Improved Methods for Cultivation of the Extremely Thermophilic Bacterium Thermotoga neapolitana

Growth medium components and cultivation conditions for the extremely thermophilic bacterium Thermotoga neapolitana were optimized. A defined marine salts medium was formulated. Trace amounts of iron stimulated growth of T. neapolitana, while zinc inhibited growth at concentrations exceeding 11.1 μM. Other trace metals had no effect on its growth. Of the vitamins tested, only biotin was required for optimal growth. A defined mineral medium containing 5 g of carbohydrates per liter as the carbon source and 0.5 g of cysteine per liter as the sulfur source and reductant supported growth. Growth was stimulated by inclusion of vitamin-free Casamino Acids. Elemental sulfur, cystine, and dimethyl disulfide in the growth medium enhanced growth. Elemental sulfur and cystine relieved growth inhibition by hydrogen. T. neapolitana formed colonies in 2 days on plates of complex medium solidified with gellan gum and in 4 days on defined medium. The efficiency of plating was determined when growing cultures were sampled both aerobically and anaerobically and plated under aerobic and anaerobic conditions. Mean plating efficiencies were improved by sampling the growing cultures under strictly anaerobic conditions. Little or no improvement was obtained by inoculating plates inside an anaerobic chamber. Plating efficiencies of approximately 80% were obtained. Polycarbonate jars with aluminum lids withstood repeated incubation at 77°C without significant deterioration of the anaerobic seal and provided the most consistent results.     

Tracheary Element Formation from Single Isolated Cells in Culture

Friable callus tissue of Centaurea cyanus L. was grown on a solidified synthetic nutrient medium (EBM-1) to produce a tissue with a low frequency of differentiated tracheary elements. Tissues were then suspended in liquid nutrient medium with agitation to produce a suspension which was filtered and the single-cell suspension resulting was used as inoculum for either cell suspension cultures or for plating of cells into solidified medium in Petri plates. Media for the suspension cultures were selected to favor cytodifferentiation of tracheary elements. Differentiated tracheary elements formed as early as 10 days and numbers of tracheary elements increased with time roughly in relation to the increase in total cell number. From plating experiments it was shown conclusively that single isolated parenchyma cells differentiated directly into single isolated tracheary elements, although this event was rare. More usual was the division of isolated cells to form small colonies and then the differentiation of one, several or all of the cells into tracheary elements. Comparisons are made between results with cell plating experiments and cell suspension cultures. Optimism is expressed for finding a cell suspension culture system for studying cytodifferentiation.     

Protoplast culture of rice (Oryza sativa L.) using media solidified with agarose

A procedure is described for the reproducible establishment of rice cell suspension cultures from callus of embryo origin. Protoplasts were readily isolated from cell suspension of four rice cultivars, including japonica and indica types, when maintained in an amino acid-based culture medium. Sustained protoplast division from two japonica genotypes has been obtained in agarose solidified culture medium. An increase in the agarose concentration from 0.6% to 1.2% (w/v) produced a marked improvement in protoplast survival, division and plating efficiency. Protoplast division and plating efficiency frequencies of up to 26% and 0.5%, respectively, were obtained at the higher agarose level. The protoplast-derived calli were similar in appearance to explant-derived morphogenic callus and produced distinct embryo-like structures.     

Plant Regeneration from Protoplast of Trititrigia (Triticum sect. trititrigia Maekey)

Trititrigia is the intergeneric hybrid which is from the hybridization between Triticum durum Desf. and Elytrigia intermedium (Host) Nevski. Protoplasts of Trititrigia were isolated from the embryogenic cell suspension derived from immature inflorescence-induced calli of the hybrid F1. The first division occured 48 hr after plating in modified KM8p culture medium. The plating efficiency of protoplasts was 2% and 12.14% when they were cultured in liquid medium and agarose solidified medium, respectively. Clusters grew vigorously under these conditions. Fresh medium with decreased osmoticum was added 20–30 days after plating. When protoplast-derived calli, 2–4 mm in the size, were transferred step by step to different differentiation media, embryoids, green spots emerged and numerous plants regenerated eventually.     

