Complex Agar Media Research Articles

Isolation, Classification and Antagonistic Properties of Alkalitolerant Actinobacteria from Algerian Saharan Soils

The Sahara, one of the most extreme environments on Earth, constitutes an unexplored source of alkalitolerant actinobacteria. In this work, we studied the diversity of alkalitolerant actinobacteria in various soils collected from different regions of the Algerian Sahara. A total of 29 alkalitolerant actinobacterial strains were isolated by using a complex agar medium. The diversity of these actinobacteria was evaluated using a polyphasic approach, which included morphological, chemotaxonomic, physiological (numerical taxonomy) and 16S rRNA gene analyses. The isolates which were assigned to the genus Nocardiopsis, shared relatively low 16S rRNA gene sequences similarities compared to closely related species suggesting that they belonged to putatively new species. All of the strains were tested for antibiotic activity against a broad range of microorganisms and screened for genes encoding polyketide synthases and non-ribosomal peptide synthetases and found to have the potential to produce secondary metabolites. Consequently, the study supports the view that extreme environments contain many novel actinobacteria, which represent an unexplored source for the discovery of biologically active compounds.

Identification of a Novel Protein with a Role in Lipoarabinomannan Biosynthesis in Mycobacteria

All species of Mycobacteria synthesize distinctive cell walls that are rich in phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). PIM glycolipids, having 2-4 mannose residues, can either be channeled into polar PIM species (with 6 Man residues) or hypermannosylated to form LM and LAM. In this study, we have identified a Mycobacterium smegmatis gene, termed lpqW, that is required for the conversion of PIMs to LAM and is highly conserved in all mycobacteria. A transposon mutant, Myco481, containing an insertion near the 3' end of lpqW exhibited altered colony morphology on complex agar medium. This mutant was unstable and was consistently overgrown by a second mutant, represented by Myco481.1, that had normal growth and colony characteristics. Biochemical analysis and metabolic labeling studies showed that Myco481 synthesized the complete spectrum of apolar and polar PIMs but was unable to make LAM. LAM biosynthesis was restored to near wild type levels in Myco481.1. However, this mutant was unable to synthesize the major polar PIM (AcPIM6) and accumulated a smaller intermediate, AcPIM4. Targeted disruption of the lpqW gene and complementation of the initial Myco481 mutant with the wild type gene confirmed that the phenotype of this mutant was due to loss of LpqW. These studies suggest that LpqW has a role in regulating the flux of early PIM intermediates into polar PIM or LAM biosynthesis. They also suggest that AcPIM4 is the likely branch point intermediate in polar PIM and LAM biosynthesis.

Evaluation of Agar and Grain Media for Mass Production of Conidia of Dactylaria higginsii.

Growth and sporulation of Dactylaria higginsii were quantified on complex agar media containing biological materials (group 1) and chemically defined agar media (group 2), as well as on grains, and the inoculum produced on these various substrates was tested for virulence on Cyperus rotundus. The fungus grew well between 25 and 30°C on potato dextrose agar (PDA), with 27°C being the optimum temperature. Generally, conidial production was highly variable and lower on complex agar media than on chemically defined media. Addition of purple nutsedge leaves to PDA did not increase colony growth or conidial production when compared with una-mended PDA. Conidial production was lowest on brown rice compared with white rice or white rice with nutsedge leaves. Peak production on grain media occurred from day 12 in test 1 (2.4 × 106 spores/g of grain) and on day 16 in test 2 (2.5 × 106 spores/g of grain). Germination rate of conidia produced on white rice was 50% compared with the near 100% germination of conidia produced on PDA or on white rice amended with potato dextrose broth (PDB). Conidia produced on white rice or PDA, when tested fresh or after two washings, were less virulent on C. rotundus than conidia from white rice amended with PDB. After four washings, conidia from all three media produced the same level of disease severity. White rice supplemented with PDB and PDA in trays were suitable for mass production of conidia of D. higginsii.

Toluene mineralization and growth potential of Pseudomonas putida PaW164 under toluene-limiting conditions.

