Agar Concentration in Counting Clostridium Colonies

Decreasing the agar concentration of a counting medium from the usual 1.5% resulted in larger colonies with less interference from gas in Clostridium botulinum 115B and C. sporogenes PA 3679. Optimal agar concentration was 0.65% for C. botulinum with 24-hr incubation and 0.50% for C. sporogenes with 48-hr incubation. Lower concentrations yielded growth too diffuse for counting. Motility was considered the explanation for increased colony size in softer agar. The greater the degree of motility, the greater would be the diffusibility expected, and thus the higher the agar concentration required to insure discrete colonies. For quantitating motility, evaluations were made by use of microscopic examination of liquid cultures and rate of diffusion in a semisolid medium. With both criteria, the degree of motility of C. botulinum 115B clearly exceeded that of C. sporogenes PA 3679. Small-colony variants of C. botulinum in 0.65% agar yielded only small colonies on subculture, with a corresponding decrease in degree of motility of the cells by both criteria. Colony size of the nonmotile C. perfringens ATCC 3624 was unaffected by lowered agar concentrations.     

Induction of benzoquinone formation by activated carbon in Lithospermum erythrorhizon cell suspension cultures

Cultured cells of Lithospermum erythrorhizon which were capable of producing red naphthoquinone (shikonin) derivatives on Linsmaier-Skoog's agar medium stopped synthesizing these compounds when grown in liquid medium without agar. However, when the liquid medium was supplemented with a small amount of activated carbon, the cells produced a new orange benzoquinone derivative, echinofuran B, which may be considered an abnormal metabolite of geranylquinol, the key intermediate in the biosynthesis of shikonin. A similar effect of activated carbon was also observed with a variant cell line incapable of producing shikonin derivatives even on the agar medium. By contrast, the callus cultures grown on the agar medium as well as the dried roots of the intact plant were found to contain a small amount of echinofuran C, another new benzoquinone related to echinofuran B, in addition to shikonin derivatives.     

High concentrations of horse serum inhibit growth of corn stunt spiroplasma.

Corn stunt spiroplasma (CSS) grew faster and achieved higher titers in liquid or agar medium containing 5 or 10 percent horse serum than it did in medium containing 20 percent horse serum. When growth in liquid medium was initiated with a small inoculum, CSS achieved excellent growth in the presence of 5 percent serum but did not grow in medium containing 0 or 20 percent serum. Addition of arginine to liquid or agar medium supplemented with 20 percent serum stimulated CSS growth, but addition to that containing 5 percent serum did not.     

Comparative Study of Two Methods for Detection of Clostridium perfringens in Ground Beef

The tryptose-sulfite-cycloserine agar pour plate method was superior to selective enrichment in liquid sulfite medium for isolation of small numbers of Clostridium perfringens from frozen ground beef.     

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 10⁶ cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 10⁵ cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 10⁶ cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.     

SIVB 2003 Congress Symposium Proceeding: Plant Growth and Sugar Utilization in an Agitated,Thin-Film Liquid System for Micropropagation

