Spontaneous mutation of polysaccharide production in Alcaligenes faecalis var. myxogenes 10C3

Alcaligenes faecalis var. myxogenes 10C3, which produces large amounts of succinoglucan and small amounts of curdlan, was genetically unstable and mutated spontaneously to a form producing more curdland than succinoglucan when stocked on nutrient agar slants. The mutation occurred in the absence of cell division when the cells were incubated in saline and was enhanced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methane sulfonate, or ultraviolet light. Mutant strains were genetically stable and did not revert spontaneously for at least 1 year when stocked on nutrient agar slants.     

Oxidase testing from Kligler's iron agar and triple sugar iron agar slants

Many members of the familyVibrionaceae have been implicated as causative agents of diarrhea. Most of these organisms are non-lactose fermenters, and all are oxidase-positive. If the oxidase test could be reliably performed on growth from the surface of Kligler's iron agar and/or triple sugar iron agar slants, it would aid in the screening of potential stool pathogens. Forty-six isolates from the generaAeromonas, Plesiomonas, andVibrio were inoculated onto Kligler's iron agar and triple sugar iron agar slants, incubated overnight, and tested for oxidase activity. All 46 isolates produced alkaline over acid, with or without gas, Kligler's iron agar slants and were oxidase-positive. On triple sugar iron agar slants, 13 isolates produced these same patterns, and all were oxidase-positive. Acid over acid, with gas, triple sugar iron agar slants were produced by 18 isolates, and all were oxidase-positive. Acid over acid, without gas, triple sugar iron agar slants were produced by 15 isolates, and all were oxidase-negative. These negative oxidase tests were due to low pH. Oxidase tests performed from the surface of Kligler's iron agar and triple sugar iron agar slants used to screen stool isolates were reliable, provided the slants were acid over acid with gas, or alkaline over acid with or without gas. Kligler's iron agar is recommended with this procedure, since most potential stool pathogens of both theEnterobacteriaceae and theVibrionaceae will produce an alkaline over acid, with or without gas, slant, and false negative oxidase tests will be minimized.     

Survival, growth and pathogenicity of Gaeumannomyces graminis var. graminis with different methods of long-term storage

The fungal plant pathogen Gaeumannomyces graminis var. graminis was preserved with 12 different storage methods. Five strains, each with unique morphological and pathological characteristics, were used for comparison of the methods. The storage treatments included potato-dextrose agar slants, with or without mineral oil, stored at either 4 C, 28 C or ambient temperature; colonized agar plugs placed in glycerol solution at either -75 C or -20 C; colonized agar plugs placed in sterile deionized water at either 4 C or ambient temperature; and mycelial growth on intact or precut pieces of filter paper, desiccated and stored at ambient temperature. Survival was evaluated at 6, 12, 24, 36, 48 and 120 mo. The three best treatments for survival were PDA slants, with or without mineral oil, and colonized agar plugs stored in water, all at ambient temperature. All five fungal strains were recovered from all four replicates at each sampling date for agar plugs stored in water at ambient temperature. The worst treatments were agar slants and agar plugs in water stored at 4 C and agar plugs stored in glycerol at -20 C. Morphological characteristics were not affected by storage treatments. In general, there were minimal or no effects on growth and pathogenicity for all strains for all storage treatments with survival. Colonized agar plugs stored in water at ambient temperature provides an economical storage method (materials and labor) that does not need an electrical power for long-term maintenance.     

Rapid test for determination of urea hydrolysis

A urease test for the rapid determination of urea hydrolysis is described in which diluted urea agar concentrate was used in small amounts with dense inoculum of the test organisms. The method was evaluated and compared with Christensen's urea agar slants by using 728 clinical isolates of gram-negative bacteria. Of the 325 strains of urease-positive Proteus-Providencia-Morganella, 282 (87%) gave positive results within 5 min with the rapid test. Urease activity of 97% of these organisms became evident within 30 min. All 287 isolates which showed no urease activity on Christensen's urea agar also remained negative by this test.     

