Growth of Peptococcus and Peptostreptococcus: effect of variations of culture media on efficiency of recovery

Reference strains and clinical isolates of Peptococcus and Peptostreptococcus spp. were evaluated for their growth response in supplemented thioglycolate-yeast extract media. Supplements used included various combinations of hemin, menadione, sodium bicarbonate, and Tween 80. Parallel studies were done to compare the efficiency of recovery of viable cells grown in thioglycolate-based media and Wilkins-Chalgren broth and agar. In addition, the effects of age of the medium and medium storage on viable cell yields for reference strains were determined. Reference strains grown in freshly prepared thioglycolate-yeast extract medium supplemented with sodium bicarbonate produced a 10-fold greater increase in the number of viable cells recovered after 24 h of incubation than did the same organisms cultivated in Wilkins-Chalgren medium. The efficiency of recovery of organisms when either mid-logarithmic- or mid-stationary-phase cells were used to prepare standardized inocula was similar. The results suggest that thioglycolate-yeast extract medium supplemented with sodium bicarbonate is more productive than Wilkins-Chalgren medium for the cultivation of anaerobic gram-positive cocci and may represent a suitable alternative for antimicrobial susceptibility testing of these organisms.     

Enrichment, Isolation, and Cultural Characteristics of Marine Strains of Clostridium botulinum Type C

Terrestrial strains of Clostridium botulinum type C, designated 468 and 571, were used to screen various media for growth and sporulation at 30 C. Of the various formulations tested, only egg meat medium fortified with 1% additions of yeast extract, ammonium sulfate, and glucose (FEM medium) gave good growth and satisfactory sporulation. FEM medium was used to recover four marine type C isolates from inshore sediments collected along the Atlantic, the Gulf of Mexico, and the Pacific coasts of the United States. The isolation techniques involved repeated transfer of cultures showing type C toxin in FEM medium and purification by a deep tube method. The medium used for purification was beef infusion-agar supplemented with 0.14% sodium bicarbonate and 0.1% l-cysteine hydrochloride. l-Cysteine was adopted in preference to sodium thioglycolate, because some lots of the latter were definitely inhibitory for growth. The addition of bicarbonate markedly increased viable spore counts of both the marine and terrestrial strains. Various cultural and biochemical characteristics of the marine and the terrestrial strains were compared. With the exception of some variations in their fermentation patterns, both groups showed similar characteristics. Of 23 fermentable compounds tested, the terrestrial strains attacked only glucose and mannose. The marine strains fermented glucose, mannose, galactose, and ribose actively; dextrin, inositol, maltose, and melibiose were weakly fermented.     

Recovery of spores of Clostridium botulinum in yeast extract agar and pork infusion agar after heat treatment.

Yeast extract agar, pork infusion agar, and modifications of these media were used to recover heated Clostridium botulinum spores. The D- and z-values were determined. Two type A strains and one type B strain of C. botulinum were studied. In all cases the D-values were largest when the spores were recovered in yeast extract agar, compared to the D-values for spores recovered in pork infusion agar. The z-values for strains 62A and A16037 were largest when the spores were recovered in pork infusion agar. The addition of sodium bicarbonate and sodium thioglycolate to pork infusion agar resulted in D-values for C. botulinum 62A spores similar to those for the same spores recovered in yeast extract agar. The results suggest that sodium bicarbonate and sodium thioglycolate should be added to recovery media for heated C. botulinum spores to obtain maximum plate counts.     

Recovery of spores of Clostridium botulinum in yeast extract agar and pork infusion agar after heat treatment.

Yeast extract agar, pork infusion agar, and modifications of these media were used to recover heated Clostridium botulinum spores. The D- and z-values were determined. Two type A strains and one type B strain of C. botulinum were studied. In all cases the D-values were largest when the spores were recovered in yeast extract agar, compared to the D-values for spores recovered in pork infusion agar. The z-values for strains 62A and A16037 were largest when the spores were recovered in pork infusion agar. The addition of sodium bicarbonate and sodium thioglycolate to pork infusion agar resulted in D-values for C. botulinum 62A spores similar to those for the same spores recovered in yeast extract agar. The results suggest that sodium bicarbonate and sodium thioglycolate should be added to recovery media for heated C. botulinum spores to obtain maximum plate counts.     

