Culture of Clostridium pasteurianum in Defined Medium and Growth as a Function of Sulfate Concentration

Clostridium pasteurianum strain W-5 was selected as an anaerobe which may be grown from large inocula in defined media with sulfate as its primary sulfur source. Since it is important to keep inocula small in minimizing transfer of sulfur sources, culture conditions were optimized. The medium devised decreased lag period and generation time when compared with other media, but growth could not be induced consistently with 6 x 10(6) cells per ml or less. Addition of trace elements, chelating agents, reducing agents, metabolites, and spent medium from various stages of growth did not stimulate growth from small inocula. Generation time was 85 min on inoculation with 10(7) or more cells per ml taken from young stocks, but the lag period decreased somewhat with larger inocula. On the other hand, generation time and lag period increased with age of the inoculum. The total yield of cells increased when buffer capacity was increased. Growth of C. pasteurianum W-5 was dependent upon sulfate at relatively low sulfate concentrations, and the organism is thus suitable for study of sulfur metabolism. No evidence of a maintenance requirement for sulfate was detected.     

Sucrose-mediated giant cell formation in the genus Neisseria.

Growth of Neisseria perflava, Neisseria cinerea, and Neisseria sicca strain Kirkland in media supplemented with sucrose (0.5 to 5.0% w/v) resulted in the formation of giant cells. Response to sucrose was specific in that a variety of other carbohydrates did not mediate giant cell formation. Giant cells appeared only under growth conditions and did not lyse upon transfer to medium lacking sucrose or upon resuspension in hypotonic media. Reversion of giant to normal cells occurred when giant cells were used as inocula and allowed to multiply in media lacking sucrose.     

Growth and Metabolism of L Cells in a Chemically Defined Medium in a Controlled Environment Culture System: I. Effects of O2 Tension on L-Cell Cultures

Six water-jacketed 500-ml Bellco spinner flasks were equipped to monitor and control environmental variables to study their effects on the growth and metabolism of mammalian cells. Studies with automated control of pO(2) levels of l-cell cultures, grown at pH 6.9 +/- 0.1, showed that dissolved O(2) tensions of ca. 9% were optimal for cell growth. At pO(2) values of 5 and 20%, maximum cell yields as well as growth rates were reduced by approximately 20%. Peak yields of L-cell cultures exceeded 5 x 10(6) cells/ml when grown for 4 days without medium renewal from inocula of ca. 10(6) cells/ml in a defined medium sparged with 5% CO(2) and maintained at 9% dissolved O(2) tension. The redox potentials of L-cell cultures reflected the pO(2) levels in the medium and ranged from -45 to +160 mv (versus calomel reference) for O(2) values ranging from 2 to 20% dissolved oxygen tension. Increased utilization of glucose per cell occurred in the presence of increased pO(2), whereas minimal accumulation of ammonia occurred with a pO(2) value maintained at 9%.     

Poliovirus and echovirus survival in Tetrahymena pyriformis culture in vivo

Axenic cultures of Tetrahymena pyriformis, strain I MT IV, grown in a defined medium at room temperature, were used to study interactions of these protozoa with vaccination strain L Sc 2ab of poliovirus type 1, vaccination strain P 712 of poliovirus type 2 and with type 30 echovirus, strain 480/78. T. pyriformis cultures in media containing 10(3.0) TCD50/1 ml of type poliovirus, 10(3.0) TCD50/1 ml of type 2 poliovirus or 10(2.5) TCD50/1 ml echovirus 30 and in virus-free medium did not differ one from another in their growth and die-away kinetics during the 21 days of observation. Two-day T. pyriformis cultures were infected with poliovirus 1 (initial concentration 10(3.2) TCD50/1 ml), and poliovirus 2 and echovirus 30 (initial concentrations 10(3.0) TCD50/1 ml). Viruses were titrated in test tube cultures of BGM cells. The supernatant fluid, standardized sediment and samples of control virus suspension free of protozoa were titrated after 0, 2, 6, 10, 13, 18, 28 and 30 days. Most of the virus in culture was found associated with the sediment, both in the period of active growth and during the die-away phase of T. pyriformis protozoa. The virus in sediment was present at higher titres and its survival time was longer than in virus in liquid phase. Thirteen days after the first contact between T. pyriformis and virus the sediment and supernatant fluid of the old protozoan culture and the T. pyriformis-free control viral suspension were taken and used as inocula for new two-day T. pyriformis cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cultivation of Parasitic Leptospires: Effect of Pyruvate