A simple and effective plating method to screen polycyclic aromatic hydrocarbon-degrading bacteria under various redox conditions

Agar plates with a polycyclic aromatic hydrocarbon (PAH) layer have been used to screen for microorganisms that degrade PAHs, leaving clear zones around colonies; however, there are several problems with previous methods such as undesired contamination in the fume hood and difficulty in controlling the amount of PAH on the plates. In this study, we developed a modified screening method to address the drawbacks encountered with previous screening methods. A uniform white layer of PAHs was generated by spreading PAHs dissolved in volatile solvents over a surface of solidified agar medium, followed by the evaporation of the solvents. An inoculation was then performed by spreading a molten agar medium containing microbial samples over the solidified agar medium with a PAH layer. Subsequently, the white PAH layer migrated to the surface of the molten agar medium. This essential modification enabled us not only to solve problems of the previous screening methods but also to prepare an agar plate with a PAH layer without a complicated experimental scheme in the anaerobic chamber. After solidification of the molten agar medium and incubation of the plates, clear zones were successfully detected around colonies with aerobic and anaerobic PAH-degrading microbial cultures.     

Promotion of septation in irradiated Escherichia coli by a cytoplasmic membrane preparation

Septation can be promoted in an X-irradiated lon mutant of Escherichia coli K-12 by the addition of an E. coli B/r cytoplasmic membrane preparation to the postirradiation plating medium. The promotion of septation was not associated with an inhibition of growth rate. Two distinct cytoplasmic membrane-associated properties were necessary to promote septation. One of these, the cytochrome-based electron transport system, produced anaerobic conditions by the reduction of oxygen dissolved in the medium. The second system, functioning independently from the first, altered substances found in the peptone and yeast extract components of the postirradiation plating medium. When both systems were operative, significant repair of the cell division mechanism occurred.     

产H_2O_2的乳酸杆菌分离培养基的改良

对MRS培养基的使用方法进行了改良。将MRS(含0.25 mg/ml TMB)融化后倾倒入平皿,待其凝固后,在琼脂表明加入200μl(0.01 mg/ml)HRP,涂匀后进行乳酸杆菌划线分离。37℃、厌氧培养48~72 h。结果表明,产生H2O2的乳酸杆菌呈蓝色菌落,和原来的培养方法相比,改良的方法简便经济、HPR的使用量为原来的1%,分离产H2O2的乳酸杆菌的效果良好。     

Isolation and culture of daylily mesophyll protoplasts

Mesophyll protoplasts were isolated from leaf tissues of a diploid daylily (HemerocallisxRed Magic) by enzymatic digestion with a solution containing 0.5% Pectolyase Y-23, 0.1% Cellulase R-10, 0.1% Driselase, 0.6 M sorbitol and half-strength MS inorganic salts. When cultured on MS medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA, the protoplasts underwent sustained division to produce multicellular colonies. The optimal plating density for cell division was 0.5 × 10⁵ protoplasts/ml. The highest plating efficiency was obtained in cultures grown in media solidified with 0.2% Gelrite. Under these conditions, formation of colonies occurred from 14% of cultured protoplasts. Calli were recovered from 9 colonies only after the cultures were treated with a conditioned medium. Intact plants were regenerated from protoplast-derived calli through organogenesis.Abbreviations BA 6-benzylaminopurine - FDA fluorescein diacetate - GA₃ gibberellic acid - MS medium Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration

Summary In vitro-grown apical meristems of wasabi (Wasabia japonica Matsumura) were successfully cryopreserved by vitrification. Excised apical meristems precultured on solidified M S medium containing 0.3M sucrose at 20°C for 1 day were loaded with a mixture of 2M glycerol and 0.4M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) for 10 min at 25°C prior to a plunge into liquid nitrogen. After rapid warming, the meristems were expelled into 2 ml of 1.2M sucrose for 20 min and then plated on solidified culture medium. Successfully vitrified and warmed meristems remained green after plating, resumed growth within 3 days, and directly developed shoots within two weeks. The average rate of normal shoot formation amounted to about 80 to 90% in the cryopreserved meristems. This method was successfully applied to three other cultivars of wasabi. This vitrification procedure promises to become a routine method for cryopreserving meristems of wasabi.Abbreviations BA 6-benzylaminopurine - DMSO dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS medium Murashige and Skoog medium (1962) - PVS2 vitrification solution     

Evaluation of chromogenic medium (ORSAB) for routine identification of methicillin resistant Staphylococcus aureus (MRSA)

Performance of chromogenic medium (ORSAB) for routine detection of methicillin resistant S. aureus (MRSA) was evaluated on 510 specimens collected from patients suspected of MRSA infection or colonization. Addition of ORSAB plates to the routine protocol allowed MRSA identification in 24 hours from samples plating. In 18 samples MRSA colonies were identified only on ORSAB plates, those cases would have been missed by routine protocol alone.     