Expression of a new porin 'OmpE' by strains of Salmonella enteritidis

Abstract Strains of Salmonella enteritidis expressed a novel porin with a subunit size of 35.5 kDa as shown by SDS-PAGE. This protein was expressed as a major outer membrane protein (MOMP) when grown on nutrient agar, McConkey agar or blood agar, or in Tris-succinate medium; but was only produced in trace amounts when strains were grown in nutrient broth. This OMP was produced by all strains of S. enteritidis examined, regardless of phage type, and expression was not related to the possession of a 38-MDa mouse-virulence plasmid or the ability of strains to make long-chain lipopolysaccharide. This new porin has been tentatively called OmpE.     

Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil.

The conjugative plasmid pIJ101 and its conjugative nondeletion derivatives pIJ303 and pIJ211 were tested for their transferability between strains of Streptomyces on laboratory media and in the soil environment. Their roles in the mobilization of the cloning vector plasmid pIJ702, a nonconjugative deletion derivative of pIJ101, were also examined. Biparental and triparental crosses were performed on agar slants and in sterile soil between the plasmid donor Streptomyces lividans and several recipient Streptomyces strains previously isolated from soil. Conjugative plasmids were transferred to seven recipients in slant crosses and to three recipients in soil. Plasmids isolated from recipients showed restriction fragment patterns identical to that of the original plasmid in S. lividans. Plasmid pIJ303 was transferred less frequently in soil than on slants, and the frequency of transfer was higher at 30 degrees C than at the other temperatures examined. Transconjugant Streptomyces strains differed in their ability to maintain pIJ303. The nonconjugative plasmid pIJ702 was mobilized on agar slants into S. coelicolor 2708, which already contains a self-transmissible plasmid. Plasmid pIJ702 was also mobilized into S. flavovirens, Streptomyces sp. strain 87A, and S. parvulus on slants and in sterile soil after triparental crosses with two donors, one containing pIJ702 and the other containing either pIJ101 or pIJ211. The presence of a conjugative plasmid donor was required for the transfer of pIJ702 to S. parvulus 1234, S. flavovirens 28, and Streptomyces sp. strain 87A. Plasmid pIJ702 was always transferred in its normal, autonomous form. Chromosomal recombination also occurred in transconjugants after the transfer of pIJ702. This is the first report of gene transfer between Streptomyces strains in soil.     

Relative stability of biological properties in encapsulated strains of Staphylococcus aureus after freezing and freeze-drying

The relative stability of the biological properties of three encapsulated strains of Staphylococcus aureus was compared after preservation for 1 year in two different vehicles, 10% glycerol and 15% honey and at two different temperatures, ?30 and ?80 °C. A third method of preservation was by lyophilization in 10% skim-milk plus 0.1% glutamic acid and 2% honey. Comparison with control stock cultures maintained by bimonthly subcultivation on brain heart infusion (BHI) agar slants indicated that viability of the organisms was best preserved in 15% honey. When freezing and freeze-drying were compared, superiority was achieved by the latter. Quantitative activities of acid phosphatase, DNase, and coagulase remained constant in all subcultures. Also, while no loss of virulence for mice was observed with these methods, some did occur with the stock subcultures on BHI agar slants. Concerning relative salt tolerance of the strains in these preparations, the lyophilized organisms surpassed the frozen ones. However, when lyophilizing time was prolonged, yellow pigmentation corresponding to β-carotene decreased. Finally, both frozen and lyophilized organisms maintained stable characteristics of growth type in serum-soft agar.     

Stability of the antibiotic formation trait of the tobramycin producer Streptomyces cremeus subsp. tobramycini

The stability of the tobramycin-producing organism was studied by the property of the antibiotic production. The organism was stored under conditions of different exposures to light and subculture on slants. The culture was also subjected to long-term storage with various methods. The organism was stable in preserving its antibiotic activity for 1.5 months when stored on the Gauze organic agar No. 2. Subcultures of the organism on this medium provided preservation of the antibiotic activity throughout 4 passages. The culture storage on millet provided preservation of the antibiotic activity for 6 months.     