Sodium pyruvate (100 mug/ml) is a useful addition to the Tween 80-albumin medium for the cultivation of parasitic serotypes. It is most effective in promoting growth from small inocula and growth of the nutritionally fastidious serotypes.     

Dichloran-glycerol medium for enumeration of xerophilic fungi from low-moisture foods.

A low water activity (alpha omega) medium (0.95 alpha omega) containing 18% (wt/wt) glycerol and 2 micrograms of dichloran per ml was developed for enumerating the fungal flora of dried and semidried foods. The medium, designated DG18, was shown to be significantly better than Christensen malt salt agar when both media were tested with foodstuffs and with pure culture inocula. The need for a medium of reduced alpha omega for enumerating xerophilic fungi from low-moisture foods was demonstrated by comparing fungal counts obtained on both high-alpha omega and low-alpha omega media.     

Growth conditions required for bacteriocin production by strains of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The influence of growth parameters on the production of bacteriocins (aureocins) by five strains of Staphylococcus aureus was investigated. These aureocins have a broad spectrum of activity and can inhibit important human and animal pathogens. All strains produced large inhibition zones upon the indicator strain when they were grown in rich media such as brain-heart infusion (BHI), N2GT and 2 × YT. Bacteriocin production was not influenced by the initial pH of the medium (6.0–8.0). At lower temperature (28 °C), there was a marked reduction in bacteriocin production. Incubation of the producers under anaerobiosis affected profoundly the production of two related bacteriocins, aureocins MB92 and 146L, and slightly increased the production of aureocins A53 and 215FN. Production of aureocins MB92 and 215FN was apparently abolished in media containing 2.5 g and 3.5 g Nacl/100 ml, respectively. Although production of the remaining aureocins was observed in all NaCl concentrations tested (0.5–7.5 g/100 ml), the larger inhibition zones were detected in media containing up to either 1.5 g (for aureocins A70 and 146L) or 2.5 g NaCl/100 ml (for aureocin A53). Aureocin 215FN could not be detected in the culture supernatant. For the remaining aureocins tested, the highest levels of bacteriocin production occurred in either the late-log or early stationary growth phase of cultures grown in BHI medium at 37 °C. Changes in environmental conditions can, therefore, have detrimental effects on the production of active aureocins. Such factors are relevant when considering the potential biotechnological applications of these substances and when testing new S. aureus isolates for bacteriocin production.     

A Growth Initiation Factor for Host-Independent Derivatives of Bdellovibrio bacteriovorus

Host-independent (H-I) derivatives of Bdellovibrio bacteriovorus 109 Davis could not be isolated when concentrated suspensions of host-dependent (H-D) cultures, washed free of spent medium, were plated on host-free media. However, H-I colonies did appear when spent broth was incorporated into the isolation medium, indicating the presence of a factor in the spent medium essential for the growth of H-I cells. This growth factor (GIF) was also present in cell-free extracts of Escherichia coli and a variety of other microorganisms including H-D and H-I derivatives of strain 109 Davis. GIF was heat stable, non-dialyzable, and present in both soluble and particulate fractions of extracts. Heating of extracts at 70 C for 10 min resulted in 10- to 40-fold stimulation in GIF activity, and evidence for a heat-labile inhibitor was obtained. Colonies appearing on host-free medium in these experiments were shown to be those of typical H-I derivatives by isolation and subsequent host-independent cultivation of these organisms. GIF was a conditional requirement dependent on age and size of inoculum for all H-I derivatives characterized. Although GIF stimulated the growth of washed exponential phase cells transferred to fresh medium, it was not essential for growth. However, it was essential for the initiation of growth of washed stationary phase cells from small inocula transferred to fresh medium. It is proposed that GIF is required to initiate growth of metabolically quiescent cells.     