内生枯草芽孢杆菌JL4在葡萄叶上的定殖及其对葡萄霜霉病的防治

为了明确枯草芽孢杆菌JL4在葡萄叶表面和内部的定殖情况,研究定殖与防治效果的关系,采用电击转化的方法将含有GFP基因的质粒pGFP78导入枯草芽孢杆菌JL4中,并得到成功表达GFP 的生防菌JL4-gfp,测试了标记菌株的稳定性及其对葡萄霜霉病菌的抑制作用.采用叶片喷雾法接种,用抗生素平板稀释分离回收,检测生防菌JL4-gfp在葡萄叶片的定殖情况,并将采回的叶片在室内接种葡萄霜霉菌孢子囊悬浮液进行生防测定.结果表明: 标记菌株在经过10次传代培养后,仍具有良好的发光表型,能稳定表达GFP蛋白,并且标记菌株JL4-gfp对葡萄霜霉菌保持了原有的抑菌作用;用抗生素平板稀释分离回收,检测到JL4-gfp菌株在葡萄叶片表面的定殖量在接种后的0、3和7 d分别为3.6×10⁵、2.7×10⁵和3.1×10³ CFU·g^-1;叶片内部的定殖在接种3 d后达到最大(9.6×10⁴ CFU·g^-1),然后下降,14 d后已经检测不到接种菌株;室内生防测定结果显示,喷雾后3 d对葡萄霜霉病的防治效果达88.0%以上,但7 d后则无明显防效.JL4-gfp的定殖量与其防治葡萄霜霉病的效果呈正相关,其有效定殖量临界值为10⁵ CFU·g^-1.     

Agarose/agar assay system for the selection of bacteriocin-producing lactic fermentation bacteria

The direct selection of bacteriocin-producing lactic fermentation bacteria was possible by plating diluted cultures of Pediococcus acidilactici on mixed agarose agar layers with the amount of each component incrementally adjusted to 1.2% (w/v). Between 0.5 and 1% agarose, the increased flexibility of the solidified support layer allowed its removal from Petri dishes without tearing and its smooth layering on the surface of 1.5% (w/v) standard agar medium seeded with Listeria innocua as the test organism. Selection of bacteriocin-producing clones was based on the size of inhibition zones visible in the bottom agar layer under colonies growing on the agarose/agar top layer. The lack of contact with the test organism permitted the transfer of superior clones from the surface of the agarose/agar layer directly into an appropriate nutrient medium.     

Optimum conditions for growth of cyanobacteria on solid media

The colony forming ability of single cells or very short filaments of 7 strains of cyanobacteria was tested on media solidified by agar or by agar substitutes (Gel Gro or Gel Rite). In addition, the effect of various methods for preparation of agar media on colony forming ability was measured. High efficiency colony formation for most of the strains required that the agar be autoclaved separately from the salts in the medium. The addition of thiosulfate, but not buffer, significantly increased the plating efficiency of most strains.     

An Anaerobic, Intrachamber Incubator for Growth of Methanosarcina spp. on Methanol-Containing Solid Media

To simplify the incubation of Methanosarcina spp. on solid agar medium, a two-port, manual, rectangular air lock was modified to serve as an anaerobic incubator. In one operation, it is possible to incubate 153 petri plates, the equivalent of 11 standard anaerobic jars, with plating efficiencies identical to those of traditional protocols.     

Differential carbohydrate media and anaerobic replica plating techniques in delineating carbohydrate-utilizing subgroups in rumen bacterial populations.

A basal (BC) medium devoid of added carbohydrates, a complete (CC) medium containing nine carbohydrates were developed for enumerating rumen bacteria. The colony counts on the BC medium were 85 to 100% of those obtained on the CC medium. These colonies were pinpoint size (less than or equal to mm in diameter) but increased in size (2 to 5 mm in diameter) when carbohydrates were subsequently added. With the CC medium or other media tested, the colony counts were 20 to 50% higher on plates than on roll tubes and were about 35% of the direct cell counts. The lower colony counts on roll tubes were shown to result primarily from the loss of viability due to heat stress. The DC media were found by plating techniques to be suitable for differentiating mixed rumen bacterial populations into subgroups based upon carbohydrate utilization as shown by differences in subgroup profiles found within solid and liquid fractions of rumen contents, within rumen contents from animals fed high-forage and high-grain diets, and by correct colony formations by pure cultures of rumen bacteria on appropriate DC media. With simple modifications and use of an anaerobic glove box, replica plating methods and the CC and DC media were found to be a suitable means of rapidly determining the range of utilizable carbohydrate energy sources of rumen bacteria.     

