detected with the bag method.


Analysis of acetoin and diacetyl in bacterial culture supernatants by gas-liquid chromatography. The acetoin and diacetyl contents of culture supernatants of Voges-Proskauer-positive "viridans" streptotocci, Klebsiella pneumoniae and Staphylococcus aureus, were determined by a gas liquid chromatographic procedure, in which supernatants were extracted with diethyl ether and diacetyl was measured on columns of 10% (wt/wt) polyethylene glycol 400 (PEG 400) at 73 C. Acetoin was converted to diacetyl, before analysis, by a simple oxidation procedure with ferric chloride and without a distillation step. Streptococcal culture supernatants were shown by this method to contain only acetoin; supernatants of K. pneumoniae and S. aureus contained both acetoin and diacetyl.


Comparison of three tests for the serological diagnosis of lymphocytic choriomeningitis virus infection. Levels of lymphocytic choriomeningitis virus antibody were assayed in 62 infected persons. The three tests used were indirect fluorescent antibody (IFA), complement fixation, and neutralization in mice. The sera first became positive by the IFA test, and IFA titers rapidly rose to a relatively high level, with the sera remaining positive long after the antibody detectable by complement fixation had disappeared. The IFA test appeared to be specific. The sera became positive last by the mouse neutralization test; with this test, antibody first appeared several weeks after infection. Virus-infected cells were stable when stored at -60 C, allowing diagnostic sera to be tested promptly by the IFA test. The IFA test for lymphocytic choriomeningitis antibody should increase the number of serological diagnoses, since it is not only rapid and specific, but detects cases not diagnosed by the other methods.


Evaluation of antibody coating of yeasts in urine as an indicator of the site of urinary tract infection. Antibody coating of yeasts (Candida sp. and Torulopsis sp.) found in urine specimens was investigated to ascertain whether the presence of such coating might identify the site of urinary tract infection. Washed yeast cells obtained by centrifugation of fresh urine specimens were reacted with fluorescein-conjugated goat antihuman immunoglobulins (Ig) G, A, and M and examined by fluorescent microscopy. IgG was found on the surface of all species of yeast encountered in all urine specimens evaluted, whereas there was variability of IgA AND IgM coating. Antibody coating with IgG, IgA, AND IgM was also demonstrated on yeasts from other body sites (sputum, gastrostomy, oral, etc.). Control experiments confirmed the specificity of the reactions. Thus, it appears that yeasts from any body site are coated with antibodies. These results are in contrast to recent work with bacteria which showed that the presence of antibody-coated (IgG) bacteria indicates upper urinary tract infection (pyelonephritis) while bacteria are not coated with antibodies in lower urinary tract infection. Since all yeasts from all body sites tested were found to be coated with antibody regardless of the clinical situation, the presence of surface antibody has no diagnostic value in identifying the site of urinary tract infection with yeasts.


Detection of fungi in clinical specimens by phase-contrast microscopy. During 1973 and 1974, the following fungi were detected in clinical specimens by using phase-contrast microscopy: Blastomyces dermatitidis, 5; Coccidioides immitis, 3; Cryptococcus neoformans, 11; other yeasts 918; dermatophytes, 863; Mucor species, 1; and Aspergillus fumigatus, 16. This technique allows rapid detection and, in many instances, immediate identification of fungi in clinical specimens.


The classification of depressive disorders. II. A review of historical and physiological classification studies. A review of recent classification studies of depressive disorders based on historical data and physiological indices was made. It was found that historical variables, such as age of onset and family background, are potentially useful to distinguish depressive subgroups, although these groupings did not differentiate consistently unipolar from bipolar depressives. While the physiological indices do not support the present psychiatric nosology, single physical signs were found to differentiate subgroups within the effective disorders. The implications of these findings for a different research model were discussed.


A comparison of muscle relaxation training and music in the reduction of state and trait anxiety. A comparison was made between muscle-relaxation training and relaxing music in the reduction of state and trait anxiety. Ss were 108 anxious female college students, half of whom received three sessions in muscle-relaxation training and half of whom received three sessions of relaxing music. Results indicate that both types of treatment significantly reduce state anxiety, but neither reduces trait anxiety. Results support Spielberger's state-trait anxiety theory and hold some implication for treatment.