Toluene-induced cells of Pseudomonas putida PaW164 (pWWO-164) were monitored for growth potential, maintaining the TOL plasmid, and potential toluene mineralization activity in toluene-amended and nonamended soil. A follow-up study was done in a carbon-free mineral salts solution to obtain further information on physiological changes that occur during starvation. These studies showed that there was a larger decline in colony forming units (CFUs) recovered on a toluate- or benzoate-defined mineral salts medium than on a complex agar medium, a greater percent decrease of CFU than of potential mineralization activity, no decrease in direct counts, and no loss of the TOL plasmid during starvation. Toluene-induced cells also showed an increasing lag time and a decreasing potential for mineralization of (14)C-toluene with starvation. In contrast, the lag time for mineralization of glucose was longest at the onset of starvation and reached a minimum by 3 days; thereafter, the potential for glucose mineralization remained high.

Mycoplasma pneumoniae Infections: Diagnosis Based on Immunofluorescence Titer of IgG and IgM Antibodies

Mycoplasmas are small bacteria (0.3 to 0.8 μm in diameter and up to 150 μm in length) that are unique because they have no cell wall. Two genera, Mycoplasma and Ureaplasma (associated with genital infections), are of clinical importance to humans. M. pneumoniae is primarily a respiratory tract pathogen that involves the nasopharynx, throat, trachea, bronchi, bronchioles, and alveoli. Symptomatic infections attributable to this organism most commonly occur in children and young adults (ages 2 to 19 years). The organism has also been associated with infections of the cardiovascular, dermatologic, and central nervous systems. Other mycoplasmas may be present, especially in the upper respiratory tract; however, they are considered part of the normal microbial flora of the oral cavity and do not cause symptoms or disease. Infections with M. pneumoniae occur worldwide throughout the year, especially in temperate climates. Epidemics occur at 4- to 6-year intervals in both civilian and military populations.1Denny FW Clyde Jr, WA Glezen WP Mycoplasma pneumoniae disease: clinical spectrum, pathophysiology, epidemiology, and control.J Infect Dis. 1971; 123: 74-92Crossref PubMed Scopus (177) Google Scholar, 2Noah ND Urquhart AM Epidemiology of Mycoplasma pneumoniae infection in the British Isles, 1974-9.J Infect. 1980; 2: 191-194Abstract Full Text PDF PubMed Scopus (14) Google Scholar The infection often seems to be a sporadic, endemic illness in families or closed communities because it has a relatively long izncubation period (2 to 15 days), because its prolonged shedding in nasal secretions may result in additional infections during a protracted interval, and because asymptomatic infections are common. M. pneumoniae infections may be diagnosed by cultivation of the organism on complex agar medium or by serologic methods. Because of the slow growth of the organism, partly reflecting fastidious nutrient requirements, culture techniques have been expensive, tedious, and somewhat unproductive. In the clinical microbiology laboratory at the Mayo Clinic, the rate of isolation of the organism has ranged from 0 to 43% from throat and sputum specimens in individual years.3Dorman SA Wilson DJ Smith TF Comparison of growth of Mycoplasma pneumoniae on modified New York City and Hayflick media.Am J Clin Pathol. 1983; 79: 235-237PubMed Google Scholar The time interval between isolates, however, may be several months because of the sporadic occurrence of these infections. Moreover, the last five isolates of M. pneumoniae in our laboratory necessitated more than 20 days of incubation time before a report could be made. Perhaps genetic probes could produce more timely results with acceptable sensitivity and specificity relative to cultivation of the organism. Titration of antibodies in acute and convalescent phase serum specimens is usually done with use of the complement fixation reaction. Currently, however, cells that contain M. pneumoniae antigenic substrate are commercially available (Zeus Technologies, Inc., Raritan, New Jersey), and these facilitate performance of the indirect immunofluorescence test4Carter JB Carter SL Acute-phase, indirect fluorescent antibody procedure for diagnosis of Mycoplasma pneumoniae infection.Ann Clin Lab Sci. 1983; 13: 150-155PubMed Google Scholar and separate measurement of antibodies of IgG and IgM classes. Of 14 paired (acute and convalescent phase) serum specimens, in each pair of which the presence of an M. pneumoniae infection was confirmed by a rising titer of complement fixation, 11 had fourfold or greater increases in titer, as measured by the indirect immunofluorescence test. One of the three paired serum specimens that did not have a diagnostic serologic increase in titer had IgM class antibody present in both acute and convalescent phase serum samples. Importantly, IgM antibodies, indicative of a current infection, were demonstrated in 13 of the 14 patients. In five patients, IgM antibodies were detected in the acute phase serum sample. Twenty serum specimens submitted for cytomegalovirus serology were tested for antibodies to M. pneumoniae by the indirect immunofluorescence test. Seventeen of the 20 specimens (85%) had low levels of IgG antibody directed against the organism; of these, 15 had titers of 1:10 or less. None was positive for IgM antibodies to M. pneumoniae. Exposure to this organism is widespread—in one analysis, more than 90% of asymptomatic, apparently well, Mayo Clinic blood donors had IgG antibodies to M. pneumoniae, as detected by the indirect immunofluorescence test (Fig. 1). The indirect immunofluorescence test for M. pneumoniae is sensitive and specific and can distinguish between current (IgM antibody) and past (IgG antibody) infections. Nonetheless, examination of both acute and convalescent phase (7- to 10-day) serum specimens is urged, because the IgM antibody may not be demonstrable early in some cases and a rising titer confirms the diagnosis of M. pneumoniae infection.

