Induction and cultivation of a stable L-form of Bacillus subtilis

The induction of L-forms of Bacillus subtilis from protoplasts is described. The method involved the frequent subculture of the unstable L-form on a growth medium supplemented with lysozyme and horse serum. A stable culture, which did not revert when lysozyme and horse serum were omitted from the medium, was obtained after 13 subcultures. This culture could be grown on solid and in liquid medium by routine microbiological methods. Long-term storage of these cells was achieved by freeze drying and maintenance in glycerol at -70 degrees C. The cultural adaptability of the L-form is described and discussed with respect to methods of cultivation and growth.     

Serum inhibits penicillin-induced L-form growth in Staphylococcus aureus: a note of caution on the use of serum in cultivation of bacterial L-forms.

Heat-inactivated horse serum inhibited penicillin-induced L-form colony formation in Staphylococcus aureus when included in an osmotically stabilized culture medium. Most, perhaps all, L-form colonies that appeared with low frequencies on the serum-supplemented medium were of the penicillin-independent, stable type. This relationship must be taken into account when use of serum is considered for L-form cultivation.     

Morphology, growth and reversion in a stable L-form of Escherichia coli K12

An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0.34 M and 1 mM, respectively. No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0.34 M-NaCl. On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaCl.     

Primary culture and maintenance of steroid-secreting human placenatal monolayers.

A simple method is described for primary culture and for maintenance of hormone-producing cells from normal human placenta. A consistent yield of cells was obtained and an average survival of 3 to 4 months in culture using 1 mm3 explants from the most vascular area of the placentas. These explants were placed in a variety of culture media in 30 ml flasks and incubated at 37 degrees C in an atmosphere of 5% CO2 and 95% air. The best yields in terms of cell growth were observed with Eagle's MEM (minimum essential medium) with supplements of horse serum and fetal calf serum or human cord serum. (Ham's F-10 with supplement of horse serum and fetal calf serum supports growth for the longest period and media containing human cord serum had the best yield of steroids.     

Induction of L-Phase Variant from Protoplast of Staphylococcus aureus

Protoplasts of Staphylococcus aureus 209P and Cowan 1 were induced by treatment with lysostaphin. These protoplasts were sensitive to detergent, a low concentration of sodium chloride and low temperature. Almost all protoplast cells spread on CLYS agar medium (casein hydrolysate, yeast extract, Na-lactate, and NaCl) formed typical L-form colonies. Horse serum (0.25%) and Mg²⁺ (109 mm) are essential factors for formation of the L-form colonies of 209P. In the case of Cowan 1, Mg²⁺ was not required. The active factor(s) in horse serum was heat-resistant and protein in nature.     

Design of a Microculture Chamber to Observe Cell Division of Bacterial L-Forms in Liquid Medium

A culture chamber is described that permits phase-contrast microscopic observations of the growth of stable L-form cells of Bacillus subtilis 168 in liquid medium.     

Primary culture and maintenance of steroid-secreting human placental monolayers

Summary A simple method is described for primary culture and for maintenance of hormone-producing cells from normal human placenta. A consistent yield of cells was obtained and an average survival of 3 to 4 months in culture using 1 mm³ explants from the most vascular area of the placentas. These explants were placed in a variety of culture media in 30 ml flasks and incubated at 37°C in an atmosphere of 5% CO₂ and 95% air. The best yields in terms of cell growth were observed with Eagle’s MEM (minimum essential medium) with supplements of horse serum and fetal calf serum or human cord serum. (Ham’s F-10 with supplement of horse serum and fetal calf serum supports growth for the longest period and media containing human cord serum had the best yield of steroids.) Presented in part at the 25th meeting of the Tissue Culture Association, June 5, 1974, Miami Beach, Florida. Supported by a contract from the Agency for International Development (2491/csd) and a grant from the National Institutes of Health (CA-12455).     

Induction, growth and antibiotic production of Streptomyces viridifaciens L-form bacteria

AIMS: To induce, cultivate and investigate the characteristics of L-form bacteria derived from the filamentous actinomycete Streptomyces viridifaciens. METHODS AND RESULTS: L-forms were induced in a liquid medium supplemented with lysozyme and penicillin. A stable culture which no longer required inducing agents but could still revert, was obtained by the twelfth subculture. The specific growth rate of stable L-forms was faster (0.751) than unstable L-forms (0.361). After the exponential growth phase, the cell diameter continued to increase, as did the percentage of vacuoles. Morphologically, the L-forms appeared as spherical bodies with no signs of differentiation and were sensitive to osmotic stress, indicating removal of the cell wall. The L-forms produced secondary metabolites although much lower levels of antibiotic were assayed in the L-forms compared with the cell walled forms. CONCLUSION: Stable L-form bacteria were induced from S. viridifaciens and their growth characterized. The L-forms produced secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Stable Streptomyces L-forms were induced and have potential as biocontrol agents.     