Immunofluorescent detection of alpha1-antitrypsin in paraffin embedded liver tissue. Alpha1-antitrypsin was detected by indirect immunofluorescence in frozen sections of liver biopsies from patients with clinically and biochemically proven alpha1-antitrypsin deficiency. The antigen could also be demonstrated in those liver specimens of the same patients which were fixed in Bouin's fluid and embedded in paraffin. The cellular localization and the brightness of the fluorescence were the same in both frozen and paraffin sections. Four additional biopsies from three other patients were selected on the basis of PAS-positive diastase-resistant inclusions reported in the hepatocytes. All these biopsies showed bright fluorescence in the cytoplasm of the liver cells although one of the biopsies was stored for as long as eight years. Specific fluorescence was constantly found in the periportal hepatocytes with varying degrees of positivity. No fluorescence was observed in the six control biopsies from patients with various other liver diseases. These findings prove that paraffin embedded specimens are suitable for immunofluorescence detection of alpha1-antitrypsin and that a retrospective study on old paraffin blocks is possible.


Variants of Escherichia coli giving the appearance of mixed growths in urine. Urines from patients with symptoms of urinary tract infection yielded mixed growths of different colony types of Escherichia coli. The different colony types were found to be variants of single infecting strains caused by mutation or by phase action. It is suggested that care should be exercised in the interpretation of apparently mixed growths from urine.


Microbial flora of the lower genital tract during pregnancy: relationship to morbidity. Nineteen genera and groups of micro-organisms were isolated from the lower genital tract of 280 women at their first antenatal visit. Chlamydia, viruses, and T-strain mycoplasmas were not sought, and only routine methods of anaerobic culture were used. Growth was recorded as scanty, moderate or heavy. The population studied was grouped according to age, parity, gestational stage at booking, presence and degree of severity of lower genital tract morbidity, past history of vulvovaginitis, and suspicion of lower genital tract morbidity as evidenced by a request for a report on the microbiological findings. The frequency of isolation of the various microbes in health and in disease is given. The grading of Gram-stained smears bore no relation to the isolation rates of lactobacilli, but there was a significant increase (p less than 0-001) in the isolation rates of each of the following: Mycoplasma hominis, Bacteroides spp., Trichomonas vaginalis, Gram-variable cocco-bacilli, and anaerobic streptococci in those patients with smears in which lactobacilli were adjudged to be absent. The isolation of faecal streptococci was increased (p less than 0-001) in women aged more than 34 years. Escherichia coli (p less than 0-05) and anaerobic and microaerophilic streptococci (p less than 0-02) were isolated more frequently from those booking after the 25th week of pregnancy. The incidence of M. hominis (p less than 0-02) and of anaerobic streptococci (p less than 0-05) increased between the first and third trimesters. No significance positive correlations were established between the isolation rates of the various microbes and objective assessment of lower genital tract morbidity or the demonstration of pus cells, but lactobacilli were isolated less frequently (p less than 0-01) from those with morbidity. The isolation of Candida albicans (p less than 0-02), T. vaginalis (p less than 0-05), and M. hominis (p less than 0.05) was increased in patients in whom vulvovaginitis was suspected, and that of T. vaginalis (p less than 0-05) was increased in those with a past history of vulvovaginitis. The study indicates that, other than the pathogens T. vaginalis and C. albicans, only M. Hominis could be suspected, on statistical grounds, of being associated with disease of the lower genital tract during early pregnancy.


Evaluation of a dehydrated test strip for the detection of yeasts. Use of a dehydrated test strip for the detection of yeasts is compared with traditional culture on Sabouraud's agar containing 50 mug/ml chloramphenicol. While the selective medium of the strip is satisfactory for the isolation of species of Candida, Torulopsis glabrata grows only very slowly. The strip has the advantage of a long storage life without deterioration, but a high cost may preclude general usage. The numbers of yeasts collected by a bacteriological swab disadvantages of the selective medium, and the value of direct microscopy in the examination of vaginal swabs are discussed.


