Use Of Liquid Medium Research Articles

On the use of a multiple beam optical sensor for in situ curvature monitoring in liquids

Several methods have been developed since the early 1900 to extract thin film stresses from the curvature of the substrate to which it is attached. One robust method particularly suitable for in situ curvature monitoring is the multiple beam optical sensor, which consists in measuring the change in relative spacings between parallel laser beams reflecting off the curved substrate. Although the technique is already well established for curvature monitoring in low pressure, gaseous environments, its use in liquid media has not yet received similar attention. Moreover, in the majority of the published work so far, spot spacings have been assumed to depend linearly on curvature. In this paper, it is first shown that this assumption may induce significant errors, particularly at large curvature. A more accurate set of equations is proposed. Next, the relationship between spot spacings and curvature is established when the substrate of interest is in a liquid, and a constitutive formula is proposed in that case as well. Finally, some practical aspects of the multiple beam technique for performing curvature measurements in a liquid are discussed. Various factors disturbing the measurement resolution are identified, with a specific interest for thin film anodizing, and a cell design is proposed to minimize their effect.

Exhibition of persistent and drug-tolerant L-form habit of Mycobacterium tuberculosis during infection in rats

Abstract A model for studying mycobacterial L-form formation in vivo was established to demonstrate the ability of M. tuberculosis to behave as a drug-tolerant L-form persister. Rats were infected by intranasal (i.n.) and intraperitoneal (i.p.) routes with 1×108 cells/ml of M. tuberculosis. At weekly intervals during a period of five weeks, samples from lung, spleen, liver, kidney, mesenterial and inguinal lymph nodes, broncho-alveolar and peritoneal lavage liquid were plated simultaneously on Löwenstein-Jensen (LJ) medium or inoculated into specially supplemented for L-forms Dubos broth (drug-free and drug-containing variants). The use of liquid media enabled isolation of mycobacterial L-form cultures during the whole period of experiment including the last two weeks, when tubercle bacilli were not isolated on LJ medium. An unique feature of mycobacterial L-forms was their ability to grow faster than the classical tubercle bacilli. Isolation and growth of L-form cultures in primary drug-containing media demonstrated their drug-tolerant properties. Electron microscopy of liquid media isolates showed that they consisted of morphologically heterogenous populations of membrane-bound and of variable sized L-bodies that completely lack cell walls. The identity of the isolated non-acid fast and morphologically modified L-forms as M. tuberculosis was verified by specific spoligotyping test. The results contribute to special aspects concerning the importance of mycobacterial L-form phenomenon for persistence and latency in tuberculosis, phenotypic drug tolerance, as well as for diagnosis of difficult to identify morphologically changed tubercle bacilli which are often mistaken for contaminants.

Evaluation of Combination of BACTEC Mycobacteria Growth Indicator Tube 960 System and Ogawa Media for Mycobacterial Culture

The combined use of liquid media and solid media is recommended for mycobacterial culture. We evaluated diagnostic performance of combination of BACTEC Mycobacteria Growth Indicator Tube (MGIT; Becton Dickinson, USA) and 2% Ogawa media (Korean Institute of Tuberculosis, Korea) for recovery of mycobacteria. In September 2007, 1,764 specimens from 1,059 patients were cultured with MGIT and Ogawa. Acid fast bacilli (AFB) smear was fluorochrome-stained. The isolates were identified into Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) with PCR using Seeplex TB Detection Kit (Seegene, Korea). Recovery rate, time to detection (TTD), contamination rate, mixed growth rate and species distribution were analyzed. Two hundred thirty-five specimens (13.3%) from 165 patients (15.6%) were positive for mycobacterial culture. Recovery rates of mycobacteria from the group using both media, MGIT only, and Ogawa only were 13.3%, 12.1%, and 7.8%, respectively. While MGIT recovered 98.9% of MTB and 79.7% of NTM, Ogawa recovered 65.9% of MTB and 54.1% of NTM. TTDs of total mycobacteria/MTB/NTM in MGIT and Ogawa were 10.6/11.4/9.7 days and 31/29/33 days, respectively. MGIT TTDs of total mycobacteria/MTB/NTM from AFB-positive specimens were significantly shorter than those of AFB-negative specimens; 8.2/9.5/4.4 days vs 11.6/12.7/10.7 days. Contamination and mixed growth rate of MGIT were 9.6% and 3.7%. Primary culture of Ogawa recovered 1 MTB and 1 NTM among the 170 MGIT-contaminated specimens and 38 mycobacteria among 66 specimens that showed mixed cultures of MGIT. MGIT warrants sensitive and rapid isolation of mycobacteria. However, the combination of MGIT and Ogawa is more desirable to recover mycobacteria in the case of contaminations or mixed cultures.