Effects of long-term storage on plasmid stability in Bacillus anthracis

The plasmid profiles of 619 cultures of Bacillus anthracis which had been isolated and stored between 1954 and 1989 were analyzed using the Laboratory Response Network real-time PCR assay targeting a chromosomal marker and both virulence plasmids (pXO1 and pXO2). The cultures were stored at ambient temperature on tryptic soy agar slants overlaid with mineral oil. When data were stratified by decade, there was a decreasing linear trend in the proportion of strains containing both plasmids with increased storage time (P < 0.001). There was no significant difference in the proportion of strains containing only pXO1 or strains containing only pXO2 (P = 0.25), but there was a statistical interdependence between the two plasmids (P = 0.004). Loss of viability of B. anthracis cultures stored on agar slants is also discussed.     

5-Fluoroorotate-resistant mutants of Salmonella typhimurium.

Spontaneously occurring mutants of Salmonella typhimurium resistant to 5-fluoroorotate (5-FOA) were isolated. One class of mutant showed marked derepression of pyrimidine biosynthetic enzymes and had the unusual property of being unable to grow on nutrient agar. However, when the osmotic strength of nutrient agar was increased, the mutants were able to grow. The genetic basis for the osmotic fragility and elevated pyr enzyme synthesis was the result of mutations affecting pyrH, encoding the enzyme uridine 5'-monophosphate kinase.     

In Vitro Induction of a Herpes-Type Virus in “Summer-Phase” Lucké Tumor Explants

A herpes-type virus was induced in explants of "summer-phase" Lucké tumor maintained on agar slants at 7.5 C for 13 to 14 weeks.     

Lysine Decarboxylase Activity in Broth and Agar Media

Four lysine decarboxylase media were studied by testing them with 305 Enterobacteriaceae and 42 nonfermenting bacilli. A comparison was made between lysine decarboxylase broth medium (Moeller base) and Johnson's semisolid agar without lactose and Bachrach's broth medium and lysine-agar slants which contain lactose. The nonlactose media, lysine decarboxylase broth and the semisolid medium of Johnson, were the best media for use with all of the bacteria studied. The exclusion of lactose from lysine decarboxylase medium seems desirable to extend the usefulness of this medium among members of the Enterobacteriaceae. When the results with lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, a 6% difference existed between the results obtained with lysine decarboxylase broth and Johnson's semisolid agar. When the results with Bachrach's broth and lysine-agar slants with lactose were compared, a 1% difference existed between Bachrach's broth and the agar slant method. At times, reading and interpretation were difficult because of intermediate degrees of color change. The inability of Pseudomonas aeruginosa or Herellea to utilize glucose under the anaerobic condition of the medium makes the lysine decarboxylase test an undesirable procedure for these organisms. Of the four test media used, the lysine-lactose-agar slants seemed to be the least desirable because of the more frequent occurrence of indistinct color reactions and shifts in color.     

Modeling growth and succinoglucan production by Agrobacterium radiobacter NCIB 9042 in batch cultures

Wild-type Agrobacterium radiobacter NCIB 9042 has been cultivated in batch cultures on a synthetic medium which was adapted for growth and succinoglucan production. Experiments were carried out in a 4-L stirred-tank aerated reactor. Glucose, biomass, polysaccharide, protein, and inorganic- and organic-nitrogen concentrations were measured, and oxygen consumption and CO(2) production rates were obtained by a gas-balance technique. Nitrogen balance shows that inorganic nitrogen is entirely recovered into proteins. The carbon balance is satisfied with in +/-5%. Stoichiometric equations for biomass growth and succinoglucan synthesis were established. The biosyntheticpolymer pathways including ATP and cofactor consumption were investigated. From previous studies, a (P/O) value of 1.66 is selected for oxygen sufficient cultures. The actual ATP requirements of 25.4 mmol ATP/g succinoglucan (38.5 mol ATP/mol succinoglucan), determined by a metabolic analysis, is 2.39 times the stoichiometric value. Experimental results were modeled by a system of differential equations. The exponential growth phase was described by a nitrogen-limited Monod equation. Subsequent succinoglucan synthesis followed a slightly modified Luedeking-Piret relation partitioning internal and external polysaccharide. Experimentally determined coefficients are compared with published results for continuous culture of A. radiobacter NCIB 11883.     

Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil

The conjugative plasmid pIJ101 and its conjugative nondeletion derivatives pIJ303 and pIJ211 were tested for their transferability between strains of Streptomyces on laboratory media and in the soil environment. Their roles in the mobilization of the cloning vector plasmid pIJ702, a nonconjugative deletion derivative of pIJ101, were also examined. Biparental and triparental crosses were performed on agar slants and in sterile soil between the plasmid donor Streptomyces lividans and several recipient Streptomyces strains previously isolated from soil. Conjugative plasmids were transferred to seven recipients in slant crosses and to three recipients in soil. Plasmids isolated from recipients showed restriction fragment patterns identical to that of the original plasmid in S. lividans. Plasmid pIJ303 was transferred less frequently in soil than on slants, and the frequency of transfer was higher at 30 degrees C than at the other temperatures examined. Transconjugant Streptomyces strains differed in their ability to maintain pIJ303. The nonconjugative plasmid pIJ702 was mobilized on agar slants into S. coelicolor 2708, which already contains a self-transmissible plasmid. Plasmid pIJ702 was also mobilized into S. flavovirens, Streptomyces sp. strain 87A, and S. parvulus on slants and in sterile soil after triparental crosses with two donors, one containing pIJ702 and the other containing either pIJ101 or pIJ211. The presence of a conjugative plasmid donor was required for the transfer of pIJ702 to S. parvulus 1234, S. flavovirens 28, and Streptomyces sp. strain 87A. Plasmid pIJ702 was always transferred in its normal, autonomous form. Chromosomal recombination also occurred in transconjugants after the transfer of pIJ702. This is the first report of gene transfer between Streptomyces strains in soil.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. VII. CELLULAR VARIATION

Variation in long-continued cultures of Haplopappus gracilis and Daucus carota has been investigated. A strain of carrot tissue was isolated that grew with a compact habit, in contrast to the highly friable habit of the parent strain. Its dividing cells were arranged quite differently than in the parent strain. Earlier work had shown that Haplopappus cultures could be reversibly altered in their pigmentation and form, by changing the culture medium. This was confirmed, and it was further shown that pronounced changes in nitrogenous compounds also occurred in response to factors in the medium. However, strains of Haplopappus were isolated which differed persistently from the parent strain, even when they were maintained under the same conditions. The variant strains, grown in the same medium, showed differences in their content of nitrogenous compounds. Stock cultures also changed spontaneously with time with respect to their content of nitrogenous substances. Acriflavine, at low concentration, inhibited the growth and formation of colonics by cells plated on nutrient agar, but, by prolonged exposure to sublethal amounts of the drug, resistant strains were isolated. Certain of the spontaneous variant strains were found to differ from each other and from the parent strain in their chromosome complements in ways that are described and to which the observed changes in morphology and metabolism of the cultures may be attributed. The variations that may occur in the free cells in culture are contrasted with the greater uniformity of the cells as they exist in the plant body.     

Effects of Long-Term Storage on Plasmid Stability in Bacillus anthracis

The plasmid profiles of 619 cultures of Bacillus anthracis which had been isolated and stored between 1954 and 1989 were analyzed using the Laboratory Response Network real-time PCR assay targeting a chromosomal marker and both virulence plasmids (pXO1 and pXO2). The cultures were stored at ambient temperature on tryptic soy agar slants overlaid with mineral oil. When data were stratified by decade, there was a decreasing linear trend in the proportion of strains containing both plasmids with increased storage time (P < 0.001). There was no significant difference in the proportion of strains containing only pXO1 or strains containing only pXO2 (P = 0.25), but there was a statistical interdependence between the two plasmids (P = 0.004). Loss of viability of B. anthracis cultures stored on agar slants is also discussed.     

Slime Production by Pseudomonas aeruginosa

Adequate experimental conditions for slime production by Pseudomonas aeruginosa were investigated using a cellophane plate method. Definite slime production was observed on heart infusion agar, brain heart infusion agar, yeast extract agar and synthetic agar, but not on nutrient agar. The addition of phosphate to the nutrient agar above 0.05% caused visible slime formation. Incubation at 37 C resulted in a higher yield of slime than at 25 C. Longer incubation seemed more favorable for slime production, while the pH reaction of the test media did not effect the slime yield. All the test cultures of P. aeruginosa produced large amounts of slime by this procedure. Cultures of Pseudomonas fluorescens, other Pseudomonas spp. and certain vibrios also produced slime under these experimental conditions.     