Growth characteristics and ultrastructure of protoplast type L-forms from streptomycetes.

L-form colonies from S. hygroscopicus, S. griseus and S. levoris were isolated after incubation of lysozyme protoplasts on an osmotically stabilized complex agar medium. Unstable and stable L-forms grow on solid and in liquid media. L-form colonies are 5-10 times smaller than normal colonies and show a typical morphology for each species. In ultrathin sections L-form cells are characterized by nucleoid areas with typical core-like structures, by a ribosome-rich cytoplasm with different inclusion bodies, and by a cytoplasmic membrane. Because there are no cell wall structures L-forms of the three Streptomyces species belong to the protoplast type. Analysis of cell size and cell shape shows a variation in diameter and a cell propagation by regular and irregular division- and budding-like processes. Many L-form cells contain more than two chromosomes. The results are discussed with regard to the cellular organisation of streptomycetes and the nature of the stable L-form.

Growth characteristics and ultrastructure of protoplast type L‐forms from Streptomycetes

AbstractL‐form colonies from S. hygroscopicus, S. griseus and S. levoris were isolated after incubation of lysozyme protoplasts on an osmotically stabilized complex agar medium. Unstable and stable L‐forms grow on solid and in liquid media. L‐form colonies are 5–10 times smaller than normal colonies and show a typical morphology for each species. In ultrathin sections L‐form cells are characterized by nucleoid areas with typical core‐like structures, by a ribosome‐rich cytoplasm with different inclusion bodies, and by a cytoplasmic membrane. Because there are no cell wall structures L‐forms of the three Streptomyces species belong to the protoplast type.Analysis of cell size and cell shape shows a variation in diameter and a cell propagation by regular and irregular division‐ and budding‐like processes. Many L‐form cells contain more than two chromosomes.The results are discussed with regard to the cellular organisation of streptomycetes and the nature of the stable L‐form.

Germination of macroconidia and growth of Sporidesmium sclerotivorum in vitro.

Macroconidia of Sporidesmium sclerotivorum, a mycoparasite of Scleroninia spp., were induced to germinate by aqueous and ethanolic extracts of sclerotia of sclerotinia minor. Paper chromatography of sclerotial extracts indicated the presence of several amino acids and carbohydrates, chiefly glucose. Glucose was identified as the principal germination stimulant in ethanolic extracts. Glucose, fructose, mannose, cellobiose, sucrose, maltose, trehalose, soluble starch, and glycerol at 0.1% (w/v) stimulated macroconidia to germinate in 3-6 days at 25 degrees C. Crude sclerotial extracts, and glucose combined with inorganic and organic nitrogen sources, supported germination of greater numbers of macroconidia than glucose alone. Yeast extract, Casamino acids, peptone, and several carbon substrates alone did not support germination. Macroconidia germinated well (greater than 30%) over the range of pH 3-7; maximum germination (greater than 80%) occurred at pH 5.0-5.5. Mycelial growth in a glucose - Casamino acids - mineral salts medium was also greatest in the range of pH 5.0-5.5, but growth fell off sharply below pH 4.5 and above pH 6.0. The fungus grew slowly on several complex agar media adjusted to pH 5.5.