Microcycle Conidiation and Spore-Carrying Capacity of Colletotrichum gloeosporioides on Solid Media

The effect of spore inoculum density, medium concentration, and temperature on slime-spot formation, spore yield, and mycelium production by Colletotrichum gloeosporioides on agar media were studied with a simple microplate assay. A steady-state spore yield (spore-carrying capacity) independent of inoculum density was reached only on media that supported good fungal growth and sporulation. The spore-carrying capacity was reached earlier, the denser the inoculum. On standard mycological media a high inoculum density (2.5 × 10⁶ spores per ml) resulted in a slimy mass of conidia forming a slime spot, a phenomenon associated with greatly reduced mycelium formation and indicative of microcycle conidiation. In contrast, for a similar inoculum density, enhanced mycelial growth preceded sporulation and overrode slime-spot formation on highly concentrated media; a very low medium concentration resulted in much less mycelium, but spore production was also decreased. Exposure to suboptimal growth temperatures of 36 to 48°C for up to 8 days did not induce microcycle conidiation from inocula that did not form a slime spot at 28°C.     

Growth ofChlamydia psittaci strain menigopneumonitis in mouse L cells cultivated in a defined medium in spinner cultures

Summary L cells were grown in spinner cultures in a defined medium consisting of Waymouth medium MB752/1 (19) supplemented with 2 mg of fatty acid-free bovine serum albumin (BSA) per ml and 5 μg of oleate per ml (WO₅ medium). Growth in WO₅ medium was comparable to spinner L cell growth in two serum-containing media. The optimal concentration of oleate in the WO medium was 5 to 10 μg per ml. The use of 20 to 80 μg of oleate per ml of medium resulted in lower peak populations and earlier declines in viable cell counts. Cell death occurred rapidly in WO₁₆₀ medium. Cell growth in WO medium containing 5 to 80 μg of oleate per ml was well above the level of growth observed when no oleate was present in the medium. Since the total lipid and fatty acid compositions of the BSA used in this study have been characterized by the authors, the WO medium may be considered a defined medium. L cells have been continuously maintained in spinner cultures in WO₅ medium for over 50 passages with no major variation in the growth pattern. A 1000-fold increase inChlamydia psittaci strain meningopneumonitis, with a peak titer of 9.3×10⁷ plaque-forming units per ml, was observed when the chlamydial agents were grown in spinner L cells in WO₅ medium. This investigation was supported by Public Health Service Research Grant HE 08214 from the Program Projects Branch, Extramural Programs, National Heart and Lung Institute; The World Health Organization; and The Hormel Foundation.     

Isoniazid Uptake in Relation to Growth Inhibition of Mycobacterium tuberculosis

(14)C-isoniazid (INH) was used to study the relationship between drug uptake or binding by Mycobacterium tuberculosis and growth inhibition of the organism, which is dependent upon the concentration of drug and the duration of exposure. When strain H37R(a), grown in modified Sauton's liquid medium, was treated with 0.1 mug of INH per ml for 2 to 6 hr, followed by 10 mug of nicotinic hydrazide (NH) per ml to block further INH uptake, growth was retarded but not completely inhibited upon continued incubation. NH itself did not retard growth. However, cells treated in a similar manner with INH alone grew normally when diluted 1:100 in fresh, drug-free media. Uptake data showed that bacilli exposed to 0.1 mug of INH per ml accumulated 5.5, 9.7, and 12 mmug/mg of dry cells at 2, 4, and 6 hr, respectively. Other experiments suggested that once isoniazid is bound, it is not rapidly lost when NH is added or when the cells are diluted in fresh media. In the presence of 1.0 mug of INH per ml, tubercle bacilli took up 10 to 37 mmug/mg of dry cells in 20 to 90 min. These cells were not markedly inhibited when diluted 1:40 in fresh NH-containing media and incubated for 6 days. Growth inhibition of tubercle bacilli by INH depends on the uptake of sufficient drug, but the evidence obtained in this study suggests that the absolute concentration of bound INH is not as important in the action of the drug as is the maintenance of a critical cellular concentration for a requisite period of time.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO COPPER AND MERCURY SALTS