THE ISOLATION AND CULTURE IN VITRO OF THE QUIESCENT CENTER OF ZEA MAYS

Using 3-day-old seedling roots of Zea mays L., cv. Kelvedon 33, it was possible to remove the root cap by a simple surgical manipulation without damage to the root proper. By a further small cut, the quiescent center (QC) itself was isolated. This double-convex lens-shaped tissue piece 100 X 250 μm is composed of 1000–1500 cells representing only 0.25 mm³ in volume. The explant was demonstrated unequivocally by ³H-thymidine incorporation before excision and then by autoradiography to be composed of the specific cells usually designated the quiescent center. Using sterile techniques, the QC's were placed on nutrient agar slants and allowed to grow in culture. Of a number of nutrient media tested, only a medium supplemented with organic nitrogen components, indoleacetic acid, kinetin and inorganic nutrients plus sucrose (S2M + K -2,4-D) was effective in eliciting development. Thirty to 40 percent of the 150 isolated QC's grown on this medium formed elongated roots, up to 2 cm in length in 3–4 weeks. Roots developing on agar medium showed in their proximal portion a vascular pattern with 5–6 metaxylem elements or variations of this pattern, but as the root elongated, the vascular pattern was progressively reduced in complexity at the more distal end to a small central group of metaxylem elements. When agar-grown roots were transferred after one week in culture to a liquid nutrient medium of the same composition, the initially reduced vascular pattern evident in the proximal tissues became progressively more complex in the distal portion of the root and after 2 cm of elongation, showed an essentially normal primary vascular tissue pattern characteristic of the seedling root.     

The impact of long-term contact of Achromobacter sp. 4(2010) with diesel oil – Changes in biodegradation,surface properties and hexadecane monooxygenase activity

A bacterial strain was isolated from soil that was contaminated with diesel oil and was used in our experiments. The strain was then phenotypically, biochemically and genetically tested and named as Achromobacter 4(2011). In order to examine the impact of long-term contact with diesel oil of bacterial cells, the strain was stored under different conditions – on standard nutrient agar plates and on agar plates with 50 μl diesel oil as a sole carbon and energy source. The results clearly indicated that longer contact with diesel oil led to changes in both the bacterial surface and biochemical properties, as well as the hexadecane monooxygenase activity. Moreover, the fatty acid profiles also changed, leading to an increased content of saturated fatty acids. In addition, the rates of biodegradation of diesel oil were higher even when supplemented with the surfactants – rhamnolipids and saponins. This work demonstrates that prolonged contact of microorganisms with diesel oil can lead to many changes, not only in biodegradation potential, but also in their surface and genetic properties.     

Nutritional studies of histoplasma capsulatum

Summary A chemically defined medium, composed of inorganic salts, glucose, asparagine, cystine, and a vitamin supplement, has been devised for growth of the yeast phase ofHistoplasma capsulatum. Growth in this medium was abundant and compared favorably with that in media containing complex natural material. Conversion of each of the 20 strains examined was accomplished by one or more passages on agar slants of the medium and incubation of the cultures at 37° C. Yeast phase cultures on this medium have been stored for 6 months or more at approximately 4° C without conversion or loss of viability. Of the 20 strains examined for vitamin requirements of the yeast phase, all were partially deficient for thiamine; nine for inositol; five, either partially or completely deficient for niacin; and one, completely deficient for biotin.No specific amino acid was required for growth of the yeast phase, but an organic source of sulfur and one of nitrogen were essential. Cystine and cysteine were equally effective for growth of the yeast phase when supplied on an equivalent sulfur basis and very little difference in growth occurred in media which contained equal amounts of nitrogen in any one of the following compounds: asparagine, glutamic acid, aspartic acid, and proline,Of the 20 strains, all but one, which requires biotin, were capable of continued growth in the mycelial phase when subcultured on an agar medium containing only inorganic salts and dextrose, but growth was improved significantly by asparagine or casein hydrolysate.This investigation was supported in part by a PHS research grant (AI-03524) from the National Institute of Allergy and Infectious Diseases, Public Health Service.