Callus culture and an unconventional pattern of sporophyte regeneration in Drynaria quercifolia—A medicinal fern

Callus induction and regeneration studies were carried out on a medicinal fern, Drynaria quercifolia native to Asian countries. It is a seasonal fern that regenerates only during the monsoons. Callus was induced on Knop’s (1865) medium supplemented with 20 gl−1 sucrose, 8gl−1 agar, and either 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-amino-3,5,6-trichloropicolinic acid (picloram), or indole-3-butyric acid at different concentrations. Morphogenetic callus obtained on 5 mgl−1 2,4,5-T was subcultured onto solid and liquid media (shaken flask and discontinuously stirred bioreactor cultures) for callus proliferation and regeneration studies. A significant amount of sporophyte regeneration was observed on solid medium containing 10 mgl−1 6-(δ, δ-dimethylallylamino) purine (2iP). Sporophyte regeneration from callus followed an atypical pattern of development. Leafy structures of single-cell thickness with a microrhizome were formed as sporophyte initials. Prolonged cultures of these structures resulted in the formation of juvenile sporophytes in vitro. The use of liquid media resulted in increased biomass in culture. The present study is the first report of a successful system for callus production and regeneration of sporophytes from leafy structures in ferns. The method can be successfully applied for generation of biomass of D. quercifolia, throughout the year.

Laboratory diagnostic aspects of drug resistant tuberculosis.

Multi-drug resistant strains of Mycobacterium tuberculosis (MDR-TB) are present world wide, and in many areas constitute a serious threat to the efficacy of TB control programs. The most effective strategies to limit further spread of MDR-TB are rapid detection of drug resistance followed by prompt and effective therapy. Routine laboratory diagnosis of drug resistance in TB requires a viable, pure culture of M. tuberculosis. Use of liquid media has decreased the turn around time for susceptibility test results however, because of the slow growth of M. tuberculosis these assays can still take 10 to 14 days. Alternatively, an increased understanding of the molecular basis for resistance to the antituberculosis drugs can greatly contribute to further decreasing turn around time. Based on this information, more precise and rapid molecular testing can be developed and lead to more appropriate and timely treatment regimens. In this review, we discuss methods for, and problems encountered in, performing TB drug susceptibility assays. Descriptions of routine protocols will be followed by recent developments in molecular detection of drug resistance.

Proliferation and maintenance of embryogenic capacity in elm embryogenic cultures

This paper investigates maintenance and proliferation of somatic embryogenesis systems for Ulmus minor and U. glabra. Proliferation occurred with subculture of embryogenic calluses. The calluses were mainly formed by friable nodules composed of meristematic cells organized into proembryogenic cell masses (PEMs) and thin-walled vacuolated parenchymatic cells. Cotyledonary embryos, with procambial strands and differentiation of their vascular tissues as well as visible root meristems, were identifiable after 18d of culture on a proliferation medium with 0.44 μM benzyladenine (BA). The shoot meristem was only occasionally well developed. Somatic embryo multiplication from elm embryogenic calluses is a clearly asynchronic system, and PEMs as well as embryos at all stages of development are observed simultaneously at the end of subculture period. Factors affecting the proliferation of elm embryogenic callus, such as culture medium, carbon source and genotype, were studied. Basal medium (MS) or medium supplemented with 0.44 μM BA produced the highest number of somatic embryos. Somatic embryo production was higher with sucrose or glucose than with maltose, and significant differences were also found among the four embryogenic lines tested. The use of liquid medium with filter paper support is an essential step for the survival of isolated somatic embryos during the germination stage. The addition of 0.22 μM BA′ to liquid MS medium was the best treatment for germination and plantlet conversion of elm somatic embryos.

Long-term survival of Phytophthora infestans in liquid media prepared from autoclaved seeds

Five substrates (rye, corn, pea, lima bean, and pea bean) were assessed for their ability to sustain the viability of two isolates of Phytophthora infestans in liquid culture. One isolate belonged to the US-1 genotype (A1 mating type) and the other to the US-8 genotype (A2 mating type). Media based on corn and rye were consistently the best for maintaining the viability of both isolates over time. The survival of the isolate of the US-1 genotype on all five media declined dramatically after 12 months in storage at room temperature (21 degrees C). Recovery of the isolate of the US-8 genotype remained possible after 30 months of storage at room temperature from corn, rye, and pea bean media but not from lima bean or pea media. Isolates of both genotypes grown on a rye kernel medium and kept at 4 degrees C were still recovered after 30 months in culture, although the percentage of successful transfers (mean survival) had declined. According to linear regression analysis, the US-8 isolate grown in sterile culture at 21 degrees C showed better survival over time than the US-1 isolate. To maintain isolates of P. infestans for 1 to 2.5 years without transfer, the use of liquid media based on corn or rye is recommended.