The susceptibility of Botrytis cinerea to copper sulphate in liquid media increased when the volume, and therefore the depth, of the medium in culture bottles exceeded certain values; when the volume was 40 ml. the maximum concentration allowing growth was 300 p.p.m.
By growing mycelium in media containing progressively higher concentrations of copper sulphate a strain was produced which grew at a concentration of 750 p.p.m.
In high concentrations of copper sulphate growth always started at the edge of the liquid, and inocula grew only if they were placed in this position.
In germination tests spores from the resistant strain were more resistant to copper sulphate than were spores of the parent strain.
The resistance of mycelium, and to a lesser extent of spores, was retained after growth of the resistant strain for six months in fungicide-free media.
Spore and mycelial inocula grew in much higher concentrations of copper sulphate when nutrient media were solidified with agar.
The strain resistant in liquid media was no more resistant than the parent strain on agar media.
The resistance of the fungus was not increased after growth for long periods on agar containing high concentrations of copper sulphate. The resistance of the strain resistant in liquid media was not lost after growth on agar media for 3 months.
Attempts to produce strains more resistant than the parent to mercuric chloride were unsuccessful.
The results obtained with phenyl-mercuric acetate were essentially similar to those obtained with copper sulphate, but relatively much more resistant strains were produced.     

Solute stresses affect growth patterns, endogenous water potentials and accumulation of sugars and sugar alcohols in cells of the biocontrol yeast Candida sake

AIM: To evaluate the effect of modifications of water activity (aw 0. 996-0.92) of a molasses medium with different solutes (glycerol, glucose, NaCl, proline or sorbitol) on growth, intracellular water potentials (psi(c)) and endogenous accumulation of polyols/sugars in the biocontrol yeast Candida sake. METHODS AND RESULTS: Modification of solute stress significantly influenced growth, psi(c) and accumulation of sugars (glucose/trehalose) and polyols (glycerol, erythritol, arabitol and mannitol) in the yeast cells. Regardless of the solute used to modify aw, growth was always decreased as water stress increased. Candida sake cells grew better in glycerol- and proline-amended media, but were sensitive to NaCl. The psi(c) measured using psychrometry showed a significant effect of solutes, aw and time. Cells from the 0.96 aw NaCl treatment presented the lowest psic value (- 5.20 MPa) while cells from unmodified media (aw = 0. 996) had the highest value (- 0.30 MPa). In unmodified medium, glycerol was the predominant reserve accumulated. Glycerol and arabitol were the major compounds accumulated in media modified with glucose or NaCl. In proline media, the concentration of arabitol increased. In glycerol- and sorbitol-amended media, the concentration of glycerol rose. Some correlations were obtained between compatible solutes and psi(c). CONCLUSIONS AND SIGNIFICANCE: This study demonstrates that subtle changes in physiological parameters significantly affect the endogenous contents of C. sake cells. It may be possible to utilize such physiological information to develop biocontrol inocula with improved quality.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5.2 with HCl and incubated at 30 degrees C, inocula containing less than 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give greater than 10(8) bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10-20 weeks. Citric acid concentrations of 10-50 mmol/l at pH 5.2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10(6). The citric acid also reduced the concentration of free Ca2+ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5.2 could be prevented by the addition of Ca2+ or Mg2+ and greatly reduced by Fe2+ and Mn2+. The addition of Ca2+, but not of the remaining divalent metal ions, restored the concentration of free Ca2+ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca2+. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5.2. The effect of citric acid and Ca2+ at pH 5.2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

