Inoculation Of Medium Research Articles

Presevvation of Xanthomonas campestris in Brassica oleracea seeds

Brassica oleracea seeds were sterilized by gamma radiation and sodium hypochlorite washing. Xanthomonas campestris was inoculated into the seeds by incubating, under vacuum, a suspension of the bacteria with the seeds. After thorough washings with sterile distilled water, the seeds retained about 13 000 cells per seed. The seeds were maintained at 4°C during 21 months, during which the viability of the bacteria and their capacity to produce xantham gum in shake flasks, were evaluated. Bacterial viability showed oscillations but after 20 months it was 10% of the initial. When these seeds were used as a pre-inoculum for a culture to produce xanthan, the final polymer concentration increased slightly with time of seed storage and the final broth viscosity was fairly constant. The specific polymer production (per weight of final bacterial cells) increased about three-fold after 21 months of experimentation. The method, besides being able to produce xantham in quantity and quality, has the advantages of an easy inoculation procedure, no need for transfers, less contamination risk and improved growth rate of the bacteria in the inoculation medium.

Production of mature bovine pancreatic ribonuclease in Escherichia coli

The coding sequence for the bovine pancreatic ribonuclease (RNase) precursor has been cloned and produced in Escherichia coli using the polymerase chain reaction (PCR) technique. A PCR amplification has been carried out utilizing as template the recombinant plasmid, pQR138, which contains the coding sequence for the RNase precursor, and primers that allow for the addition of new sequences at the 5' and 3' ends of the coding sequence. The resultant fragment contains two coding sequences, one for a hexapeptide and the other for pre-RNase. This fragment has been cloned into the expression vector, pKK223.3, under the control of the tac promoter, to form a two-cistron vector. Upon induction with IPTG, E. coli cells harboring this construct generate a bicistronic mRNA which upon translation produces a hexapeptide and pre-RNase. The RNase precursor is efficiently translocated into the periplasmic space of E. coli. Upon translocation, the signal sequence is removed generating mature RNase. Formation of the disulfide bridges in RNase is facilitated by the oxidative environment of the periplasm and a fully active protein is obtained. RNase produced in E. coli has been purified to homogeneity by cation-exchange chromatography, and the removal of the signal sequence has been verified by N-terminal sequencing. The total process from inoculation of media to obtaining pure and fully active recombinant RNase is achieved in 48 h

Monoclonal Antibody Produced against Partially Purified Sporozoite Antigen Inhibits Invasion of Cells by Sporozoites of Avian Eimeria Species

Previous studies showed that molecules of in vitro-cultured primary turkey kidney cells bound to 23-, 40-, and 60- to 65-kDa antigens of sodium dodecyl sulfate-solubilized sporozoites of Eimeria adenoeides. Similar binding to antigens of three other species of avian Eimeria, E. tenella, E. acervulina, and E. meleagrimitis, is now reported. Strips containing the most avidly bound sporozoite antigen (approximately 40 kDa) were excised from the sodium dodecyl sulfate-polyacrylamide gels on which E. adenoeides antigens had been electrophoretically separated. The strips were homogenized and injected into mice to produce hybridoma cell lines. Twelve cell lines secreting monoclonal antibodies (McAb) that reacted with E. adenoeides sporozoites were detected. One of these McAb, H11C3, reacted with structures in the anterior tip of sporozoites of E. adenoeides and five other species of avian Eimeria. When included in the inoculation medium, this McAb significantly inhibited invasion of cultured kidney cells by sporozoites of E. adenoeides and E. tenella. In contrast, when the sporozoites were pretreated with McAb H11C3 and then washed free of the antibody, no inhibition of invasion was observed.

Effect of age, amount of inoculum and inoculation medium composition on lactic acid production from glucose byLactobacillus casei subsp.rhamnosus

Effect of composition of the medium used for the inoculum cultivation, of the age and amount of the inoculum was investigated using a 3-L glass fermentor with a working volume of 1 L. The highest productivity of the culture was obtained when using a 20% (V/V) 1-d inoculum grown in the MRS medium. Yields of lactic acid were 88-97%, while the L(+)-isomer represented about 80% of the total product.