Evidence for the Production of Ethylene by the Mycelium of Agaricus bisporus and its Relationship to Sporocarp Development

The increased production of ethylene that occurs during the development of sporocarps of Agaricus bisporus results from increased formation by the compost but not by the casing layer. The increase depends on the presence of sporocarps up to a critical point in their development. It is concluded that the mycelium of A. bisporus in the compost is responsible for this ethylene production because no significant bacterial flora was found in the compost from fruiting cultures, and the mycelium growing on complex agar media also liberated ethylene. Mycelium growing on defined synthetic medium also evolved ethylene if methionine was supplied. From the results of the different assays, there was no evidence for a regulatory role of ethylene in growth or development of A. bisporus.

Some properties of an unidentified halophile: growth characteristics, internal salt concentration, and morphology.

An unidentified halophile isolated from plates of a complex agar medium containing 4.25 M NaCl showed optimum growth in broths containing 0.5-1.0 M NaCl but exhibited a wide range of growth from 0.045-4.5 M. The organism can be classified as a facultative halophile with wide salt tolerance. Logarithmic phase cells grown in media containing 0.5 M NaCl were rod-shaped in long chains which changed to smaller, single, or paired cells in stationary growth. The internal Na+ and K+ concentrations were 0.05 M and 0.34 M for logarithmic phase cells and 0.29 and 0.32 M for stationary phase cells. In 4.3 M NaCl media the cells were rod-shaped throughout the growth cycle, occurring primarily in pairs. The internal Na+ K" concentrations in cells in logarithmic phase growth were 0.62 M and 0.58 M while in stationary phase growth these values were 1.01 M and 0.66 M respectively. In contrast, logarithmic phase cells of the extreme halophile Halobacterium cutirubrum had internal Na+ and K+ concentrations of 0.80 M and 5.32 M when grown in 3.3 M NaCl. The internal Na+ and K+ concentrations, therefore, in the unidentified halophile do not resemble those found in H. cutirubrum but are much closer to those present in Escherichia coli.

Cytological Studies on Tissue Culture of Pinus cembra

AbstractCallus tissue of Swiss stone pine, Pinus cembra L. var. sibirica Loud., has been grown successfully for 9 months through 7 transfers. Excellent initial growth was obtained from hypocotyl segments of 7‐day‐old seedlings. A complex agar medium was used supplemented with 2 mg/l 2,4‐D, 0.05 mg/l kinetin, 1 g/l Edamin and 10% v/v coconut milk. No deterioration of growth was observed on this medium after numerous transfers. The callus tissue did not produce shoots or roots, but showed an ability for cytodifferentiation. Tracheids or tracheid‐like structures were formed in every passage, but the tracheids of the primary callus culture differed markedly from those of the subcultures. The tracheids of the primary culture probably already existed in the hypocotyl, whereas those of the subcultures were formed in vitro. The callus tissue was mainly diploid during the period studied, and more than 86% of the mitoses were diploid. A few mitoses with tetraploidy or a higher ploidy also occurred, but the tissue did not show any tendency to polyploidization.

Colonial variation in Actinobacillus mallei.

An examination of colonies of 51 strains of Actinobacillus mallei grown on a complex agar medium containing heart infusion broth, yeast extract, glucose, and glycerol indicated a high degree of heterogeneity in respect of colonial morphology both within and between strains. A strain possessing high virulence for hamsters (I.P. LD50 for hamsters < 20 cells) was almost completely homogeneous, and colonies of this strain, when viewed microscopically using an oblique lighting technique, were buff in color and had a dull, slightly rough surface and even edges with a slightly cross-hatched appearance. The designation "typical" was given to colonies of this type. Other colonial types designated as "smooth", "intermediate", and "dense, yellow" were isolated from subcultures of this strain. Additional colony types were found upon examination of other strains; these include a "wrinkled variant and several forms of granular and rough colonies. Prolonged incubation of stationary broth cultures of several strains led to the establishment of new colonial types in several strains tested, and ""wrinkled" and "white, opaque, glistening" colonies were isolated from cultures inoculated with "typical" and "intermediate" forms. The relationship of these findings to the earlier work of Mochida is discussed.