The effect of a chlorhexidine-containing gel on oral cleanliness and gingival health in young adults. Students at a vocational school participated in a double-blind, cross-over study to test the effect of brushing with a 1% chlorhexidine-containing gel once a day. In the control period, a placebo gel was used. Each treatment period lasted for 4 weeks. This was subsequently followed by 4 weeks of ordinary toothbrushing. Any toothbrushing additional to the therapeutic brushing was performed with a standard commercial dentifrice without abrasives. In addition to the treatment groups, containing a total of 24 subjects, another group of 12 subjects received standardized oral hygiene instruction. Oral hygiene and gingival condition were assessed in all groups throughout the study. The chlorhexidine-containing gel seemed to have no effect on gingivitis and only a slight inhibitory effect on plaque formation. Furthermore, an increase in the degree and frequency of staining of the tooth surfaces related to the use of active gel were observed.


A study of cochlear innervation patterns in cats and rats with the Golgi method and Nomarkski Optics. Cochlear innervation patterns were studied in infant cats and rats with the rapid Golgi method. Examination of thick serial sections and surface preparations with the differential interference contrast microscope (Nomarski optics) allowed direct visualization of individually impregnated spiral ganglion cells, complete with their peripheral processes and endings in the organ of Corti. Individually impregnated efferent fibers could  be recognized as heavily varicose axons that project radially to endings beneath inner and outer hair cells after taking a tangential course in the intraganglionic spiral bundle. It was often possible to visualize unimpregnated hair cells in contact with the impregnated endings of both types of fibers. There are at least two types of spiral ganglion cells in the cochlea of the infant cat and rat. One type innervates only inner hair cells by means of radial fibers. These ganglion cells constitute the overwhelming majority of ganglion cells impregnated in our preparations, and each cell typically innervates two inner hair cells. Hence, these ganglion cells establish nearly "point-to-point" connections between the auditory nerve and the organ of Corti. The other type of ganglion cell innervates  outer hair cells by means of long spiral fibers; each cell typically innervates many outer hair cells through the numerous angular enlargements and short end branches of its spiral fiber. In addition, a few of these spiral fibers also send branches to inner hair cells by means of short collaterals; it remains to be seen if such fibers also occur in mature cochleas. Efferent fibers have been traced to inner and outer hair cell regions. The simplest pattern is formed by fine beaded axons with only a few branches ending mainly beneath inner hair cells. More complex patterns are formed by larger axons with many branches ending beneath inner or outer hair cells. Many efferent fibers send branches to both inner and outer hair cells. Electrophysiological studies so far have not demonstrated different populations of units that clearly correspond to the spiral and radial fibers. Therefore, the physilogical differences between inner and outer hair cell innervation remain undefined.


The central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-beta-hydroxylase as a marker. A sensitive immunofluorescence technique was used to describe systematically the distrubution of dopamine-beta-hydroxylase (DBH)-containing cell bodies, non-terminal fiber pathways, and terminal fields in the brain of the male albino rat. DBH is the enzyme that catalyzes the conversion of dopamine to noradrenaline, and as such is useful as an anatomical marker for noradrenaline and possibly adrenaline neurons. The enzyme is not present in dopamine- or indolamine-containing neurons. Ten micron frozen sections (1-in 20 series) were prepared in the frontal, sagittal, and horizontal planes from the olfactory bulb to the upper cervical segments of the spinal cord; adjacent sections in each plane were stained for DBH and for cells (toluidine blue=azure II). An atlas consisting of 40 projection drawings of selected frontal sections illustrates the results of the investigation. DBH perikarya are confined to three groups in the pons and medulla: the well defined locus coeruleus, a more diffuse but continuous subcoeruleus group that arches through the pons and ventral medulla, and a third dorsal medullary group centered in the dorsal motor nucleus of the vagus. A single principal adrenergic fiber system distributes a great many of the axons from these neuron groups to a majority of nuclear areas in the brain. In the pons and medulla two components of the fiber system may be distinguished. A medullary branch may be followed from the posterior aspect of the subcoeruleus group dorsally and then anteriorly through the lateral tegmental field and ventral aspect of the vestibular complex to a position subjacent to the locus coeruleus, where it is joined by a subcoeruleus branch consisting of a large number of fibers coursing among cells along the length of the subcoeruleus group, and by fibers arising from the locus coeruleus. Anterior to the locus coeruleus the principal adrenergic bundle courses as a single fiber tract immediately ventrolateral to the central gray in the mesencephalon and in the zona incerta and substantia innominata in the diencephalon. At the level of the septal area separate bundles reach the cortex dorsally over the genu of the corpus calosum via the medial septal-diagonal band nuclei and the lateral septum and ventrally between the olfactory tubercle and caudate-putamen. In the medulla and pons adrenergic fibers undoubtedly course in both directions. Anterior to the most rostral pontine cell bodies, however, all fibers presumably ascend. Along the course of the bundle distinct branches emerge to innervate circumscribed terminal fields. In addition, certain regions of the brain such as the reticular formation and pontine gray receive diffuse DBH innervation derived from less clearly defined pathways. A small number of areas in the brain contain little or no detectable DBH. These include the caudate-putamen, nucleus accumbens, globus pallidus, olfactory tubercle, subthalamic nucleus, substantia nigra, pretectal area, third, fourth and sixth cranial verve nuclei, and the trapezoid body nucleus.