Isolation of thecal cells: an assessment of purity and steroidogenic potential

In the present investigation, the responsiveness of rat thecal cells, prepared by means of an optimised discontinuous Percoll density gradient centrifugation procedure and cultured under serum-free cell culture conditions, to different concentrations of follitropin (FSH), basic fibroblast growth factor (FGF-2 or bFGF), and lutropin (LH) has been examined. The estradiol (E(2)) and progesterone (P(4)) contents of the cell culture medium were simultaneously determined with aliquots collected after different times of exposure to these regulatory proteins, either individually or in combination. The results confirm that no E(2) could be detected in the cell culture medium of the rat thecal cells prepared and cultured in this manner following all of these different treatments, and hence no contamination of the thecal cell preparations by granulosa cells was evident. The effects of FGF-2 and LH on the steroidogenic and cytodifferentiational properties of these rat thecal cells under serum-free cell culture procedures were also examined. The production of P(4) in the Percoll-purified rat thecal cell cultures receiving different treatments of FSH, and/or FGF-2 did not differ from the basal cell cultures, and no E(2) was detected from the same culture media. In contrast, LH (20 or 50 ng/ml) was found to enhance the production of P(4) (P<0.05) in the serum-free cell culture media. The stimulation of P(4) production was greater at higher LH concentration (50 ng/ml) (P<0.05). Concurrent treatment of LH (20 or 50 ng/ml) and FGF-2 (1-100 ng/ml) showed that FGF-2 inhibited the production of P(4) by LH-stimulated thecal cell cultures (P<0.05). The inhibition by FGF-2 was greater when LH was at a lower concentration (EC(50)<1 ng/ml at LH-20 ng/ml vs. EC(50)>1 ng/ml at LH-50 ng/ml). The results of the present study thus indicate that rat thecal cells isolated by this optimised Percoll density centrifugation procedure maintain a very high steroidogenic potential and specificity. Consistent with the absence of contaminating granulosa cells, these rat theca cell preparations do not respond to FSH treatment in terms of E(2) production. However, these rat theca cell preparations can be stimulated by LH to express their differentiated status in serum-free medium and respond to growth factors such as FGF-2.     

Effect of Type of Carbohydrate on Growth and Protein Synthesis by Tetrahymena pyriformis

SYNOPSIS. In chemically defined media at carbohydrate concentrations ≧ 0.5% (w/v) Tetrahymena pyriformis W multiplied more rapidly, developed larger cells, and achieved greater growth as measured by optical density when carbohydrate was provided as dextrin rather than glucose. In media containing 0.3 mg/ml of amino acid nitrogen, growth increased with glucose concentration from 0.1 to 1%, did not change significantly to 3%, and was sharply inhibited at higher glucose levels. With dextrin, maximum growth paralleled carbohydrate concentration from 0.1 to 3%. At higher N levels the inhibitory concentration of glucose was lowered, but growth in dextrin media was not affected except at N concentrations that were inhibitory independent of carbohydrate source. At 1% carbohydrate levels, total cell protein per ml of culture was 60% greater, protein per cell approximately 50% greater, and cells were 1.5 to 2 times larger in media with dextrin than with glucose. Comparable differences in protein synthesis were observed at 2% carbohydrate levels and efficiency of conversion of substrate-N to protein-N was greater in the medium with dextrin than glucose.
Growth as measured by optical density in media with 0.3 mg/ml of N and 1 or 2% (w/v) of dextrin was not significantly reduced by the simultaneous presence of 1 or 2% glucose. This observation appeared to negate osmotic pressure as an explanation of reduced growth in the presence of glucose. At higher osmolar concentrations osmotic pressure appeared to be a major determinant of overall growth but not of cell size.     