The inhibition of Staphylococcus hyicus on a selective medium for staphylococci

Growth of Staphylococcus hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains was found to be severely inhibited when broth cultures of these organisms were streaked on Schleifer and Kramer's staphylococcal (SK) medium. Of the selective agents contained in SK medium, potassium thiocyanate was found to be inhibitory towards both subspecies of Staph. hyicus and sodium azide had an additional inhibitory effect on Staph. hyicus subsp. chromogenes. Of six different media supplements examined, sheep blood and Tween 80 were found to improve the growth of both Staph. hyicus subspecies when added to SK medium. These findings were confirmed in subsequent work where the supplemented SK media were used to isolate potential enterotoxin-producing organisms from simulated raw milk (Staph. aureus, Staph. hyicus subsp. hyicus, Staph. hyicus subsp. chromogenes, Staph. intermedius). SK medium supplemented with sheep blood proved more effective in allowing satisfactory recovery of Staph. aureus, Staph. hyicus subsp. hyicus and Staph. intermedius. However, neither supplement enabled satisfactory recovery of Staph. hyicus subsp. chromogenes to be achieved.     

The inhibition of Staphylococcus hyicus on a selective medium for staphylococci

Growth of Staphylococcus hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains was found to be severely inhibited when broth cultures of these organisms were streaked on Schleifer and Kramer's staphylococcal (SK) medium. Of the selective agents contained in SK medium, potassium thiocyanate was found to be inhibitory towards both subspecies of Staph. hyicus and sodium azide had an additional inhibitory effect on Staph. hyicus subsp. chromogenes. Of six different media supplements examined, sheep blood and Tween 80 were found to improve the growth of both Staph. hyicus subspecies when added to SK medium. These findings were confirmed in subsequent work where the supplemented SK media were used to isolate potential enterotoxin-producing organisms from simulated raw milk (Staph. aureus, Staph. hyicus subsp. hyicus, Staph. hyicus subsp. chromogenes, Staph. intermedius ). SK medium supplemented with sheep blood proved more effective in allowing satisfactory recovery of Staph. aureus, Staph. hyicus subsp. hyicus and Staph. intermedius. However, neither supplement enabled satisfactory recovery of Staph. hyicus subsp. chromogenes to be achieved.     

The Role of Carbon Dioxide as an Essential Nutrient for Six Permanent Strains of Fibroblasts

The results of these studies demonstrate that carbon dioxide is required for the growth and maintenance of strains of fibroblasts derived from human tissues, strains FS4-705 and U12-705, from mouse tissue, strain L-705, and from rabbit tissues, strains RM3-56, RS1-56, and RT-56 in a chemically defined medium containing phosphite buffer in place of bicarbonate and supplemented with dialyzed serum and dialyzed embryo extract. Under these conditions, the cells fail to proliferate at a significant rate and begin to degenerate within 5 to 10 days when the flasks are not stoppered. Sufficient carbon dioxide is produced by the cells to promote growth as indicated by the fact that maximal proliferation is obtained in the same phosphite media when stoppered flasks are employed. With the exception of RS1-56, all the remaining strains tested can be propagated serially in open flasks containing phosphite medium prepared with whole serum and embryo extract. The rate of growth under these conditions, however, is only one-half to one-third that obtained in stoppered flasks containing phosphite medium or the conventional bicarbonate medium.     

Nutritionally variant streptococci from patients with endocarditis: growth parameters in a semisynthetic medium and demonstration of a chromophore.

Nutritionally variant streptococci have been characterized in the past by their growth as satellite colonies and by their nutrient requirements of cysteine or vitamin B6 for growth in complex media. To further understand the growth characteristics of these strains, we studied fresh isolates from patients with endocarditis by using chemically defined medium enriched with 2% Todd-Hewitt dialysate. Under anaerobic conditions, growth yields of the strains in this medium were comparable to those obtained from a complex medium supplemented with vitamin B6, whereas under aerobic conditions, most of the strains had higher growth yields in the semisynthetic medium. Furthermore, the requirement for cysteine and vitamin B6 in the semisynthetic medium was no greater than that of other Streptococcus species. Electron microscopic studies demonstrated normal cell wall structures in organisms grown in the semisynthetic medium as compared with abnormal and irregular cell wall thickening in organisms grown in supplemented complex medium. Finally, these strains appeared to contain a common component when grown in the semisynthetic medium as demonstrated by the appearance of a chromophore after boiling the bacteria at pH 2. Therefore, the demonstration of a medium which permits adequate growth with a normal ultrastructure of nutritionally variant streptococci will permit the further study of this group of important streptococci.     

