Solid Growth Media Research Articles

Cryopreservation of fungal spores using porous beads

A simple method is described for the cryopreservation of conidia, stored adhering to small porous beads in a robust polypropylene vial. For recovery, a single bead is removed from the vial and streaked onto a solid growth medium. Preparative work is minimised so the method is rapid. The storage life of an isolate can be increased greatly if a ‘seed lot’ system is employed.

Biodegradation of Polymer Coatings

AbstractConventional paint removal methods include chemical stripping with VOCs, blasting with plastic media, and delamination with high pressure water. These methods have many limitations, in that they are labor intensive, pose human health risks, are relatively expensive and pose significant waste disposal problems. However, polymeric coatings are known to contain structural components, such as ester, amide and urea linkages, that can be degraded biologically. We are working to develop a stable, enzyme-based, non-toxic paint stripping strategy that will be environmentally safe and cost effective.The specific objectives are to identify and characterize microbial systems capable of degrading polymeric coatings, to develop a quantitative degradation assay and to optimize activity levels for subsequent purification and concentration of the biological products required for rapid degradation of coatings.A water-dispersed colloid of an ester-based polyurethane polymer has been used in solid growth medium to screen about 100 different bacteria for microbial degradation activity. Those with demonstrable activity have been grown in the presence of epoxypolyamide paint- and polyester polyurethane paint-coated aluminum coupons. We have demonstrated delamination under certain conditions and have developed a spectrophotometric method for quantitating degradation activity as a function of dye release.

Degradation of benzene compounds by yeasts in acidic soils

Attemps were made to demonstrate the role of yeasts in the degradation of benzene compounds under natural soil conditions. Yeasts were isolated from acidic sandy soil supplied with benzene compounds. For this purpose the ‘slant culture method’ was used. Growth on the benzene compounds took place on solid growth media at 10°C. Several yeast species were isolated: Leucosporidium scottii, Rhodotorula aurantiaca, Rhodotorula mucilaginosa, Trichosporon dulcitum, Trichosporon moniliiforme and Schizoblastosporion starkeyi-henricii. Cryptococcus humicolus and Cryptococcus laurentii were isolated from liquid enrichment cultures. All these strains assimilated several benzene compounds in pure culture.

A Simple Procedure for the Cryopreservation of Hydrated Embryonic Axes of Pisum sativum

Pea seeds were imbibed for 4 hours or set to germinate for 72 hours, after which the axes were excised. A protocol for rapid freezing of this material was established by determining the most suitable pre-treatments that might effect vitrification of the samples. The procedures included redehydration and variation in exposure time to different combinations of glycerol with other cryoprotectants (DMSO, sucrose, sorbitol and proline). Irrespective of the imbibition time, successful retrieval from liquid nitrogen (47.5% of 4-hour-imbibed and 25% of 72-hour-imbibed axes) was obtained by use of a cryoprotectant miXture of glycerol and sucrose, followed by a halving of the moisture content, rapid immersion in liquid nitrogen, and rapid thawing in warm liquid medium followed by plating out onto solid growth medium. Post-freezing treatments such as removal of cryoprotectants by washing or addition of proline and! or activated charcoal to the growth medium did not improve the recovery of the 4-hour-imbibed material. The results indicate that using simple, rapid and inexpensive pre-treatments, relatively large and partially hydrated plant material has the potential to be conserved in liquid nitrogen.

An automated method for DNA preparation from thousands of YAC clones

We describe an automated method for the preparation of yeast genomic DNA capable of preparing thousands of DNAs in parallel from a YAC library. Briefly, the protocol involves four steps: (1) Yeast clones are grown in the wells of 96-well microtiter plates with filter (rather than plastic) well-bottoms, which are embedded in solid growth media; (2) These yeast cultures are resuspended and their concentrations determined by optical density measurement; (3) Equal numbers of cells from each well are embedded in low-melting temperature agarose blocks in fresh 96-well plates, again with filter bottoms; and (4) DNA is prepared in the agarose blocks by a protocol similar to that used for preparing DNA for pulsed-field gels, with the reagents being dialyzed through the (filter) bottoms of the microtiter plate. The DNA produced by this method is suitable for pulsed-field gel electrophoresis, for restriction enzyme digestion, and for the polymerase chain reaction (PCR). Using this protocol, we produced 3000 YAC strain DNAs in three weeks. This automated procedure should be extremely useful in many genomic mapping projects.

An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins.

