Isolation of L-forms of Bacillus subtilis which grow in liquid medium

L-forms of Bacillus subtilis can be isolated by treatment of the parent strain sequentially with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme and selection of the surviving protoplasts on semisolid medium containing 2,000 units of penicillin per ml. Some of these clones can be adapted to grow in liquid cultures containing 1.2 m NaCl. This method will aid in the isolation of cell wall mutants which require hypertonic medium for growth.     

Ultrastructure of Brucella abortus L-forms induced by penicillin in a liquid and in a semisolid medium

Brucella abortus L-forms were induced by 5.0 or 10.0 mug of penicillin/ml in a broth medium containing 0.3 m sucrose, and in a semisolid medium containing 10% calf serum and 20.0, 40.0, or 60.0 mug of penicillin/ml. After 96 hr of incubation, L-forms of various sizes and shapes were observed. Basic structures of the L-forms were similar whether induced in liquid or semisolid medium. L-forms had two "unit" membranes, each consisting of two outer dense layers separated by a lucent layer. A few large, irregularly shaped organisms in penicillin-treated broth cultures had additional surface material and were referred to as "transitional" forms. In contrast with L-forms, the bacterial cells were fairly uniform in size and shape, were smaller, and had a more complex cell wall structure. Small bodies limited by a "unit" membrane were present within and around numerous L-forms from liquid and semisolid medium cultures. Other internal membranous structures were also seen in some L-forms. Most Brucella L-forms described in this paper reverted to bacteria in the absence of penicillin and were structurally characteristic of unstable L-forms.     

Growth and adhesion of Enterococcus faecium L-forms

Abstract Comparisons of growth and surface colonisation of Enterococcus faecium L-forms and their cell-walled forms were undertaken to produce information about their ability to form sessile cells. The growth of L-forms in liquid culture was slower than that of the parent. This was reflected in their longer lag phase and slower specific growth rates: 0.16 h⁻¹ for the L-form and 0.81 h⁻¹ for the parent. Although E. faecium L-forms attached to a silastic rubber surface, the attached population density was 10–100-fold less than that of the parent. Confluent biofilms on the silastic surfaces were not observed for either bacterial form. Comparison of the attachment of E. faecium L-form and parent may provide important information on how bacteria overcome host defence mechanisms and antibiotic treatment.     

Cultivation characteristics of Legionella pneumophila in a liquid medium

The comparative analysis of liquid media for the cultivation of Legionella pneumophila, sterilized by filtration and autoclaving, has shown that, in contrast to media prepared on the basis of yeast extract, the capacity of media based on proteose peptone for supporting the growth of Legionella is not influenced by the method of their sterilization. The possibility of cultivating this organism in liquid media without ferric pyrophosphate has been demonstrated.     

Staphylococcal Growth and Enterotoxin Production in Meat

Preliminary attempts were made to explain the association of staphylococcal food poisoning with cooked rather than uncooked meats. The abilities of various meats to support the growth of an enterotoxigenic staphylococcus, and the production of enterotoxin A, were determined. The production of enterotoxin was detected by means of serological procedures. Little or no growth was obtained when the inoculum was mixed with raw ground beef. When the surfaces of raw and cooked meats were inoculated, however, good growth was obtained with the production of enterotoxin.     

Growth and division of spiroplasmas: morphology of Spiroplasma citri during growth in liquid medium.

The helical mycoplasma Spiroplasma citri was examined by electron microscopy with a newly developed transfer technique which preserves the helical morphology of the organism. The smallest viable cell was found to be a two-turn (elementary) helix. During the logarithmic phase of growth, organisms increased in length and divided by constriction, liberating two-turn elementary helices. The most frequently dividing parental helix was one with approximately four turns, yielding two elementary helices. Influence of pH and temperature on the morphology of the organism was also investigated. In unbuffered medium, growth of the organism produced a significant decrease in pH and a consequent formation of abnormal morphological forms and cell lysis. At 37 degrees C, cell division was inhibited, leading to a progressive disappearance of two-turn helices and an increase in the average length of other helices. Finally, helices were never seen to arise from round bodies at any stage of the growth cycle.     

The physical characteristics of erythrocyte settling in a liquid medium

Erythrocyte sedimentation was observed in chicken blood with the aid of a microscope. It was determined that the velocity of an ellipsoid shaped cell (chicken erythrocyte) sedimenting in a dilute suspension of cells can be predicted by Stokes' equation; i.e. it obeys Stokes' Law using the calculated ‘effective radius’ which is defined.

Sedimentation of cells in suspensions having greater than 0·003 per cent packed cell volume was characterized by reversing currents resulting from an interaction between the erythrocytes and the liquid medium. Eliminating these currents did not affect the overall erythrocyte sedimentation rate, however.     

Growth characteristics and salt requirement ofDeleya halophila in a defined medium

Growth characteristics ofDeleya halophila (CCM 3662^T), were determined using a defined medium.Deleya halophila presented its optimal growth at 7.5% (wt/vol) total salts when it was grwon at incubation temperatures of 32° and 42°C; when the temperature was lowered to 22°C, it had optimal growth at 5% (wt/vol) total salts. This bacterium had an absolute requirement for the Na⁺ cation; it could not be replaced by other cations. NaBr, Na₂SO₄, or Na₂S₂O₃ could be substituted for NaCl in the growth medium, but, when MgCl₂, KCl, LiCl, NaI, NaF, or NaNO₃ was substituted for NaCl, the medium did not support growth. Growth rates of the strain were diverse when NaCl was partially replaced by other sodium salts. Finally,D. halophila suffered loss of viability when the culture was diluted into different low NaCl concentrations (0, 0.5%, and 1%, wt/vol) at various incubation temperatures.     