Isolation and identification of fecal bacteria from adult swine.

An examination of the fecal microflora of adult swine was made with regard to the efficiency of several roll tube media in enumeration and recovery of anaerobes, the effects of medium constituents on recovery, and the isolation and identification of the predominant kinds of bacteria. Total number of organisms by microscopic bacterial counts varied among fecal samples from 4.48 X 10(10) to 7.40 X 10(10) bacteria/g (wet weight). Comparison of different nonselective roll tube media indicated that about 30% of the fecal bacteria could be recovered with a rumen fluid (40%, vol/vol) medium (M98-5). Recoveries of 21 and 15%, respectively, were obtained with M10 and rumen fluid-glucose-cellobiose agar (RGCA) media. Rumen fluid, Trypticase, sugars, and CO2 gas phase were important components required for maximum recovery with this medium. Similar high recoveries of anaerobes were also obtained with M98-5 containing swine cecal extract of place in rumen fluid or M10 plus swine cecal extract. Significantly lower recoveries were observed with RCGA, media supplemented with swine fecal extracts, reinforced clostridial medium, brain heart infusion agar, and prereduced blood agar. Ninety percent of the bacteria isolated from roll tube media were gram positive and consisted of facultatively anaerobic streptococci, Eubacterium sp., Clostridium sp., and Propionibacterium acnes. The remainder of the flora (8%) included several other species of anaerobes and Escherichia coli. Rumen fluid (or volatile fatty acids), Trypticase, and yeast extract additions to basal media stimulated the growth of anaerobic strains. Variation in the relative proportions of the predominant fecal microflora was observed. This work indicates that satisfactory enumeration, isolation and cultivation of the predominant microflora in swine feces can be obtained when strict anaerobic culture methods and a rumen fluid medium are used.     

Conventional laboratory agar media provide an iron-limited environment for bacterial growth

Abstract The outer membrane proteins of Escherichia coli and Pseudomonas aeruginosa grown in a number of conventional laboratory media were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) High-molecular-weight proteins similar to those produced by these strains in an iron-limited chemically defined medium were detected in cells grown on the surface of various agar media. In contrast, these proteins were not produced or were only poorly expressed by the corresponding broth cultures or by cells grown an agar supplemented with iron. A catecholic substance could be detected in DST agar extracts subsequent to bacterial growth which was produced to a lesser extent in IST agar and in broth cultures.     

Clear Media for the Recovery of Pasteurella tularensis

Peptic digests of blood and hemoglobin were investigated as substitutes for the blood used in the preparation of glucose-cysteine-blood agar plating medium for the recovery of the virulent Schu strain of Pasteurella tularensis. Digest media so prepared were found to be satisfactory for the quantitative recovery of freshly grown cells but not for cells stored longer than several days. The addition of appropriate quantities of human plasma, bovine sera, or soluble starch rendered the digest media appropriate for use with stored cultures. The peptic digest-plasma (PDP) and peptic digest-starch (PDS) media were evaluated and found satisfactory for the quantitative recovery of P. tularensis Schu from freshly prepared and stored cultures, and from aerosols produced therefrom. With cultures stored longer than 6 weeks, the starch modification (PDS) was not as satisfactory as, and the plasma variation (PDP) was better than, glucose-cysteine-blood agar (GCBA) for the recovery of the organisms. PDP was superior to either GCBA or PDS medium for the recovery of the weakly virulent Jap 4 and Niieg-blue strains of P. tularensis.     

Effect of pH on viability of Entodinium caudatum, Entodinium exiguum, Epidinium caudatum, and Ophryoscolex purkynjei in vitro

Cultures of Entodinium caudatum, Entodinium exiguum, Epidinium caudatum, and Ophryoscolex purkynjei were grown and transferred in poorly buffered media prepared using different concentrations of sodium bicarbonate and a nitrogen gas phase. By transferring every 12 or 24 h, culture pH was gradually decreased until the protozoa disappeared. The cultures were transferred by placing half of the culture into an equal volume of fresh medium, resulting in pH fluctuations similar to those in the rumen, resulting from fermentation, eating, and saliva production. All four species appeared to maintain their concentrations around pH 5.8, but numbers decreased as pH values fell below 5.6. The four species were similar in that they all survived above pH 5.3. These results differ from previous reports in which Entodinium species appeared to be more tolerant to low pH than all other species of rumen ciliates. No adaptation to low pH was observed in Epidinium caudatum cultures after recovery from pH 5.4 medium containing only one or two viable cells.     