Summary Agitated layers of liquid medium were created on platform shakers in jars with 25–30 ml of medium (similar to conventional agar culture) rotating at 90 rpm. Thin films were scaled up in larger rectangular vessels on tilted shelves that periodically rock. In jars of liquid medium with a density of 180 explants per liter, multiplication rates of Hota tokudama var. ‘Newberry Gold’ were optimal with a media sucrose concentration of 5% [both with and without 1 μM benzyladenine (BA)]. Endogenous levels of soluble sugars were directly related to the concentration of sucrose in the medium. Three Hosta cultivars (‘Striptease’, ‘Minuteman’, and ‘Stiletto’) with plant densities of 40–200 explants per liter of medium were tested in larger, agitated, thin-film vessels in media with 5% sucrose and directly compared to agar medium. Higher rates of multiplication were observed in liquid than agar with the magnitude of the difference dependent on explant density. Pooled results for the three varieties with 200 explants per liter showed multiplication rates of 1.7x and 2.3x for agar and thin-film liquid, respectively. At 40 explants per liter, the multiplication rate was increased to 2.1x for agar and 3.4x for thin-film liquid. Sugar uptake was greater in liquid than agar and was greater in the higher densities, with the magnitude of the effect dependent on plant variety. Increased vessel size in the liquid, thin-film system and greater sugar uptake allowed more, larger plants to be harvested. Alocasia macrorrhizos was cultured in growth medium containing 1μM BA and 5% sucrose with plant densities in the range of 33–330 explants per liter. Dry weight and multiplication rate were greater in the liquid system than agar with the magnitude of the difference dependent on plant density. With approximately 165 explants per liter, and greater at the initiation of culture, plant density limited growth in both agar and liquid thin-film systems. In a multiplication medium (3 μM BA and 3 μM ancymidol) plant size was reduced by 50% and 60% (fresh weight) in liquid and agar, respectively. Initial density in the range of 165–330 explants per liter did not limit growth with the smaller plants in liquid or semisolid multiplication medium. Sugar uptake was greater in liquid than agar. While ample sugar was present in media for growth at any density on agar, sugar depletion was limiting growth at highest densities with the larger plants in liquid growth medium. In semisolid agar medium, sugar uptake by plants was more rapid than diffusion across the agar medium, resulting in non-equilibrium conditions following the culture cycle. In agitated, liquid medium, a greater transfer of sugars to plant tissue was related to accelerated growth.     

Filter centrifugation as a sampling method for miniaturization of extracellular fungal enzyme activity measurements in solid media

A novel sampling method to evaluate extracellular fungal enzyme activities was developed and the validity tested for agar media. The method is based on centrifugation of small agar pieces taken from growing fungal solid-state cultures. Centrifuge tubes that allow spinning liquid out from small samples containing, for example, the hyphal front of a growing mycelium are essential for the protocol. Centrifugation recovers a liquid phase from the samples, which contains soluble material including many enzymes. The recovery of two added model enzymes, namely laccase and manganese peroxidase (MnP), from agar media was sufficient (ranging from 50 % to 75 %) but the addition of humic material into agar decreased the observed MnP activity significantly to approx. 25 % of the stock solution. Using growing cultures, the presence of humus as well as Scots pine sawdust on Hagem’s agar plates induced the production of laccase and peroxidase in certain fungi, which indicates that the method is suitable for screening enzyme activities on different growth media or with variable additives or growth conditions. The use of the presented sampling method for functional enzyme fingerprinting of different fungi may be a promising tool for investigating the behaviour and ecological role of forest soil fungi. This method also allows obtaining spatial data from very small and defined areas of solid fungal cultures, e.g. from microcosms.     

Efficiency in thin-film liquid system for <Emphasis Type="Italic">Hosta</Emphasis> micropropagation

Three varieties of Hosta (Striptease, Minuteman and Stiletto) at four densities (40, 80, 120 and 200 explants per litre) were micropropagated on semi-solid agar and a thin-film liquid system with intermittent wetting of plant tissue. The mechanics of wetting by a small wave front required a larger rectangular vessel (11 × 27=297 cm ²) compared to the common cylindrical baby food jar (18 cm ²). Plants multiplied more rapidly in the agitated thin-film system than on agar. Lower plant densities increased rates of multiplication in liquid, but had little or no effect on multiplication rate on agar. Increasing plant density lowered the overall multiplication rate, but yielded greater numbers of plants per vessel. Yield, tabulated for utilization of shelf-space in growth room, was greater at all densities in rectangular vessels of liquid than conventional jars of agar media. Increased plant density lowered the sugar residual in media following the culture cycle and liquid media had less residual sugar than agar media. A liquid medium with 50 g l^–1 sucrose was concentrated enough so that sugar depletion did not limit growth, even at the highest densities. The liquid system allows the technician to skip the step of manually spacing and orienting the freshly cut bud tissue at the transfer station. Harvesting 75–100 plants per vessel from the large rectangular vessels resulted in most efficient use of technician time. Plants from liquid and agar acclimatized to greenhouse. Increased multiplication, space utilization, sugar availability and worker efficiency was demonstrated to be greater in thin-film liquid than more conventional agar-based system.     