Comparison of the efficacy of egg yolk extract and horse serum for growth of Mycoplasma pneumoniae

The usefulness of chicken egg yolk extract as a substitute for horse serum in culture media for Mycoplasma pneumoniae was investigated. As a growth-supporting factor in the growth medium for M. pneumoniae and some other mycoplasmal species, the primary isolation medium for M. pneumoniae, and the metabolism-inhibition test medium for diagnosis of M. pneumoniae infection, egg yolk extract may be an excellent substitute for horse serum. The particular superiority of egg yolk extract to horse serum is that egg yolk extract, unlike horse serum, did not show any inhibitory effect on the growth of M. pneumoniae.     

Growth Medium Containing Cyclodextrin and Low Concentration of Horse Serum for Cultivation of Helicobacter pylori

The growth of Helicobacter pylori, a Gram-negative microaerophilic bacterium, is often difficult and requires complex media with the supplementation of 5% to 10% blood or blood derivatives. We have found that Brucella broth supplemented with 1% heated horse serum and 0.1% β-cyclodextrin supports the good growth of H. pylori. The degree of growth and production of urease and vacuolating cytotoxin in this medium were equal to those in the medium supplemented with 5% horse serum. This medium was found to be suitable for both the routine laboratory culture and primary isolation of H. pylori from biopsy samples.     

Preparation of protoplasts and whole cell ghosts from Mycobacterium smegmatis.

Cell wall-deficient forms of Mycobacterium smegmatis were produced in growth medium containing D-cycloserine and horse serum. These cells were transformed into protoplasts with EDTA and lysozyme. Subsequent lysis by nucleases followed by osmotic shock produced membrane vesicles (whole cell ghosts).     

Growth of mouse vaginal epithelial cells in culture: Effect of sera and supplemented serum-free media

Summary Normal mouse vaginal epithelial cells isolated from ovariectomized ca. 35-d-old BALB/cCrgl mice were grown in primary culture using collagen gel metrix and a serum-free medium composed of a 1∶1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 (D:H) medium supplemented with insulin (IN), epidermal growth factor (EGF), cholera toxin, transferrin, and bovine serum albumin V (BSA). Three-dimensional cellular outgrowths occurred inside the collagen gel matrix. The contribution of each factor to cell growth was examined by individual addition to the basic D:H medium and by individual deletion from the complete serum-free medium. When added individually, only IN promoted growth. Deletion of IN from the complete serum-free medium markedly, diminished growth; deletion of EGF or BSA slightly diminished growth. When horse, fetal bovine, or chicken serum was added to the basal D:H medium, only with increasing doses of horse serum was there enhanced cell growth. The effect of 17?-estradiol and diethylstilbestrol on the growth of cells was also tested, using a suboptimal medium of D:H supplemented with BSA and IN, or a minimal medium supplemented with IN alone. During the 8-d time period, addition of estrogen did not enhance cell growth in either medium. To date, we have been unable to demonstrate a mitogenic effect of estrogen; rather a dose-dependent inhibition of proliferation is seen. This investigation was supported by grants CA-05388 and CA-09041, awarded by the National Institutes of Health, DHHS, Bethesda, MD.     

Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: application to studies of lymphoblast proliferation in vitro.

We have compared several methods for reducing calcium and magnesium concentrations in tissue culture medium, with the objective of producing selective deficiency effects on the growth of mouse (L5178Y) and human (P1R) lymphoblasts. In experiments in which calcium- and magnesium- "free" McCoy's medium was supplemented with 15% horse or fetal calf serum, enough calcium and magnesium was provided by serum to support normal lymphoblast growth rate. Either dialysis or chelating-resin treatment of horse or fetal calf serum reduced calcium and magnesium contents approximately 100-fold. Use of dialyzed sera resulted in reduced growth rate, although in most cases the reduction in growth could be attributed to other effects of dialysis on serum, inasmuch as growth in those experiments was not restored to normal by the addition of calcium and magnesium to the medium. In contrast, the reduction of lymphoblast growth rate that occurred when resin-treated serum was used was always attributable to removal of calcium and magnesium, as normal growth always occurred in cultures to which calcium and magnesium were added. To demostrate a growth-inhibiting effect on either mouse or human lymphoblasts by severe reduction of either calcium or magnesium in the presence of normal amounts of the alternative cation, it was necessary to (a) expose McCoy's Ca-Mg-"free" medium to chelating-resin to reduce further the residual cation concentrations; (b) wash cells from stock cultures in a medium devoid of calcium and magnesium prior to inoculation into experimental cultures; (c) reduce the proportion of serum in the final medium from 15 to 5%; and (d) add 100 muM EGTA to cultures. Under these conditions, growth of both cell types was completely abolished in the presence of normal magnesium but in the absence of added calcium, and markedly reduced in the presence of normal calcium but in the absence of magnesium. These modifications did not compromise growth in cultures containing normal concentrations of both ions.     