百日咳菌の効率的・簡便な臨床分離法の検討

Commercially available Amies transport medium with charcoal and three isolation media were tested to assess their efficiency in the clinical isolation of B. pertussis. First, the isolation rates of B. pertussis were compared between direct inoculation of nasopharyngeal specimens and inoculation after stored in Amies transport medium for 8 hours or less. The comparative isolation rates were 81% both (35 of 43 specimens from 29 patients) for direct inoculation and transport medium. Second, nasopharyngeal specimens were incubated for 5 days at 35 degrees C on the three media; Bordet-Gengou Medium (BG) with 5 micrograms of CEX per ml, Cyclodextrin Solid Medium (CSM) with 5 micrograms of CEX per ml, and Charcoal Agar with 40 micrograms of CEX per ml. The organism was detected from 44 nasopharyngeal specimens from 20 patients on at least one of the three tested media. The comparative isolation rates were 91% (40 of 44) on BG with 5 micrograms of CEX per ml, 93% (41 of 44) on CSM with 5 micrograms of CEX per ml, and 91% (40 of 44) on CA with 40 micrograms of CEX per ml. Although no differences in the isolation rates were observed among the three media, the appearance of the colonies was earlier by one day on BG than the rest of the two media. The detection of B. pertussis was occasionally easier on CA than the rest of the two because of its higher suppression for nasopharyngeal flora. CSM has its advantage in that it does not need any blood and can be prepared at anytime. Also, the shelf life of these three media proved to be at least one month when stored at 10 degrees C. We conclude that the clinical isolation of B. pertussis was highly successful with the following simple procedure: nasopharyngeal specimens stored in Amies transport medium and inoculated on one of the three media, BG, CSM, or CA, and then incubated for 5 days at 35 degrees C.

Specimen transport containers are not created equal

Specimen Transport Containers Are Not Created Equal Ideal specimen culture procurement and inoculation of media should occur at the bedside. However, this is neither practical nor efficient in a hospital setting, and, therefore, specimen transport devices were developed. The most effective transport systems rely upon preservation of the actual specimen taken from the patient. However, most specimens other than sterile body fluids or tissues are sent to the microbiology laboratory on cottonor rayontipped swabs. Following receipt in the laboratory, specimen material on these swabs is inoculated onto or into culture media. Based on the quantity and identity of organisms present, decisions are made by the laboratory professional staff (i.e., determining on which organisms to perform identifications and susceptibility tests) and the medical staff (i.e., evaluating the culture report and deciding initiation or modification of therapy). This system is less than ideal for several reasons. First, organisms must be preserved without replication in the transport system. Second, some fastidious microorganisms may not survive in transport systems designed for routine microbiological examination. And third, many investigators have shown that swabs trap organisms or are toxic and prevent them from being recovered from culture (3, 7). In our laboratory, we observed an increased recovery of streptococci from transport swabs by rinsing them in 1 mL of sterile saline and culturing an aliquot of the saline rinse (Table 1). In addition to isolating organisms in greater quantity, separation of organisms on culture plates exhibiting a mixed flora is easier. Specimen Table 1 Isolation of beta hemolytic streptococci a from 135 throat cultures c

Differentiating astroglia in nervous tissue histogenesis/regeneration: studies in a model system of regenerating peripheral nerve.

The role of astroglia in nervous system histogenesis/regeneration (morphogenesis) was studied as a function of cell age. The effect of inoculated astroglia at different cell maturation stages on axonal growth was examined in a peripheral nerve regenerating model system. This model system consists of rat sciatic nerve stumps that regenerate through an empty silicone chamber (Lundborg et al.: Journal of Neuropathology and Experimental Neurology 41:412-422, 1982). Rat astroglial cell populations grown in cell culture were suspended either in a liquid (physiological solution) or in a solid (isotonic collagen gel) medium and inoculated within the silicone chamber at the time of surgery. Immature and mature cell populations, at ages corresponding to 9-69 postnatal days (P9-P69), were inoculated, and their effect on neural growth was analyzed by histological, immunocytochemical, and ultrastructural techniques, 2-6 weeks later. Astroglial cells differentially modulated the process of nerve regeneration, an effect that is a function of the cells' developmental stage. P19 astroglia and older cells inhibited the regeneration process, encapsulating the axons in a glia-limitans-like structure. Immature astrocytes (P9) did not seem to interfere with the regeneration process; nerve outgrowth in their presence resembled and was comparable to the ones obtained in the presence of inoculated Schwann cells. The differential effects of the developing astroglia were not significantly changed by the inoculation media, e.g., liquid or solid, and were already pronounced 2 weeks after neural transection. It is suggested by the results of the study that the role and function of astroglia in nervous system morphogenesis are changing with cell differentiation. Adult astrocytes seem to downregulate axonal growth; presumably, their function is to confine the neurites within designated structural and functional boundaries.