Agar fractions could protect apple shoots cultured in liquid media against hyperhydricity

Apple shoots were grown in a Murashige and Skoog liquid proliferation medium containing 4.4 10 -6 M BA, and supplemented with various fractions of agar. Hydrolysed agar from Difco was able to overcome hyperhydricity when its concentration was increased to 0.7%. Among the fractions isolated from this hydrolysed agar, only oligosaccharides were found to reduce the occurrence of this developmental abnormality. The most anti-hyperhydric fraction had a molecular weight slightly less than 1900 daltons and contained methylated and sulphated galactose derivatives. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth of Mouse L Cells in Shaken Culture and Mengovirus Plaque Formation on L Cells Suspended in Agar

Procedures are described that require a minimum of equipment and maintenance for growing mouse L cells suspended in liquid medium and for plaquing mengovirus on L cells suspended in agar. Viability of L cells during storage for 1 to 2 hr at relatively high concentrations was better in media at 30 C than at 0 C, as measured by viable counts after growth for 24 hr at 35 C. The number and size of plaques increased with increasing concentration of NaHCO(3) in the agar layers, but the relative difference in plaque size was maintained. Large- and small-plaque-size variants had similar virulence as determined by the rates of viability loss of L cells in liquid suspension cultures.     

The Processing of Mammalian Haemopoietic Cells in Thin Layer Agar Cultures for Electron Microscopy

Human and mouse haemopoietic cells cultured by the thin layer agar technique have been studied with the electron microscope. To process colonies of haemopoietic cells or individual cells which appeared in these colonies, a special technique had to be developed. The technique presented covers methods of selection, isolation, and sectioning that were devised for this purpose.

Haemopoietic cells are cultured in small plastic Petri dishes containing a culture system with 0.25% agar. Cell colonies and individual cells intended for light as well as for electron microscopic study are examined and selected microscopically with the aid of a numbered grid which is placed under the closed Petri dish.

Cells in the agar gel are fixed with glutaraldehyde which is pipetted directly onto the cultures. In order to facilitate their removal from the medium, the consistency of the agar solution is increased by evaporating liquid with controlled mild warming.

Pieces of agar containing colonies or single cells are cut out with a fine trephine and postfixed in osmium tetroxide. Agar pieces are embedded cell side up in a thin layer of Epon. After polymerization, the Epon-embedded pieces of agar are appropriately oriented at the head of flat embedding molds filled with fresh Epon. After another polymerization procedure, the top of the Epon blocks containing the cells are trimmed to a smooth surface with a glass knife.

The exact distance between the smooth surface of the blocks and the cells is measured by use of the vertical micrometer of a standard light microscope. The Epon layer around the specimen is trimmed away to expose selected cells for subsequent semi-thick and ultrathin sectioning. Sections are stained and examined microscopically.

With minor modifications the technique described also enables the processing of extremely small quantities of biological materials derived from other experiments for both light and electron microscopic observation.     

A comparison of solid and liquid media for measuring the sensitivity of heat-injured Salmonella typhimurium to selenite and tetrathionate media, and the time needed to recover resistance

Sensitivity of heat-injured Salmonella typhimurium to selenite and tetrathionate media was measured by viable counts in liquid and on agar-solidified versions of these media and on nutrient media. All solid media, including the supposedly non-inhibitory nutrient agar, were more inhibitory to injured cells than the corresponding liquid media. Catalase or pyruvate increased counts on nutrient agar to the level obtained in nutrient broth. Therefore nutrient agar plus pyruvate was the most suitable reference medium against which to compare recoveries on other media. Although recoveries of injured cells varied widely depending on the composition and physical state of the medium, this had a minor effect on estimates of repair time because resistance to all selective media was regained by the end of the lag phase.     

Enhanced embryogenesis and plant regeneration from cucumber (Cucumis sativus L.) callus by activated charcoal in solid/liquid double-layer cultures

Enhanced embryogenesis and plant regeneration methods were established in cucumber (Cucumis sativus L. cv. ‘Delilah’) using hypocotyl segments as explants. Callus formation, followed by pro-embryogenic aggregates and globular embryoids required liquid shake cultures. In liquid medium, however, many of the embryoids developed into abnormal structures — ‘neomorphs’ or succulent plantlets. Embryoids subcultured to stationary liquid or agar cultures dedifferentiated and underwent secondary embryogenesis. Neither increased osmolarity nor adding abscisic acid (ABA), zeatin or activated charcoal to the liquid medium inhibited abnormal morphogenesis. The use of double layer cultures containing activated charcoal in the lower agar layer and ABA with elevated calcium in the upper liquid phase prevented dedifferentiation and secondary embryogenesis and allowed normal organized growth of the embryoids. Hardening in vitro by partial desiccation with CaSO₄ under aseptic conditions improved the cucumber plantlet's leaf growth and their survival after transplanting to soil.     