Diagnostic Mycobacteriology Laboratory Practices

The resurgence of tuberculosis has forced clinical laboratories to improve the methods used for detection of M. tuberculosis. Current recommendations for diagnostic laboratory performance [7] include (1) daily processing of specimens (i.e., handling these specimens in the same way that all other specimens sent to the laboratory are handled); (2) inoculation of liquid media (e.g., BACTEC) for the primary culture; (3) use of nucleic acid probes or the NAP test for identifying isolates as M. tuberculosis as soon as possible; (4) determining drug susceptibility with use of liquid media; and (5) reporting results of each step to physicians in a timely manner. The immediate goals are to report identification of M. tuberculosis within 10-14 days of receipt of the specimen and to report drug susceptibilities within 15-30 days. This can be done if current technologies are fully utilized. The amplification-based systems for the identification of M. tuberculosis and the luciferase-based systems for rapid determination of drug susceptibilities should help further shorten turn-around times. The results to date demonstrate that these systems are feasible, although they must be reduced to formats that can be used routinely in clinical laboratories. The gene-amplification systems may be the most promising, and they are nearing commercial availability. If the assays function as well during routine use as they have during clinical trials, a clinical laboratory may soon be able to report confirmed M. tuberculosis infection to the physician within hours of receiving a specimen, instead of within the typical period of 2-4 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Anin vitro procedure to eradicate potato viruses X, Y, and S from Russet Norkotah and two of its strains

A tissue culture method was developed for the eradication of three important potato viruses, PVX, PVY, and PVS, from the Russet Norkotah variety and two of its strains (TXNS 112 and TXNS 278). The method combined the use of liquid medium, thermotherapy, and chemotherapy. Initially, virus-free plants were inoculated with PVX, PVY, and PVS and, after 10 d, tested quantitatively for virus titer by ELISA to determine the initial virus concentration. The apical tip and the roots were removed from the inoculated plants, and only stem sections from inoculated plants were cultured in liquid medium containing MS inorganic salts, vitamins, and ribavirin (40 µM or 80 µM). After 5 d, half of the plants were subjected to thermotherapy and half were kept at room temperature. At 6 wk, the uppermost node (5–7 mm) was removed and recultured, and plants were tested then and again after 6 wk using ELISA to identify the virus-free plants.

943 INFLUENCES OF NODAL EXPLANT POSITIONS AND MEDIA OVERLAY ON MICRO PROPAGATION OF SHEPERDIA ROTUNDIFOLIA

Limited numbers of broad-leaved evergreen species grow well in the cold, dry plains of Colorado. The roundleaf buffaloberry, Sheperdia rotundifolia Parry, is a broad-leaved evergreen species that's adaptable to xeriphytic conditions of the Western states. Since little propagation information is available for this species, we studied procedures for clonal propagation with emphasis on tissue culture procedures. In this study, we evaluated the use of liquid media overlays on rapid propagation and the influence of node position on growth. A comparison of media overlay, transfer to fresh media and a control of no transfer (remained in same media) showed limited differences among treatments. Comparison of terminal, middle and basal node sections showed considerable differences. Sections from the middle produced more branches than the terminal and basal sections, though the branches were shorter than the terminal sections.

Principles involved in the detection and enumeration of clostridia in foods

The clostridia are a group of anerobic bacteria that vary considerably in their biochemical and physiological properties. Not surprisingly, attempts to develop a single isolation medium for all species that occur in foods have not been entirely successful, and the problem is compounded by the need to recover both vegetative cells and spores, some of the latter being unable to germinate without heat activation. Most available isolation media, except some of those used in the dairy industry, include sulphite and an appropriate iron salt, so that blackening due to sulphite reduction can serve as a differential test for clostridia. The limitations of this test in solid agar media are discussed and some advantages described in relation to its use in liquid media for Most Probable Number determinations. A medium favoured for the purpose is the Differential Reinforced Clostridial Medium of Gibbs and Freame (1965). An unresolved issue is whether or not special precautions are needed to exclude oxygen during food sample preparation and dilution, preparation of media, and in conditions used for anaerobic incubation. Although such stringency may be required for maximum recovery of sub-lethally damaged cells or spores, practical constraints in food control laboratories necessitate use of relatively simple procedures for detecting clostridia routinely.