A fully defined, clear and protein-free liquid medium permitting dense growth of Neisseria gonorrhoeae from very low inocula

Neisseria gonorrhoeae is difficult to cultivate in liquid medium. Currently there are no liquid media, defined or undefined, that reliably permit growth of this bacterium from low inocula. Standard clinical laboratory broths may allow multiplication of some strains of gonococci from large inocula, but such media incorporate infusates, extracts or digests and are therefore undefined. In this study, 20 gonococci of ten auxotypes were tested in various experimental media in the development of an easily prepared chemically defined, clear and protein-free liquid medium. The final medium - GW medium - allowed the growth of three clinical isolates of gonococci from inocula of <10(3) CFU mL(-1) to >10(8) CFU mL(-1) by 24 h. None of four commercially-available broths (nutrient broth, brain heart infusion, tryptone soya broth, and Mueller-Hinton broth) tested in parallel reliably supported growth of these isolates to the same extent. GW medium should be useful for studies of the growth of gonococci under different conditions and, as the medium is clear and colorless, this can be monitored turbidometrically. GW medium may be suitable as a basal medium for biochemical identification tests, antimicrobial susceptibility determinations and antimicrobial synergy studies.     

Bovine colostrum or milk as a serum substitute for the cultivation of a mouse hybridoma

A mouse-mouse hybridoma was grown in serum-free medium supplemented with bovine milk or colostrum. Bovine colostrum supported growth of the hybridoma whereas bovine milk alone did not support cellular proliferation. For growth in medium supplemented with colostrum, the maximum cell concentration achieved was 1.4 x 10(6) cells/mL in 2.2% colostrum, which is 44% of that obtained in 9% serum. When cells were grown in media containing milk and low amounts of serum (<1%) the maximum cell concentration in 2.2% milk with 0.4% serum was 2 x 10(6) cells/ml, whereas it was only 0.2 x 10(6) cells/ml and 1.3 x 10(6) cells/ml in 2.2% milk alone and 0.4% serum alone, respectively. Similar behavior was observed for growth in media containing colostrum and low amounts of serum. The monoclonal antibody production in media containing combinations of serum and milk or colostrum was comparable to that obtained in media with higher serum concentrations. Experiments performed with conditioned media suggest that the rapid decrease in viability, after the maximum cell concentration has been reached, is partially due to the presence of some inhibitory components generated during the cell culture rather than due to depletion of some serum components.     

Induction of tryptophan oxygenase by dexamethasone in isolated hepatocytes. Dependence on composition of medium and pH.

Hepatocytes were isolated from perfused rat livers. 4 x 10-6 cells/ml were incubated at at 37 degrees C in different media in the absence and presence of a steroid hormone, dexamethasone phosphate (2 x 10-5 M). 1. Hormonal enzyme induction occurred in cells suspended in a simple salt medium, devoid of amino acids and macromolecules. This induction was completely blocked by addition of either actinomycin D (2 mu-g/ml) or cycloheximide (50 mu-g/ml). 2. Incubation of cells in media containing defatted albumin did not enhance hormonal enzyme induction, although disintegration of cells during incubation was reduced. Addition of a crude albumin fraction reduced tryptophan oxygenase induction and dextran completely blocked enzyme induction by dexamethasone. 3. An increase of dexamethasone concentration in the presence of albumin to 9 x 10-5 M was unable to raise enzyme induction further, and a still higher concentration of hormone, 3 x 10-4 M, resulted in reduced enzyme induction. 4. The hormonal induction of tryptophan oxygenase was most pronounced when the pH of the medium was between 7.0 and 7.6, with an optium at 7.3. No induction was found when the pH of the medium was either 6.6 or 7.8. The basal tryptophan oxygenase activity was much less influenced by similar pH variations. It is concluded that hepatocytes in suspension are able to carry out hormone-stimulated enzyme synthesis and that factors influencing this process may be studied under controlled conditions in such systems.