In vitro evaluation of marginal leakage of corrosion-resistant amalgam alloy. Restorations placed without coating the cavity walls with cavity varnish exhibited significant marginal leakage at all time intervals; however, there appears to be some reduction of leakage after six months. Restorations placed after coating the cavity walls with cavity varnish demonstrated no leakage at forty-eight hours and three months. Velvalloy and Dispersalloy restorations exhibited no leakage at six months, although there was a slight leakage at one year. Spheraloy restorations with cavity varnish demonstrated some leakage at six months and one year. The results of this study appear to correlate with previous studies using the same technique to determine marginal leakage. Apparently, the corrosion resistance of the dispersed phase alloy does not increase the marginal leakage, when compared with the conventional and spherical alloys.


Pontics in fixed prostheses--status report. Council on Dental Materials and Devices. It is clear that the choice and fabrication of a pontic demands as much care as any aspect of restorative planning and execution. To apply a particular pontic design arbitrarily is to disregard the work to date which appears to emphasize the individuality of each pontic situation. Existing designs appear to take into account every conceivable situation. Presently available materials seem adequate to meet requirements. The role of bacterial plaque control in the maintenance of all pontic materials and designs must be recognized. What is required is a dentist cognizant of the reasons for using a particular pontic in a particular situation and a patient highly motivated and instructed as to his responsibility in caring for this component of his prothesis.


The isolation of salmonellas from British pork sausages and sausage meat. Between 1969 and 1974, 1467 packets (3309 samples) of pork sausages and sausage meat produced by two large and two medium sized manufacturers and several local butchers were examined for the presence of salmonellas. Of these, 435 packets (786 samples) were found to contain salmonellas, but there was a wide variation in the isolation rates according to the producer. The salmonella incidence in samples from several small and two medium sized producers was low (0-11%) while the results from the two large producers investigated showed a striking difference, the rate of salmonella contamination in the product of one was low (about 2%) and in that of the other consistently high (40-60%). A comparison of liquid enrichment media, incubation temperatures and selective agar media was also carried out to determine the most efficient combination for the isolation of salmonellas from minced meat products. The results showed that (a) incubation of enrichment cultures at 43 degrees C. yielded a consistently greater number of salmonella isolations that at 37 degrees C., regardless of plating medium, (b) tetrathionate broth A (Rolfe) was superior to selenite broth as en enrichment medium at both 37 and 43 degrees C. and (c) brilliant green agar gave better results than deoxycholate citrate sucrose agar and bismuth sulphite agar as a selective medium.