A study of factors affecting anther culture of cauliflower (Brassica oleracea var. botrytis)

Experiments on three autumn-heading cauliflower genotypes (2 hybrids and a genotype selected from a population) were conducted to study different factors affecting anther culture. Culture conditions of the donor plants proved to be important: the best results were obtained during spring in a greenhouse where the temperature was maintained between 10 and 20°C. Overall winter and spring seemed more suitable than summer and early autumn for culture establishment. The optimal bud development stage depended on the genotype: for the hybrid 702, the greatest number of embryos for 100 plated anthers was obtained at the uninucleate pollen stage of the microspores; for V23.2 and 703, the optimal stage of the buds corresponded to the first mitotic division. Sucrose proved to be the best carbon supply for embryogenesis with an optimal concentration of 140 g l-1. The addition of a cytokinin (BAP) in the medium led to lower embryo production, and this negative effect increased when the hormone concentration in the medium increased. The use of liquid medium and a dark incubation period immediately after the high temperature treatment were favourable for embryogenesis.

Rapid method for detecting beta-lactamase producing bacteria in clinical specimens.

The use of liquid media to detect the production of beta-lactamase by beta-lactamase producing organisms has been compared with the conventional method of inoculation on to agar media. Pharyngeal cultures were obtained from 162 children treated with penicillin for acute tonsillitis. beta-lactamase producing organisms were detected within 72 h in 80 (49%) of the specimens inoculated on to agar media, while beta-lactamase production was found in 76 (47%) of the specimens after their incubation in liquid media for 24 h. Twenty one of the cultures were positive only after anaerobic incubation while in liquid media while nine were positive only after aerobic incubation. Incubation in liquid media enabled detection of beta-lactamase activity in 53 of the 76 (70%) specimens within 12 h.

Use of an Animal Membrane in the Evaluation of Chemical Repellents against the Stable Fly1

Candidate insect repellents can be applied in laboratory tests to an animal membrane commercially available under the to an ox caecum membrane commercially available under the trade name Silverlight (derived from ox caecum; available from Julius Schmid, Inc., New York City). Repellency was determined by observing the number of caged stable flies ( Stomoxys calcitrans (L.)) that fed or were prevented from feeding when the untreated side of the membrane was in contact with warmed beef blood. A count of the flies that have fed is facilitated by the use of an ultraviolet activated fluorescent dye previously added to the blood. When results with this procedure were compared with those of another method in which chemicals were applied to cloth, it was found that the degree of repellency might vary depending on the surface to which the chemical had been applied; evaporation from the cloth or better retention on the membrane may account for some of this variation. Evaluation by means of the membrane test method was found to more closely parallel the order of effectiveness obtained in field experience with repellents on dairy animals, and it may have these other advantages as well: ease and accuracy of counting fed and unfed flies; maintenance of uniform or variable applications and surface conditions; use of small amounts of chemicals; elimination of maintenance of an animal as an attractant; possible addition of chemicals to the blood for systemic repellency or other types of testing; and the possible use of liquid media other than blood for attracting flies to the treated membrane.

Observations on soil protozoa

1. It has been shown that the direct counting method for soil protozoa devised by Kopeloff and Coleman for use in liquid media gives results entirely comparable with those obtained by a dilution method.2. The factors governing the relation between the protozoa and the soil particles are those of surface action, and the capacity of various substances, sand, soil and clay, for retaining these organisms is specific and constant.3. Coarse sand is capable of withdrawing per gram approximately 145,000 amoebae and flagellates per c.c. from a suspension of any strength. Fine sand withdraws approximately 980,000 per c.c: soil and partially sterilised soil 1,650,000, ignited soil 1,500,000 and clay 2,450,000.4. These figures are constant for given material and organisms and are independent of the concentration of the suspension, the time of action, or whether the suspension contains cysts or active forms of the amoebae and flagellates investigated. Also the action is the same when the experiment is performed with a suspension of living or dead organisms.5. Experiments with the ciliate—Colpoda cucullus—show that coarse sand per gram retains 27,000 per c.c.; fine sand per gram 185,000 per c.c; soil and partially sterilised soil 280,000 per c.c; ignited soil 270,000 per c.c. and clay 450,000 per c.c.