A fusion between tsr (encoding the inner membrane protein Tsr) and phoA (encoding the periplasmic protein alkaline phosphatase, AP) generates a membrane-bound hybrid protein (Tsr-AP 2) with AP enzymatic activity. The hybrid protein is proteolytically unstable and is broken down to yield a smaller, soluble species with AP activity. We devised a genetic screen to distinguish between cells containing only membrane-bound AP and those containing soluble AP. The screen depends on diffusion of soluble AP away from cells with a leaky outer membrane to produce a halo of AP activity around colonies on solid growth medium. Several mutants lacking this halo show reduced degradation of Tsr-AP 2. One mutant is also defective in breakdown of five other abnormal periplasmic proteins but not of two cytoplasmic proteins. The mutation in this strain, degP4::Tn5, defines a locus distinct from previously identified loci that affect protein stability or protease activities. This strain may be useful for preventing the breakdown of unstable foreign proteins in Escherichia coli.

Somatic embryogenesis and plantlet regeneration in the soybean Glycine max

A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. TGM119 were immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form calli. Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was filtered to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging drop cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the calli. Calli containing somatic embryos were transferred into liquid medium and incubated under low light conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask.

Salt, pH and Temperature Dependencies of Growth and Bioluminescence of Three Species of Luminous Bacteria Analysed on Gradient Plates

Summary: Two-dimensional diffusion gradients (of NaCl and H+ concentrations) were established in solid growth medium containing glycerol and yeast extract as major carbon sources. These were used to investigate conditions favourable for growth and bioluminescence of three species of luminous bacteria during incubation at different temperatures. Photobacterium leiognathi, Photobacterium phosphoreum and Vibrio fischeri all grew over the entire salt range used [0.9–3% (w/v) NaCl] and at pH values < 7 at the most favourable temperatures (20 °C, 20 °C and 15 °C respectively); upper and lower temperature limits for growth over a 72 h period were 30 and 10, 25 and 5, and 30 and 5 °C respectively. Bioluminescence was observed at all temperatures that supported growth; in P. leiognathi emission at 10 °C was hardly detectable even after 72 h, but at higher temperatures it occurred at all NaCl concentrations. Low pH values and high NaCl concentrations favoured luminescence in the other two organisms; after 48 h light emission decreased from the high pH and low NaCl regions of the gels. These results are discussed with reference to the symbiotic (P. leiognathi, V. fisheri) or free-living (P. phosphoreum) origins of the organisms studied.

The ultrastructure of Pseudomonas aven ae— I. Paracrystalline surface layer and extracellular material

The general morphological features of the Gram-negative bacterium Pseudomonas avenae have been studied by electron microscopy. Electron micrographs from negatively stained specimens show the outermost surface layer to be composed of subunits arranged in a crystalline tetragonal array, with an average lattice spacing of 6.7 nm and unit cell of 9.7 nm when determined by image processing methods. Experiments designed to show the effects of various negative stains on the bacterial cell wall morphology suggest that the ‘convoluted’ or ‘smooth’ appearance of the wall material can be influenced by different stains when mixed with cells in liquid suspension. Various morphological products assembled as extracellular structures from subunits apparently of about the same size as those forming the cell surface crystalline lattice were observed in specimens prepared from a solid growth medium. These extracellular products ranged from tube-like assemblies with considerable variation in length, to forms which consisted of slender filaments in a coiled configuration. The axial periodicity of some of these products was 6.7 nm.

Production of chlamydospores and conidia by Trichoderma spp in liquid and solid growth media

Isolates of Trichoderma spp grew and produced chlamydospores as well as conidia in molasses-corn steep liquor (M-CSL), sucrose nitrate (SN), and glucose tartrate (GT) media. In M-SCL, isolates of T. hamatum, T. viride, and T. harzianum formed 10.4, 5.9 and 1.1 × 10 8 chlamydospores g −1 dry weight of mycelium. Fewer chlamydospores formed in SN and GT. Although T. harzianum formed the least number of chlamydospores, it produced the highest number of conidia in all three media. Molasses-corn steep liquor was superior to SN or GT in supporting development of both spore types. Spore production was not influenced by initial pH of the media or by continuous maintenance of the media at pH 4 or 7. Equal numbers of chlamydospores were formed in liquid media incubated in shake or static culture. Conidia formation, however, was stimulated in static culture. Chlamydospores and conidia of several naturally occurring isolates and induced biotypes of Trichoderma spp were abundantly produced on a variety of solid substrates moistened with liquid nutrients or water, preferably at pH 4. Bran, cornmeal, and peanut hull meal were better than eight other solid substrates for production of chlamydospores and conidia. A u.v.-induced, benomyl-resistant biotype of T. viride (T-1-R9) formed 22 × 10 7 and 18 × 10 8 chlamydospores and conidia, respectively, g −1 of bran-SN. The ratio of conidia to chlamydospores was always greater in solid than liquid media. In solid media, 10 times more conidia than chlamydospores were formed, whereas in liquid only two or three times more were formed. Chlamydospores from liquid and solid fermentations were viable ( ca 80%) and fresh chlamydospores germinated well ( ca 75%) on nutrient agar. Although dried chlamydospores were viable, as determined with tetrazolium bromide, their germination on agar was poor ( ca 8%).