Contribution to the study of live streptomycin-dependent Salmonella vaccines: the problem of reversion to a virulent form. The recovery of virulence by means of reversion of a live streptomycin-dependent (Sm D) Salmonella typhimurium vaccine was studied in CD-1 Swiss mice. Initially, a one-step Sm D mutant was obtained from a virulent streptomycin-sensitive (Sm S) S. typhimurium strain. Afterwards, two pools of streptomycin-independent (Sm I) revertants were prepared from the Sm D strain. The virulence of the Sm D strain and of the Sm I revertants was tested intraperitoneally. In the virulence testing the original suspension of the Sm I revertants, as well as their 1st and 10th passages on plain medium, medium+50 mug. streptomycin/ml. and medium+1000 mug. streptomycin/ml. were used. The results show that the Sm D mutant was avirulent, its avirulence being due to an intrinsic, genetic quality. The Sm I revertants, compared to the original Sm S strain, also displayed a lack of virulence. However, afterwards, the Sm I revertants behaved quite differently, according to their subsequent passages. Indeed, there was an increase in virulence after passages on plain medium, whereas similar passages on medium containing the drug, the virulence not only failed to increase, but disappeared almost completely. Moreover, the passages on medium containing 1000 mug. streptomycin/ml. induced a return to the status of drug-dependence. The danger of recovery of virulence by means of revertants is evaluated.


Experimental model of oral antityphoid vaccination with live streptomycin-dependent Salmonella typhimurium in C57BL/6 mice. The present experimental model offered the opportunity to study particular aspects concerning the avirulence stability of live streptomycin-dependent (Sm D) Salmonella vaccines, under conditions resembling human enteric fever. The results seem to indicate that, in practice, the risk of reversion to a virulent form during oral antityphoid vaccination with Sm D strains remains slight even after the interruption of concomitant streptomycin administration.


Comparison of two methods for assessing the removal of total organisms and pathogens from the skin. A standard hand-wash sampling technique was compared with a simple finger-streak sampling method in assessing the relative effectiveness of a number of alternative preparations used for disinfecting the surgeon's hands (alcoholic 0.5% chlorhexidine, alcoholic 0.1% tetrabrom-o-methyl phenol, a 4% chlorhexidine detergent solution, aqueous 0.5% chlorhexidine, 2% 'Irgasan' detergent solution and, as control, bar soap). There was a fairly good correlation between the results of assessment by the two methods after a single disinfection and after six disinfections, three on one day and three on the next. Significant differences were shown in 21 comparisons between treatments when the hand-wash sampling test was used, and 16 of these comparisons also showed a significant difference by the finger-streak test. Staphylococcus aureus was found in hand samplings from 5 out of 8 nurses in the Burns Unit of Birmingham Accident Hospital by the hand-wash sampling method and from 2 of the same 8 nurses by the finger-streak method; the numbers were small, and no Staph. aureus were isolated from the same hands after 1 min. wash in 70% ethyl alcohol. Similar sampling on 29 nurses in other wards showed Staph. aureus on 3 nurses (one in large numbers) by the hand-wash technique and on 1 nurse by the finger-streak test; in only 1 nurse whose hands showed Staph. aureus before disinfection was the organism found, by hand-wash sampling, after disinfection. Parallel sampling of nurses' hands after washing with soap and water and after disinfection with 95% ethanol showed larger numbers of Staph. aureus in a hospital for skin diseases than in a general hospital, and a lower incidence and somewhat lower density of Staph. aureus after ethanol treatment than after washing with soap and water; Gram-negative bacilli, on the other hand, were commoner on hands in the general than in the skin hospital, and present in much smaller numbers after disinfection with ethanol than after washing with soap and water. Antibiotic sensitivity tests showed the frequent recurrence on the hands of some nurses of multi-resistant Staph. aureus with resistance patterns similar to those found in infective lesions in some of the patients; different sensitivity patterns were usually found in staphylococci isolated from the nose. Even in wards where many patients were infected, carriage by nurses' hands of a particular strain of Staph. aureus did not seem to last for more than a few days.