Equilibrium-driven mechanism for preferential adhesion between chick embryo cells

The experiments presented here confirm the hypothesis according to which, in our experimental system of differential cell adhesion (where we studied the kinetics of the earliest period of adhesion of a suspension of chick embryo neuroblasts to layers of astroblasts or fibroblasts), the mechanism of adhesion appears to consist of two steps, the first of which is a short-term reversible phase corresponding to a binding equilibrium. In fact, adhesion of neuroblasts to each of the two cell layers occurs according to kinetic constants and attains levels which are characteristic for each of the two adhesion systems. In both systems, neuroblasts that have not adhered at equilibrium are able to adhere if inoculated over a fresh cell layer of the same type, as they do during the first inoculation; conversely, neuroblasts that have adhered to a cell layer can be made to de-adhere by substituting cell-free fresh medium to the inoculation medium containing non-adhering neuroblasts. This shows that, as predicted for a reversible equilibrium system, removal of adhering neuroblasts from the system at equilibrium provokes adhesion, and removal of non-adhered neuroblasts provokes de-adhesion. Furthermore the level of adhesion at equilibrium is, in all cases, the same. The reversibility of adhesion, which is almost quantitative during the onset of the equilibrium, gradually decreases with time, indicating the presence of a process of irreversible attachment between cells after the first reversible step. The developmental implications of the complete sequential mechanisms are briefly discussed.

Interactions of Cowpea Mosaic Virus with Cowpea Protoplasts1)

AbstractThe capability of cowpea mosaic virus to attach to and infect protoplasts of immune, hypersensitive, and susceptible cowpea (Vigna unguiculata) lines was examined by inoculating protoplasts with either purified virus or radioiodinated purified virus 125I‐CPMV. Systems were used in which plants were immune and protoplasts susceptible, plants were immune and protoplasts resistant, and plants and protoplasts were susceptible to CPMV. No differences were observed in the attachment of 125I‐CPMV to resistant and susceptible protoplasts. Polycations, proteins, or virus particles were added to the inoculation medium to neutralize potential nonspecific interactions between cells and virus particles. The various additives induced quantitative differences in binding of virus particles to protoplasts.

Joint utilization of glucose and n-alkanes in citric acid synthesis by Saccharomycopsis lipolytica

Fermentations for the overproduction of citrate and isocitrate with S. lipolytica in media containing both glucose and n-alkanes as mixed C-source have been performed. Biomass and product yields strongly depend on the C-source of the inoculation culture. If the inoculation culture had been taken from media containing glucose as sole C-source both glucose and n-alkanes were utilized for cell growth in the main culture whereas only glucose was utilized if the inoculation medium contained only n-alkanes. For idiophasic citrate and isocitrate production both glucose and n-alkanes were consumed independently of the C-source of the inoculum but that C-source was preferentially utilized which has been the C-source of the inoculation culture. These findings are reflected by the activities of the isocitrate lyase and the pyruvate carboxylase, respectively. In S. lipolytica both anaplerotic pathways are coexisting but the C-source of the inoculation culture determines the level of the specific activities even if the ratio of the cell-mass of the inoculum to the cell mass of the main culture at the end of the growth phase is about 1:35.

Gemeinsame Verwertung von Glucose und n‐Alkanen bei der Citronensäuresynthese durch Saccharomycopsis lipolytica

AbstractFermentations for the overproduction of citrate and isocitrate with S. lipolytica in media containing both glucose and n‐alkanes as mixed C‐source have been performed. Biomass and product yields strongly depend on the C‐source of the inoculation culture. If the inoculation culture had been taken from media containing glucose as sole C‐source both glucose and n‐alkanes were utilized for cell growth in the main culture whereas only glucose was utilized if the inoculation medium contained only n‐alkanes.For idiophasic citrate and isocitrate production both glucose and n‐alkanes were consumed independently of the C‐source of the inoculum but that C‐source was preferentially utilized which has been the C‐source of the inoculation culture.These findings are reflected by the activities of the isocitrate lyase and the pyruvate carboxylase, respectively. In S. lipolytica both anaplerotic pathways are coexisting but the C‐source of the inoculation culture determines the level of the specific activities even if the ratio of the cell‐mass of the inoculum to the cell‐mass of the main culture at the end of the growth phase is about 1:35.