Adaptation of a stable L-form of Bacillus subtilis to minimal salts medium without osmotic stabilizers.

An L-form isolated from Bacillus subtilis 168 was adapted to growth in a 340 mOsm minimal salts medium without the addition of osmotically protective solutes. This L-form had no chemically detectable peptidoglycan residues on its surface, but 0.8% of the dry weight of washed membranes was hexosamine. The osmotic stability and susceptibility to bacitracin and vancomycin of the L-form adapted to growth in 340 mOsm osmotically unprotected medium was twice that of the L-form grown in 2,680 mOsm medium supplemented with 1.2 M NaCl.     

An L-Form of Staphylococcus aureus Adapted to a Brain Heart Infusion Medium without Osmotic Stabilizers

An L-form derived from halotolerant Staphylococcus aureus Tasaki was adapted to growth in a brain heart infusion medium without any supplemental osmotically protective solutes (360 mOsm/kg). This L-form had no chemically detectable peptidoglycan residues on its surface. Electron microscopic observations confirmed morphologically the absence of the structures and also of other osmotically protective polymers within or exterior to the cytoplasmic membrane. The osmotic stability and susceptibility to bacitracin, d-cycloserine, and vancomycin of the L-form adapted to growth in 360 mOsm osmotically unprotective medium was higher than that of the L-form grown in 1,950 mOsm supplemented with 4.5% NaCl. The adapted L-form tended to be more sensitive to almost all of the antibiotics examined, other than the inhibitors for cell wall-synthesis, than the original L-form strain requiring osmotic protection for growth. Chemical analysis of the membrane of the adapted L-form indicated 16.3% total lipids and 20.6% proteins by dry weight of the membrane, and it contained larger amounts of lipid phosphorus (20.0 μ/mg).     

Induction of Enterococcal L-Forms by the Action of Lysozyme

Suspensions of enterococci were treated with lysozyme in the presence of osmotic stabilizers. The resulting osmotically fragile bodies prepared from Streptococcus faecium strain F24 and S. faecalis strain E1 gave rise to L-forms under optimal osmotic and nutritional conditions for treatment and subsequent growth. The most critical component of the growth medium, to obtain maximum yields, was the nature and concentration of the added salt. The two most effective salts were sodium chloride and ammonium chloride in the range of 2 to 3% (w/v) added to a suitable agar base. Ammonium chloride was more versatile, because it could be used with either sucrose or polyethylene glycol 4000 as the osmotic stabilizer for preparation and dilution of the osmotically fragile bodies. Sodium chloride would not consistently support growth of S. faecium F24 as L-forms when polyethylene glycol 4000 was used as the osmotic stabilizer during lysozyme treatment. Time-course studies of concurrent cell wall removal and L-form induction suggested that maximal induction required only cell wall damage rather than complete wall removal. This method for induction of L-forms from a suspension of enterococci is a significant improvement over other presently known methods.     

Defined Physiological Conditions for the Induction of the L-Form of Neisseria gonorrhoeae

Defined conditions are described which allowed luxuriant growth over continuous subculture of strains of Neisseria gonorrhoeae in broth and on agar. Growth was equal to or surpassed that observed in Mueller-Hinton broth or on Mueller-Hinton blood agar. The final medium adopted consisted of medium 199 and a supplemental mixture of cysteine, glucose, and various salts. Addition of sodium bicarbonate or CO(2) enrichment was not required. For solidification, only agarose allowed growth of all strains; glutamic acid stimulated growth of two strains but was inhibitory for a third. The addition of 8% polyvinylpyrrolidone (PVP), 2% purified albumin, and penicillin resulted in induction of all three strains to the L-form with frequencies up to 0.3%. At present no induction to the L-form has been achieved in the absence of albumin. Various lots of PVP proved toxic in the defined medium, and extensive dialysis was required for good growth and L-form induction. Substitution of PVP with sucrose indicated a sucrose toxicity for the parental gonococcus even on the addition of albumin. L-form induction did occur on sucrose L-medium but at significantly lower frequencies. The colonies appeared 1 week later than those on PVP L-medium but at significantly lower frequencies. The colonies appeared 1 week later than those on PVP L-medium and remained very small and poorly developed.     

Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: Application to studies of lymphoblast proliferation in vitro

Summary We have compared several methods for reducing calcium and magnesium concentrations in tissue culture medium, with the objective of producing selective deficiency effects on the growth of mouse (L5178Y) and human (P1R) lymphoblasts. In experiments in which calcium- and magnesium-“free” McCoy’s medium was supplemented with 15% horse or fetal calf serum, enough calcium and magnesium was provided by serum to support normal lymphoblast growth rate. Either dialysis or chelating-resin treatment of horse or fetal calf serum reduced calcium and magnesium contents approximately 100-fold. Use of dialyzed sera resulted in reduced growth rate, although in most cases the reduction in growth could be attributed to other effects of dialysis on serum, inasmuch as growth in those experiments was not restored to normal by the addition of calcium and magnesium to the medium. In contrast, the reduction of lymphoblast growth rate that occurred when resin-treated serum was used was always attributable to removal of calcium and magnesium, as normal growth always occurred in cultures to which calcium and magnesium were added. To demonstrate a growth-inhibiting effect on either mouse or human lymphoblasts by severe reduction of either calcium or magnesium in the presence of normal amounts of the alternative cation, it was necessary to (a) expose McCoy’s Ca−Mg-“free” medium to chelating-resin to reduce further the residual cation concentrations; (b) wash cells from stock cultures in a medium devoid of calcium and magnesium prior to inoculation into experimental cultures; (c) reduce the proportion of serum in the final medium from 15 to 5%; and (d) add 100 μM EGTA to cultures. Under these conditions, growth of both cell types was completely abolished in the presence of normal magnesium but in the absence of added calcium, and markedly reduced in the presence of normal calcium but in the absence of magnesium. These modifications did not compromise growth in cultures containing normal concentrations of both ions. This work has been supported by a Research Grant from the U.S. Public Health Service (CA 12790), from the Monroe County Cancer and Leukemia Association, and by a contract from the Atomic Energy Project at the University of Rochester, and has been assigned publication no. UR-3490-624.     

Quantitative estimation of newly synthesized radioactive proteins: use of minicolumns of antibody embedded in agarose gel or immobilized on Sepharose.

Immunochemical methods for the quantitative determination of radioactive proteins synthesized by chick embryo hepatocyte cultures are described. Cell culture medium containing secreted labeled serum proteins was concentrated and applied to agarose gels containing rabbit anti-chicken serum. Nonspecific binding in control gels was reduced to less than 2% of applied counts under conditions where more than 60% of the nondialyzable counts were precipitated in the presence of antibody. Labeled cytosol fructose-1,6-bisphosphatase (Fru-P₂ase) from cell sonicates was selectively adsorbed onto columns containing Sepharose-immobilized anti-chicken liver Fru-P₂ase. Radioactivity bound on these columns was eluted with 1% sodium dodecyl sulfate for electrophoretic analysis. Addition of dialyzed horse serum was used in both cases to minimize nonspecific binding. These techniques provide simple alternatives to direct immunoprecipitations in solution where nonspecific binding of radioactivity may be unacceptably high.     

Efficiency of procedures for induction and cultivation of Pseudomonas syringae pv. pisi L-form

The L-form of Pseudomonas syringae pv. phaseolicola has been proved to induce resistance to bean halo blight.Various procedures were tested to induce the L-form of Pseudomonas syringae pv. pisi for its potential use as biocontrol agent of pea bacterial blight. Cell-wall deficient cells were induced in a liquid medium with penicillin following a protocol described for P. s. pv. phaseolicola. Cell growth on solid induction medium developed as typical granular and vacuolated structures, and characteristic colonies were observed in the first transfer. However, there was poor growth in subsequent transfers and some reversion to the parental type. To improve the induction procedure, the following new procedures were applied: (1) viability of cells was monitored during induction. The optimum induction time in liquid medium with penicillin was lower for pv. pisi than for pv. phaseolicola. Viability of L-forms in solid induction medium with penicillin was low and decreased in time. (2) the inducer ticarcillin was combined with clavulanic acid, which prevented the reversion to the parental type and (3) a range of concentrations of penicillin and ticarcillin/clavulanic acid was applied by the spiral gradient endpoint method for calculation of minimum inhibitory concentrations (MIC). Based on the results from these tests an induction method for P. s. pv. pisi L-form is proposed and the relevance of L-form is discussed for practice.