Regeneration and frequency of tetraploid variants of Cucumis metuliferus are affected by explant induction on semi-solid medium versus the liquid/ membrane system

Ninety-eight percent of Cucumis metuliferus (PI 482439) cotyledons regenerated shoot buds after 5 weeks on basal Murashige and Skoog medium amended with 10 μm benzyladenine. Regeneration rates of explants maintained only on agar-gelled medium versus explants induced first on a liquid/membrane system were compared after weekly transfers of tissue from liquid/membrane to agar during the first 5 weeks of regeneration. The number of regenerant buds increased from six per cotyledon (on agar-gelled medium) to nine per cotyledon when explants induced on the liquid/membrane system for 3 or 4 weeks were transferred to agar-gelled medium. Shoot development, rooting and survival in the greenhouse were adequate, regardless of whether regeneration was initiated on the agar or liquid/membrane system. Tetraploid regenerants accounted for 9% of the 391 regenerants screened. Pollen morphology was a reliable screening technique to identify tetraploid plants. The frequency of tetraploid plants varied from 13.5% to 6.9% after 5 weeks of induction on the agar or liquid/membrane system, respectively. This frequency decreased to 1.5% if the explants were transferred from the liquid/membrane system to agar after the first week of induction. Transfers during this period play a critical role in eliminating tetraploid variants. Received: 17 June 1996 / Revision received: 27 August 1996 / Accepted: 10 March 1997     

Factors influencing the production of hardened glaucous carnation plantlets in vitro

Medium type, its water status and the relative humidity in the culture vessel modified carnation leaf development in vitro. Carnation shoot apices cultured on liquid or on 0.8% agar solidified media developed into plantlets having succulent and translucent leaves which are not transplantable to non-aseptic conditions. Increasing the agar and/or sucrose concentration in the medium as well as decreasing the relative humidity in the culture vessel by a desiccant promoted glaucous leaf production. Increased water status (H₂O and relative humidity) increased shoot proliferation and translucency of leaves. Decreased water status reduced shoot proliferation but induced the formation of glaucous leaves. The culture of apices for 5–6 days on liquid medium prior to their sub-culture to 1.5% agar medium improved shoot proliferation and normal leaf development. An agar slant prevented the submergence of apices in water accumulating on the medium and thus reduced leaf translucency. Survival was further increased by the transfer of plantlets in uncapped culture vessels to a desiccator for 1–2 weeks prior to transplanting to soil.     

假单胞菌荧光与非荧光铁载体对铁离子的应答差异

假单胞菌既能产荧光铁载体也能产非荧光铁载体.通过对假单胞菌在不同铁离子浓度下,在通用CAS(Chrome azroul S)检测平板、改进的蔗糖-天冬氨酸(SA)平板(MSA)上以及通用液体CAS培养基和MSA培养基内的铁载体产生情况的比较,发现在通用CAS的液体培养基上产生的主要为非荧光铁载体(pyochelin),而在改进的MSA培养基上产生的主要为荧光铁载体(pyoverdine);在铁离子的应答方面,pyoverdine较pyochelin灵敏,较低的铁离子浓度即可抑制荧光铁载体的产生,但是不能抑制非荧光铁载体.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO COPPER AND MERCURY SALTS