Ultrastructure, in vitro and in vivo, of staphylococci exposed to antibiotics

The ultrastructure of staphylococci from the peritoneal fluid of mice treated with oxacillin and from sputum of a patient treated with ampicillin was comparable to the structure of staphylococci grown on filter membranes exposed to oxacillin, but was different from the same organisms grown in broth with oxacillin. The selection of a solid phase support growth medium, such as a filter membrane for in vitro studies of drug induced morphology, appears necessary if such studies are to reflect bacterial ultrastructure in vivo.

Rosanilins: indicator dyes for chloramphenicol-resistant enterobacteria containing chloramphenicol acetyltransferase.

Rosanilin dyes such as crystal violet and basic fuchsin have been used as indicator dyes in solid growth medium for chloramphenicol-resistant enterobacterial colonies containing the enterobacterial resistance enzyme chloramphenicol acetyltransferase (CAT). On certain media containing rosanilins, cells containing CAT formed darker colonies than cells not containing CAT. Contrast was affected by the types and concentrations of complex nutrients, sugars salts, and rosanilin dyes present. When crystal violet was used as the indicator dye, contrast could not be obtained for strains whose growth was partially inhibited by crystal violet. Contrast could not be obtained between yeast colonies with and without the enterobacterial resistance enzyme, between Bacillus subtilis colonies with and without the staphylococcal resistance enzyme, or between enterobacterial colonies with and without the staphylococcal resistance enzyme. The darker coloration of enterobacterial colonies with the enterobacterial enzyme was due to the binding of dye to enzyme. Rosanilin dues have been used to score resistance phenotypes by colony color, to detect chloramphenicol-sensitive sectors in chloramphenicol-resistant colonies, and to screen for occasional chloramphenicol-sensitive cells in a resistant population during cloning by insertional inactivation of the chloramphenicol resistance gene.

A cultural method for the detection of pullulan--degrading enzymes in bacteria and its application to the genus Bacillus.

Pullulan in a solid growth medium can be precipitated selectively with ethanol. Clear zones due to pullulan hydrolysis surround microbial colonies which produce the appropriate enzyme. When this procedure was applied to 297 strains representing 26 species of the genus Bacillus, 138 (46%) of the organisms were shown to hydrolyze pullulan.

Solid-state growth of Si to produce planar surfaces

Paralleling the sophisticated control of Si and GaAs growth from fluid media, we demonstrate control of Si growth using a solid growth medium. Faceted dissolution pits were first produced in a Si substrate using standard photolithographic techniques and Al metallization. Si was then evaporated onto the cold structure, depositing as amorphous material. Upon heating, the amorphous Si migrated through the solid Al and grew rapidly in the faceted pits, typically refilling them flush with the surrounding substrate. Evidence that the rapid growth occurs only while the amorphous Si is dissolving into the Al is presented.

Mutations as possible replication errors in bacteria growing under conditions of thymine deficiency

When Escherichia coli or Bacillus subtilis cells having inhibited thymidylate synthetase activity were incubated for a long time on solid medium supplemented with a limiting concentration of thymine or thymidine (0.1–0.3 μg/ml) most of them became mutants for one or more genetic markers. This “overall mutagenesis” was detected both in Thy − bacteria and in prototrophs for thymine (Thy +) with thymidylate synthetase inhibited by the addition of 5-fluorodeoxyuridine (FUdR) to the growth medium. When thymine (or thymidine) was present in very low amounts (10 −3 μg/ml) or was totally absent, the efficiency of mutagenesis decreased some 100-fold. The solid growth medium is essential because it supports the filamentous cells grown under conditions of limiting thymine. For some of the mutants with identified deficiency their ability to revert under the action of different mutagens was studied. Most efficient was 5-bromouracil (BU). This reversion is the characteristic response of mutations due to AT → GC transitions. In addition to single mutants, many multiple mutants were induced. The repair-defective strain of E. coli pol A1 − and strains Rec A − and Exr A −, which are also defective in UV-induced mutagenesis, showed a high level of mutation induction under the conditions described. All these results are in accord with the hypothesis that overall low-thymine mutagenesis reflects the accumulation of replication errors in DNA under the conditions of a precursor deficiency.