Growth factor-induced morphological, physiological and molecular characteristics in cerebral endothelial cells

The capacity of vascular endothelial cells to modulate their phenotype in response to changes in environmental conditions is one of the most important characteristics of this cell type. Since different growth factors may play an important signalling role in this adaptive process we have investigated the effect of endothelial cell growth factor (ECGF) on morphological, physiological and molecular characteristics of cerebral endothelial cells (CECs). CECs grown in the presence of ECGF and its cofactor heparin exhibit an epithelial-like morphology (type I CECs). Upon removal of growth factors, CECs develop an elongated spindle-like shape (type II CECs) which is accompanied by the reorganization of actin filaments and the induction of alpha-actin expression. Since one of the most important functions of CECs is the creation of a selective diffusion barrier between the blood and the central nervous system (CNS), we have studied the expression of junction-related proteins in both cell types. We have found that removal of growth factors from endothelial cultures leads to the downregulation of cadherin and occludin protein levels. The loss of junctional proteins was accompanied by a significant increase in the migratory activity and an altered protease activity profile of the cells. TGF-beta1 suppressed endothelial migration in all experiments. Our data provide evidence to suggest that particular endothelial functions are largely controlled by the presence of growth factors. The differences in adhesiveness and migration may play a role in important physiological and pathological processes of endothelial cells such as vasculogenesis or tumor progression.     

Growth and morphological characteristics of vervet monkey bone marrow-derived adherent cells

Monkey bone marrow-derived adherent cells were maintained in culture for six months. Phase contrast and scanning electron microscopy showed two cell shapes: fusiform and polygonal. No difference was observed in the cyto-chemical staining of the two shapes. Both stained positively for alpha-napthyl acetate esterase, acid phosphatase, collagens I and III, and lipids and negatively for peroxidase and factor VIII antigen. A small proportion (1.5%) were alkaline phosphatase-positive. An average of 14% of the cells were phagocytic in the primary culture, but this proportion decreased progressively with passage. Fc receptors were not detected, while C3 receptors were detected in 1% of primary cultured cells in primary culture, but were not detected in subcultured cells. Adherent cells were not evident in cultures supplemented with 10 mM ammonium acetate. These findings indicate that monkey marrow-derived adherent cells are fibroblastoid in nature.     

Initiation of Staphylococcal Growth in Laboratory Media

The effects of pH and NaCl concentration on the probability of aerobic growth initiation in Brain Heart Infusion broth at 30 C by five staphylococcal strains producing enterotoxins A, B, C, and D were studied in a factorial design experiment. Statistical analysis of the data indicated: (i) significant effects of pH, NaCl, and strain on the probability of growth; (ii) diverse effects of NaCl with various pH levels and strains; (iii) essentially a linear relationship between NaCl concentration and probability of growth initiation when data for all strains were pooled; (iv) the relationship between NaCl concentration and probability of growth initiation varies from linear to sigmoid, depending on the pH of the broth, when the statistically untreated (raw) data are plotted for each strain. Equations were derived which relate the decimal reductions of the number of cells of a staphylococcal population to the concentration of NaCl and pH of broth to which the population was exposed. From the equations, the probability that one cell is capable of initiating growth can be calculated. The impact of these types of studies on the development of realistic staphylococcal standards in foods is discussed.     

Growth characteristics of Bartonella henselae in a novel liquid medium: primary isolation, growth-phase-dependent phage induction, and metabolic studies

Bartonella henselae is a zoonotic pathogen that usually causes a self-limiting infection in immunocompetent individuals but often causes potentially life-threatening infections, such as bacillary angiomatosis, in immunocompromised patients. Both diagnosis of infection and research into the molecular mechanisms of pathogenesis have been hindered by the absence of a suitable liquid growth medium. It has been difficult to isolate B. henselae directly from the blood of infected humans or animals or to grow the bacteria in liquid culture media under laboratory conditions. Therefore, we have developed a liquid growth medium that supports reproducible in vitro growth (3-h doubling time and a growth yield of approximately 5 x 10(8) CFU/ml) and permits the isolation of B. henselae from the blood of infected cats. During the development of this medium, we observed that B. henselae did not derive carbon and energy from the catabolism of glucose, which is consistent with genome nucleotide sequence data suggesting an incomplete glycolytic pathway. Of interest, B. henselae depleted amino acids from the culture medium and accumulated ammonia in the medium, an indicator of amino acid catabolism. Analysis of the culture medium throughout the growth cycle revealed that oxygen was consumed and carbon dioxide was generated, suggesting that amino acids were catabolized in a tricarboxylic acid (TCA) cycle-dependent mechanism. Additionally, phage particles were detected in the culture supernatants of stationary-phase B. henselae, but not in mid-logarithmic-phase culture supernatants. Enzymatic assays of whole-cell lysates revealed that B. henselae has a complete TCA cycle. Taken together, these data suggest B. henselae may catabolize amino acids but not glucose to derive carbon and energy from its host. Furthermore, the newly developed culture medium should improve isolation of B. henselae and basic research into the pathogenesis of the bacterium.     

Growth and ultrastructural characteristics of Citrus cells grown in medium containing NaCl

Changes in growth and structural properties of Citrus cell line Carvalhal acclimated to 100 mM NaCl in the medium were compared to unacclimated control cells and cells exposed to 100 mM NaCl. Transmission electron microscopy (TEM) showed presence of ring-shaped mitochondria, increase in the number of amyloplasts and lipid bodies, higher cell wall thickness and partitioned vacuoles in acclimated cells.