The susceptibility of Botrytis cinerea to copper sulphate in liquid media increased when the volume, and therefore the depth, of the medium in culture bottles exceeded certain values; when the volume was 40 ml. the maximum concentration allowing growth was 300 p.p.m.
By growing mycelium in media containing progressively higher concentrations of copper sulphate a strain was produced which grew at a concentration of 750 p.p.m.
In high concentrations of copper sulphate growth always started at the edge of the liquid, and inocula grew only if they were placed in this position.
In germination tests spores from the resistant strain were more resistant to copper sulphate than were spores of the parent strain.
The resistance of mycelium, and to a lesser extent of spores, was retained after growth of the resistant strain for six months in fungicide-free media.
Spore and mycelial inocula grew in much higher concentrations of copper sulphate when nutrient media were solidified with agar.
The strain resistant in liquid media was no more resistant than the parent strain on agar media.
The resistance of the fungus was not increased after growth for long periods on agar containing high concentrations of copper sulphate. The resistance of the strain resistant in liquid media was not lost after growth on agar media for 3 months.
Attempts to produce strains more resistant than the parent to mercuric chloride were unsuccessful.
The results obtained with phenyl-mercuric acetate were essentially similar to those obtained with copper sulphate, but relatively much more resistant strains were produced.     

Isolation and culture of mesophyll protoplasts from Rosa hybrida

After 1 h plasmolysis in CPW13M solution, highly viable (>75%) protoplasts were isolated from leaves of axenic shoot cultures of Rosa hybrida L. cv. Abraham Darby using an enzyme mixture containing 1.0% (w/v) Hemicellulase, 0.1% (w/v) Macerozyme, 1.0 (w/v) Cellulase RS, 0.05% (w/v) Pectolyase Y23 and 1.0% (w/v) PVP-10 and from cv. Marie Pavié using an identical mixture but with Cellulase RS and Pectolyase Y23 at 0.7% (w/v) and 0.1% (w/v), respectively. With both cvs., sustained protoplast division was achieved after plating in agarose beads with modified KM8p medium containing 1.0% (w/v) polyvinylpyrrolidone (mol. wt. 10 000; PVP-10), 8.91 μM naphthaleneacetic acid (NAA) and 4.44 μM 6-benzyladenine (BA). Protoplast-derived callus gave rise to roots after transfer to SH medium containing 14 μM 2,4-D. This revised version was published online in June 2006 with corrections to the Cover Date.     

Anchorage-independence as an index of proliferative potential and maturational age: a comparison of the growth of normal, primary explanted bovine granulosa cells in semisolid agar and in liquid culture

When seeded at low-density, normal primary explanted granulosa cells will grow to form clones of functionally differentiated cells in both semisolid agar and in liquid culture. The anchorage-independent clonogenic granulosa cell differs from the anchorage-dependent granulosa cells detected in clonal liquid culture in a number of properties. Basal cloning efficiency in liquid culture is up to 50-fold higher than in agar culture. In serum supplemented medium (20% fetal calf serum) cloning efficiency in liquid culture is unaltered in the presence of added epidermal growth factor (EGF), whereas, agar cloning efficiency is augmented six-fold when cells are incubated under identical conditions. Cells derived from primary anchorage-independent clones, when dispersed and replated, will generate secondary anchorage-independent clones and anchorage-dependent liquid clones. On the other hand, although cells derived from parallel primary anchorage-dependent clones will also generate secondary anchorage-dependent clones, generation of secondary anchorage-independent clones is not detectable. These findings suggest that the anchorage-independent clonal agar assay may be detecting a developmentally earlier granulosa cell subpopulation than is detectable in the liquid culture assay.     

Thymineless Death in Escherichia coli in Various Assay Systems: Viability Determined in Liquid Medium

Thymineless death has been studied in four different Thy(-) strains of Escherichia coli by using various assay methods including conventional plating techniques as well as one performed entirely in liquid medium. Plating on L agar resulted in a greater loss in viability than the other assay methods, but this extrasensitivity of starved cells to L-agar plating quickly disappeared upon readdition of thymine to the starved cultures. This indicated that cellular damage responsible for the additional killing on L agar is reversible. The results obtained by three other assay methods, the liquid assay, plating on nutrient agar, or plating on tris(hydroxymethyl)aminomethane-minimal agar, did not differ significantly from each other with all strains tested except strain JG 151. In this strain thymineless death was much faster when assayed in the liquid system than by plating. It is suggested that thymineless death detected on nutrient or minimal agar is not a result of plating, but that the lethal event actually occurs during the period of thymine starvation.