Chemically defined and minimal media forBacteroides gingivalis

A chemically defined medium (BGDM) has been developed specifically forBacteroides gingivalis. The medium contains 4 amino acids, 5 mineral salts, cysteine hydrochloride as a reducing agent, and the growth factors hemin and menadione. Eight strains ofB. gingivalis have been subcultured repeatedly in this medium with no apparent changes in colonial or cellular morphology. The metabolic end products of strains grown in this medium were reproducible and yielded patterns similar to those produced by cells cultured in complex media. The growth rates were about 50% slower than those of cells grown in a complex medium, and the growth rate constants ranged between 0.013 and 0.067 H^–1. When the defined medium was supplemented with protein hydrolysates such as trypticase, proteose peptone, bactocasitone, or yeast extract, at concentrations up to 1.0%, growth increased. No such growth increase was observed in the medium supplemented with casamino acids. Thus a minimal medium can be formulated by adding one of the growth-enhancing protein hydrolysates to the defined medium at varying concentrations depending upon the growth yield required.     

Enhanced recovery of injured Escherichia coli by compounds that degrade hydrogen peroxide or block its formation

Escherichia coli LSUFS was injured either by freezing at -10 degrees C or by heating at 57 degrees C for 12 min. Surviving cells were recovered on nonselective tryptone-glucose extract agar and selective violet red bile agar supplemented with compounds that degrade hydrogen peroxide or block its formation. Various concentrations of the following compounds were tested: sodium pyruvate, 3,3'-thiodipropionic acid, catalase, ascorbic acid, potassium permanganate, sodium thioglycolate, dimethylsulfoxide, ethoxyquin, n-propyl gallate, alpha-tocopherol sodium metabisulfite, and ferrous sulfate. Sodium pyruvate and 3,3'-thiodipropionic acid, when added to either medium, significantly (P greater than 0.01) increased recovery of injured cells. More than 90% of the heat-injured cells and 40 to 90% of the freeze-injured cells failed to grow on unsupplemented tryptone-glucose extract agar. Supplementation of violet red bile agar increased recovery, but the counts remained considerably lower than the tryptone-glucose extract agar counts. The repair detection procedure of Speck et al. (M. Speck, B. Ray, R. Read, Jr., Appl. Microbiol. 29:549-550, 1975) was greatly improved by the addition of pyruvate or 3,3'-thiodipropionic acid. However, when this improved repair detection procedure was applied to foods, pyruvate-supplemented media showed some false-positives. We therefore recommend that 3,3'-thiodipropionic acid be used to supplement media in the repair detection procedure.     

Chemotrophic growth of heliobacteria in darkness

Abstract Conditions are described that support anaerobic dark chemotrophic growth of heliobacteria. Growth was pyruvate-dependent and was best in well-buffered pyruvate media supplemented with yeast extract. Heliobacterial cells grown in darkness synthesized bacteriochlorophyll g and carotenoids and fermented pyruvate to acidic products, CO₂, and in some cases, H₂. All recognized species of heliobacteria as well as several newly isolated strains were capable of dark anaerobic growth, suggesting that this growth mode may be ecologically important for survival of these organisms in their soil habitat.     

Evaluation of liquid and solid culture media for the recovery and enrichment of Burkholderia cenocepacia from distilled water

Burkholderia cepacia complex (BCC) presence has been the cause of recalls of both sterile and non-sterile pharmaceutical products since these opportunistic pathogens have been implicated to cause infections to susceptible individuals. BCC are ubiquitous in nature, but in pharmaceutical settings the most common source is contaminated water systems. Some strains of BCC, previously described as Pseudomonas cepacia, were not readily detected by standard culture methods. We have explored different strategies to recover and enrich Burkholderia cenocepacia previously cultured in distilled water for 40 days. Enrichment media of varied nutrient concentrations and composition were used, including modified Tryptic Soy Agar or Broth (TSA or TSB), Reasoner’s 2nd Agar or Broth (R2A or R2AB), Brain–Heart Infusion Broth (BHIB), Mueller–Hinton Broth (MHB), and Ashdown’s (ASH) medium. Of the various broth media tested, cell growth was significantly greater in TSB and R2AB than in BHIB, MHB, or ASH broth. TSB and R2AB were also compared for their recovery efficiency. Generally, there was no significant difference between the numbers of B. cenocepacia grown on 15 differently modified TSA and five modified R2A solid media. Overall, however, diluted TSA and TSB media, and R2A and R2AB showed better recovery efficiency than TSA and TSB for inocula containing small numbers of cells. All strains persisted in distilled water for 40 days. Broth media were more effective than solid media for recovery of B. cenocepacia from distilled water. These results may assist in improving detection assays with recovery and enrichment strategies to maximize recovery of these fastidious organisms.     