Differential carbohydrate media and anaerobic replica plating techniques in delineating carbohydrate-utilizing subgroups in rumen bacterial populations

A basal (BC) medium devoid of added carbohydrates, a complete (CC) medium containing nine carbohydrates were developed for enumerating rumen bacteria. The colony counts on the BC medium were 85 to 100% of those obtained on the CC medium. These colonies were pinpoint size (less than or equal to mm in diameter) but increased in size (2 to 5 mm in diameter) when carbohydrates were subsequently added. With the CC medium or other media tested, the colony counts were 20 to 50% higher on plates than on roll tubes and were about 35% of the direct cell counts. The lower colony counts on roll tubes were shown to result primarily from the loss of viability due to heat stress. The DC media were found by plating techniques to be suitable for differentiating mixed rumen bacterial populations into subgroups based upon carbohydrate utilization as shown by differences in subgroup profiles found within solid and liquid fractions of rumen contents, within rumen contents from animals fed high-forage and high-grain diets, and by correct colony formations by pure cultures of rumen bacteria on appropriate DC media. With simple modifications and use of an anaerobic glove box, replica plating methods and the CC and DC media were found to be a suitable means of rapidly determining the range of utilizable carbohydrate energy sources of rumen bacteria.     

Plant regeneration from callus-derived protoplasts of Pelargonium x domesticum

A protocol for regenerating plants from callus-derived protoplasts of Pelargonium x domesticum (rega l geranium cv. Melissa) has been developed. Protoplasts were isolated from leaf-derived callus tissue on MS medium supplemented with 3.0 mg/l naphthalene acetic acid, 2.0 mg/l 6- benzylaminopurine, and 3.0% sucrose. This callus yielded 2.7×10⁵ protoplasts/gram of tissue after a 6 hr incubation in an enzyme solution consisting of 2.0% cellulysin, 0.5% macerase, and 0.5 M sucrose. Protoplasts were plated at 1×10⁵ protoplasts/ml in a mixture (11 v/v) of KMP8/KP liquid medium layered on the same medium solidified with 0.6% agarose. Protoplast division was initiated within 2 days, and colonies of 15 to 50 cells developed 8 wk after plating. P-calli 1–2 mm³ developed 15 wk after plating, and plants regenerated from the p-calli have been transferred to the greenhouse.Abbreviations NAA naphthaleneacetic acid - 6-BAP 6-benzylaminopurine - CW Calcofluor White - FDA fluorescein diacetate     

TELLURITE REDUCTION BY FOODBORNE BACTERIA

Using an agar overlay technique, we determined that colonies of both Gram-positive and Gram-negative bacteria could reduce tellurite. We also observed that under anaerobic conditions, the growth of Gram-positive bacteria was inhibited if tellurite was present in the plating medium.     

New approaches for shoot production and establishment of in vitro root cultures of Cleome rosea Vahl.

Alternative methods for in vitro shoot culture of Cleome rosea, a Brazilian herbaceous species with ornamental value and medicinal potential, were evaluated. A protocol for rapid in vitro multiplication of roots, a valuable source of medicinal compounds, was also developed. Stem explants were cultured in liquid media (continuous immersion and paper bridge), while root explants were cultivated in continuous immersion and on solidified media. The highest numbers of shoots, 20 ± 4.6 shoots/explant, were obtained from stem explants incubated in a continuous immersion system in a liquid medium supplemented with 2.2 μM BA. Root explants cultivated in liquid media produced only hyperhydrous adventitious shoots. However, these explants generated 5.8 ± 0.8 shoots/explant by indirect organogenesis when cultivated on solidified medium supplemented with 2.2 μM BA. In addition, root multiplication was achieved in liquid medium in the presence of α-naphthaleneacetic acid. Adventitious shoots developed on newly formed roots when inoculated on solidified medium supplemented with 2.2 μM BA. Shoot microcuttings developed roots when transferred onto solidified MS medium without growth regulators. Rooted microcuttings were efficiently acclimatized when transferred ex vitro.     

Analysis of plating technique for viability and drug-sensitivity determinations using Rosa cells

Rosa Paul's Scarlet'cell suspension cultures were used as a test system for working out a method of viability and drug-sensitivity determination based on plating efficiency. High plating efficiencies (80–95%) were obtained on a simple synthetic medium when aggregates of a mean size of c . 100 cells/unit from exponential phase cultures were plated at a density of 1500 units/plate in the middle layer (5 ml) of three layers of the agar-solidified medium (total = 30 ml). This 3-layer plating technique produces homogeneous colony growth and simplifies the microscopical evaluation of plating efficiencies. The reduction of plating efficiencies seen when the smaller aggregates of stationary phase cultures were plated was mainly due to low cell density and could be overcome by enriching the medium with various supplements. Reconstitution experiments using mixtures of inactivated and non-inactivated aggregates demonstrated that plating efficiency can be taken as a goodmeasure of viability. The described plating technique was found to be more sensitive and reliable compared to two other methods for determining p -fluorophenylalanine-sensitivity of Rosa cells.