Infection of Protoplasts from Soybean Cell Culture with Southern Bean Mosaic and Cowpea Mosaic Viruses

SUMMARY Soybean protoplasts, isolated from liquid suspension culture, were successfully infected with cowpea mosaic virus (CPMV) and with southern bean mosaic virus (SBMV). Poly-l-ornithine (PLO) was required for infection with either virus. As detected by fluorescent antibody, 70 to 90% of the protoplasts were infected by CPMV when the inoculation medium contained 0.4 m-sorbitol, 0.5 µg virus/ml, 1.5 µg PLO/ml, 10 mm-potassium phosphate buffer, pH 6.3, and 0.5 mm-CaCl2, and inoculation was performed at 23 °C. This maximum level of infection was decreased sixfold when CaCl2 was omitted. Inoculation at temperatures below 10 °C also decreased infection substantially. With SBMV a maximum of 30 to 35% of the protoplasts were infected when 0.4 m-sorbitol, 2 to 2.5 µg virus/ml, 2 µg PLO/ml, 10 mm-tris-HCl buffer, pH 8.0, 1 mm-MgSO4 and 0.5 mm-CaCl2 were present in the inoculum. Less than 5% of the protoplasts were infected when magnesium and calcium salts were omitted. The major advantage of the use of soybean cell suspension culture for plant virus studies is that it can provide a reliable and continuous source of cells for protoplast isolation. These soybean protoplasts allow synchronous infection and replication under sterile conditions without antibiotics and also a high degree of reproducibility.

The development ofOnchocerca volvulusin two temperate blackfly species,Simulium ornatumMeigen andSimulium lineatumMeigen

In view of the promise shown by temperate simuliids for laboratory colonization, the ability of Simulium ornatum and S. lineatum to support the development of Onchocerca volvulus was investigated. An injection technique is described for the introduction of O. volvulus microfilariae from a chimpanzee into female simuliids in the absence of a bloodmeal. RPMI 1640 medium, with penicillin, streptomycin and foetal calf serum, proved satisfactory as an inoculation medium. Third stage larvae were obtained in both S. ornatum and S. lineatum after seven days at 27 degrees C. The rate of development of the parasite in S. ornatum is recorded.

Candida Endophthalmitis After Intravenous Drug Abuse

Patients with endogenous Candida endophthalmitis associated with intravenous (IV) drug abuse may manifest ocular and systemic signs different from those seen in other forms of endogenous Candida endophthalmitis. There may be a sparcity of evidence of systemic candidiasis, including negative serology and normal physical examination results. Anterior uveitis and extensive vitreous involvement are common and do not necessarily have associated typical retinal lesions, which are more commonly seen in the compromised host. This may occur either because of the more transitory nature of choroidal or retinal lesions or because these patients often seek treatment at later stages. Even with a typical clinical picture, it is difficult to get culture confirmation of the diagnosis. Material obtained by vitrectomy must be concentrated before inoculation of media because of the known difficulty of culturing Candida from the vitreous cavity.

The detection of leptospirae in cattle urine

Abstract Hamster inoculation, medium inoculation and microscopical methods were used for the detection of Leptospira interrogans serovar pomona organisms in bovine urine. Urine samples were collected from both naturally and experimentally infected animals during the period when leptospiruria could be expected. Inoculation into hamsters of 0.5 ml of urine from each of 14 samples resulted in 5 positives. The inoculation of 0.025 ml of the same sample into semi-solid EMJH medium gave 10 positives. From a cumulative total of 46 urine samples, 28 were positive using dark-ground microscopy of centrifuged urine deposits and 35 were positive using media inoculation. A cumulative total of 126 urine samples were examined, after centrifugation, under dark-ground microscopy and leptospirae were detected in 60. Fluorouracil (100 mg/ml) proved to be beneficial for successful isolations in media, whereas neomycin (5 mg/ml) did not.

Abnormal forms of Trichomonas vaginalis.

Abnormal forms of Trichomonas vaginalis have been demonstrated by both conventional and scanning electron microscopy after inoculation of media with clinical material from cases of trichomonal vaginitis. Twenty-six cases of vaginitis have been studied; 10 of them showed the abnormal forms of trichomonads after growth in a modification of the medium described by Bushby and Copp (1955), while 16 showed only normal forms.