The effect of antibiotic therapy on the faecal excretion of Salmonella typhimurium by experimentally infected chickens. Chickens in groups of 40 were infected orally with a nalidixic acid-resistant mutant of Salmonella typhimurium and then fed continuously on diets containing ampicillin, chloramphenicol, furazolidone, neomycin, oxytetracycline, polymixin, spectinomycin, streptomycin or a mixture of trimethoprim and sulphadiazine. The amount of S. typhimurium excreted in their faeces was estimated at intervals by culture on brilliant green agar containing sodium nalidixate, both direct and after enrichment in selenite broth; the amount of Escherichia coli excreted was estimated by culture on MacConkey agar. The feeding of diets containing 500 mg./kg. of ampicillin, furazolidone, neomycin, polymixin, spectinomycin or streptomycin or 100 mg./kg. of trimethoprim and 500 mg./kg. of sulphadiazine for 46 days reduced to a varying degree the amount of S. typhimurium and E. coli excreted, the greatest reduction in S. typhimurium being brought about by the last treatment. The effect was less obvious when the concentration of the antibiotics in the food was decreased fivefold. An important reason for the very limited effect of some of the antibiotics was the emergence of antibiotic-resistant populations of S. typhimurium and E. coli. High concentrations of antibiotic-resistant organisms also arose in the faeces of the chickens fed diets containing tetracyclines and chloramphenicol, treatments which had no apparent effect on the amount of S. typhimurium and E. coli excreted. Much of the antibiotic resistance encountered was determined by R factors, a particular R factor usually being found in the E. coli populations of individual chickens before it was found in their S. typhimurium populations. No S. typhimurium or E. coli were isolated that possessed R factors determining resistance to polymixin, furazolidone or trimethoprim. No S. typhimurium or E. coli were isolated that were polymixin-resistant and no S. typhimurium that were furazolidone-resistant. The few trimethoprim-resistant S. typhimurium isolated were thymine-dependent. The feeding of diets containing the higher concentrations of trimethoprim/sulphadiazine, neomycin, furazolidone or ampicillin for 9 days reduced the amount of S. typhimurium excreted. After the withdrawal of these diets, the amount of S. typhimurium excreted increased to the numbers found in chickens given ordinary diets throughout; the chickens that had been given trimethoprim/sulphadiazine or furazolidone did not remain faecal excreters of S. typhimurium longer than the chickens that had been given ordinary diets. Similar results were obtained with trimethoprim/sulphadiazine when the start of the 9-day treatment period was delayed for an extra 9 days or when it was extended to 18 days.


The effect of feeding diets containing permitted antibiotics on the faecal excretion of Salmonella typhimurium by experimentally infected chickens. Groups of 45 chickens were fed continuously on diets containing 10 or 100 mg./kg. of different 'growth-promoting' antibiotics and infected orally with a nalidixic acid-resistant mutant of Salmonella typhimurium. The amount of S. typhimurium organisms excreted in their faeces was estimated by culturing them at weekly intervals and in a standard manner on plates of brilliant green agar containing sodium nalidixate, both direct and after enrichment in selenite broth. All of four groups fed diets containing 100 mg./kg. of nitrovin in three different experiments excreted much larger amounts of S. typhimurium than did groups fed antibiotic-free diets. In some, but not all, experiments, larger amounts were also excreted by groups fed diets containing 10 mg./kg. of nitrovin or 10 or 100 mg./kg. of flavomycin or tylosin. Feeding diets containing 10 or 100 mg./kg. of virginiamycin or bacitracin either did not influence or slightly increased the amount of S. typhimiurium excreted. Two groups fed continuously on diets containing 100 or 500 mg./kg. of sulphaquinoxaline for 44 days excreted smaller amounts of the S. typhimurium organisms that did groups fed antibiotic-free diets; no sulphonamide-resistant organisms of the S. typhimurium strain were isolated from the faeces.


Complex surveillance of Streptococcus pyogenes. II. Resistance in human blood and bactericidal activity receptors of field and collection streptococci. A comparative study of a series of 31 epidemic streptococci and 36 collection M protein producers showed no statistically significant difference in their resistance in human blood. Even highly resistant field strains did not produce M protein (as the main bactericidal activity receptor) under laboratory conditions, however, although under natural conditions they left an anti-M response in the exposed population. Streptococcal resistance is not generally associated with M protein, but is influenced by another factor.