Demonstration of complement-dependent and independent systems in immune inactivation of Mycoplasma meleagridis.

The growth of Mycoplasma can be inhibited in the presence of specific immune serum. The inhibition is measured by the prevention of colony formation on solid growth medium where specific antiserum and the organism interact [1-3]. This process does not require complement [1, 3, 4], and the number of inhibited viable organisms is proportional to the amount of antiserum present [1, 2]. A similar phenomenon has been studied in liquid growth medium, and variable results have been reported. One form of inactivation of Mycoplasma by serum factors is characterized by a rapid decrease of viable organisms in liquid medium. The inactivation requires complement [5-9]. The rate of the reaction is independent of the number of organisms present but is dependent on the concentration of antibody and complement [5, 7]. Such inactivation has been shown to be completed within 60 min [5, 7] and is irreversible [7]. More recently, the mechanism of the complement-requiring inactivation of Mycoplasma pneumoniae has been elucidated. The organism is lysed due to damage of its surface membrane by antibody and complement [6]. This mechanism is similar to the complementdependent damage by antibody observed with erythrocytes [10], gram-negative bacteria [11], L-phase variants [12], mammalian cells [13], and viral particles [14-16]. Although less frequently observed, there appears to be another mechanism for inactivation of Mycoplasma by immune serum in liquid medium. This process may be characterized as compl ment-independent [3, 17, 18] and seems to be based on inhibition of multiplication of the organism [18]. The reaction seems to correspond to growth inhibition on agar medium. Thus, the above evidence indicates the existence of two different systems of in-vitro inactivation of Mycoplasma by specific antibody. However, the two inactivation systems have never been shown to occur in a single system. Instead, the ra her specific nature of the test methods used mig t lead one to believe that the two systems are not distinct from each other. This study will present data on complement-dependent and independent systems of inactivation operating simulta eously, but independently, in a broth-assay system with Mycoplasma meleagridis as the test organism.

Mutants of Rhodospirrillum rubrum obtained after long-term anaerobic, dark growth.

Rhodospirillum rubrum S(1) cells were grown for more than 100 generations under strict anaerobic, dark conditions in liquid medium with sodium pyruvate. During this time, growth became nonpigmented. When cells were streaked onto the surface of solid growth medium in anaerobic bottles and placed in the dark, a few light-red colonies developed, but the majority was nonpigmented. Mutants were obtained from colonies selected on the basis of pigmentation and bacteriochlorophyll a content. The growth, ultrastructure, and light reactivity of two mutants were examined. Mutant C synthesized bacteriochlorophyll a (7.2 mumoles per mg of protein), altered membrane structures, and chromatophores during dark growth. Examination of light-induced changes of the absorption spectrum of this mutant suggested that only an electron transport pathway, which included the low potential cytochrome-like pigment C428, could be detected. Mutant C grew anaerobically in the light, whereas mutant G1 was light sensitive and produced only trace amounts of bacteriochlorophyll a (0.6 mumole per ml of protein). Poorly pigmented G1 cells contained unusual membrane structures. When dark-grown G1 colonies were placed in the light, deep-red colored papillae developed in the nonpigmented colonies. During anaerobic, dark growth with sodium pyruvate, both C and G1 synthesized poly-beta-hydroxybutyrate and produced acetate, carbon dioxide, and hydrogen gas.

Quantifying Bacteria on Poultry Carcass Skin

THERE are four sampling methods that have been used for determining numbers of bacteria on poultry meat and carcasses, and a number of variations of each method (Fromm, 1959; Kotula, 1966; and Seekins et al., 1958). The four basic methods are: 1) direct contact plating, 2) swab sampling, 3) rinse sampling, and 4) skin tissue removal.Direct contact plating involves placing the surface of a solid bacterial growth medium directly against the carcass skin, then incubating the medium in the petri plate and counting bacterial colonies. A modification of this method utilizes a moist membrane filter to remove bacteria from the carcass skin. The membrane filter is then placed on the surface of a solidified medium for incubation. This method has three serious disadvantages: a) clumps of bacteria removed from the carcass skin are counted as one colony-forming unit; b) even with moderately contaminated carcasses, individual colonies are not distinguishable…