Effects of viral protein, viral RNA, and some other polyelectrolytes on infection of tobacco protoplasts by TMV

Tobacco cv. Samsun protoplasts were inoculated with TMV in the presence of different polyelectrolytes. Retention of [ 14C]TMV was determined by radioactivity of the protoplasts or by infectivity in homogenates of samples frozen immediately after inoculation and washing. Virus yield was determined by infectivity in protoplasts following incubation for 48 hr in light at 28°. Infectious TMV, noninfectious TMV, and TMV protein (100 μg/ml) enhanced virus retention. However, the virus yield for the first substance was essentially lower. When the substances were added to the incubation medium, they decreased virus yield. Bovine serum albumin and casein hydrolysate did not appreciably affect retention; yet, they also decreased virus yield. Virus RNA, whether infective or not, strongly suppressed the virus yield when present in the inoculation medium at a concentration of 1 μg/ml or more. This effect was independent of the presence of actinomycin D in the medium. Experiments with labeled TMV revealed the low virus yield to be the result of a reduction of virus retention in inoculum containing a mixture of TMV and TMV RNA. Similar effects on virus retention and yield were produced by total RNA of animal origin and by dextran sulfate. The authors assume that the three above-mentioned polyelectrolytes block virus adsorption on the plasmalemma by binding with a virus-polyornithine complex.

Infection of Tobacco Mesophyll Protoplasts by Potato Virus X

SUMMARY Conditions favouring the infection of isolated tobacco mesophyll protoplasts by potato virus X (PVX) were studied in detail, and a procedure to effect infection in 70% of protoplasts was developed. PVX required higher concentrations of both inoculum virus and poly-l-ornithine than tobacco mosaic virus (TMV) or cucumber mosaic virus (CMV). PVX infection showed a unique response to varying pH of the inoculation medium. The time course of PVX multiplication in protoplasts resembled that of TMV. Unlike the infection by TMV and CMV, PVX infection was partially inhibited by actinomycin D. Inclusion bodies characteristic for PVX infection were present in the infected protoplasts, but their involvement in virus production appeared to be unlikely.

Quinine Inhibition of Host Cell Penetration by Toxoplasma gondii, Besnoitia jellisoni, and Sarcocystis sp. In vitro

The penetration of Toxoplasma gondii, Sarcocystis sp., and Besnoitia jellisoni into cultured cells was significantly inhibited by 0.1 mg quinine sulfate/ml in the inoculation medium. The former 2 organisms were inhibited from penetration by as little as 0.01 mg of this drug. Incubation of either organisms or cells in medium containing quinine prior to inoculation of organisms in quinine-free medium did not significantly reduce penetration. Organisms remained motile in the presence of quinine and the few that entered cells remained intracellular. Results of this study parallel those with Eimeria and indicate that quinine interferes with a mechanism of host cell penetration common to all 4 genera. In vitro methods have been used to study penetration of cells by eimerian sporozoites (Fayer et al., 1970; Roberts et al., 1971; Fayer, 1971). In the latter study, addition of quinine compounds to the inoculation medium inhibited penetration of cultured cells by Eimeria meleagrimitis and E. tenella sporozoites without producing visible cytopathological effects or rendering the organisms immotile. The present study was conducted to determine the effect of quinine sulfate on the penetration of cells by Toxoplasma gondii, Besnoitia jellisoni, and Sarcocystis sp. MATERIALS AND METHODS

Quinine Inhibition of Host Cell Penetration by Eimerian Sporozoites In vitro

Leighton tube cultures of bovine embryonic kidney cells were inoculated with Eimeria meleagrimitis sporozoites suspended in culture medium containing 0.1 mg/ml quinine alkaloid, quinine hydrochloride, or quinine sulfate. Such cultures had up to 80.2, 87.5, and 88.2% fewer intracellular sporozoites, respectively, than control cultures inoculated with sporozoites in quinine-free medium. Addition of quinine sulfate to inoculation medium reduced the number of intracellular E. tenella sporozoites by as much as 96.6% as compared with controls. The effects of quinine appeared to be restricted to the process of host cell penetration. Sporozoites incubated in medium containing each of the 3 types of quinine and resuspended in quinine-free medium entered cells in nearly the same numbers as sporozoites incubated under similar conditions in quinine-free medium. Sporozoites, inoculated in quinine-free medium, were also found in nearly equal numbers in cells which had been incubated with medium containing each of the quinine compounds and in cells incubated with quinine-free medium. Sporozoites suspended in medium containing each of the quinine compounds were observed by phase contrast microscopy in a heated microscope enclosure. Although they remained motile during the observation period, few entered cells. In vitro culture methods have been used to study penetration of host cells by eimerian sporozoites (Fayer et al., 1970) and the effect of chemotherapeutic agents on coccidia (Fayer and McLoughlin, 1970). The present study was conducted to determine the effect of the antimalarial drug, quinine, on the penetration process in vitro. MATERIALS AND METHODS