Culture of Ethiopian Strains of Borrelia recurrentis

A number of standard bacteriological media with supplements were tested for their ability to support in vitro growth of Ethiopian strains of Borrelia recurrentis. Propagation of 18 out of 21 strains occurred in Trypticase soy yeast broth to which bovine albumin (fraction V), N-acetyl glucosamine, and sodium pyruvate had been added. This medium supported a population of 10(7) organisms per ml and yielded a harvest of four to five times the original inoculum during the logarithmic phase of growth. Maximal yield varied from 1.4 x 10(7) to 3.4 x 10(7) organisms per ml. Generation time in optimal media was 11.3 h. Lesser multiplication of organisms occurred in other media tested. Strains from primary cultures were infective for the green monkey. Recovery of viable organisms from subculture has not been successful.     

Carbon metabolism regulates expression of the pfl (pyruvate formate-lyase) gene in Escherichia coli.

The anaerobic expression of pfl is reduced both in a strain mutated in the pgi gene and in a pfkA pfkB double mutant strain when cells are grown in medium supplemented with glucose. When cells are grown in medium supplemented with either fructose or pyruvate, no reduction is observed in these strains. The amount of pyruvate in the cells may be responsible for the reduced expression of pfl in the strains mutated in the genes encoding the glycolytic enzymes. Because of the lowered oxygen concentration in the medium, the expression of pfl is induced when an exponentially growing culture enters the stationary phase. This induction is increased when the Casamino Acid concentration is raised 10-fold or when the medium is supplemented with NaCl. Superhelicity of DNA is decreased in a pgi mutant strain grown in medium supplemented with glucose. The superhelicity is also changed, but the opposite way, in a wild-type strain grown in medium supplemented with Casamino Acids at a high concentration or 0.3 M sodium chloride. Our data show that changes in superhelicity do not affect the aerobic expression of pfl but might be important for the anaerobic induction of pfl.     

Hypothalamic Extract Influences a Blood-Brain Barrier Model of Porcine Brain Capillary Endothelial Cells

The purpose of this study was to investigate the influence of hypothalamic extract, astrocyte co-culture, and astrocyte-conditioned medium on the barrier function of an in vitro model of the blood-brain barrier. Porcine brain capillary endothelial cells were grown on polycarbonate membranes suspended between two chambers of media, representing the capillary lumen and brain interstitium. Endothelial cells grown alone and cocultured with astrocytes were cultured in growth medium with or without 50 g/mL hypothalamic extract. An additional treatment consisted of endothelial cells cultured in growth medium that was first conditioned by astrocytes. Coculture consisted of a noncontact model with astrocytes attached to the bottom of the abluminal chamber. Barrier function of the endothelial cells was tested on days 1 through 9 post-seeding by measuring permeability to macromolecules (albumin) and small ions (electrical resistance). Resistance to the passage of macromolecules and small ions was greatest for endothelial cells grown without astro-cytes in growth medium supplemented with hypothalamic extract. This barrier was maximal during days 4 through 7 post-seeding and was significantly less permeable than the barrier formed by endothelial cells grown in un-supplemented growth medium, in coculture with astrocytes, or in astrocyte-conditioned medium. These results demonstrate that a noncontact coculture with astro-cytes did not enhance the integrity of this in vitro BBB model employing porcine brain capillary endothelial cells, but barrier function was increased when the model's medium was supplemented with hypothalamic extract.     

Three-dimensional culture of human tumor cells under standardized medium conditions

The 3-dimensional culture of human tumor spheroids under standardized medium conditions may reveal information on specific biological parameters that could be masked in serum-supplemented media. Spheroids derived from human tumor cells are growth retarded in media free of serum. Ex-Cyte IV is a substance derived from human blood that can be used to improve growth in tissue culture. In this study the growth of spheroids from four different human tumor cell lines was studied when grown in medium free of serum, medium supplemented with varying concentrations Ex-Cyte IV, and medium supplemented with foetal calf serum (FCS). The parameters used for comparisons were growth rate, growth enhancement, clonogenicity and cell cycle distribution.The four cell lines showed different growth rates in serum-free medium, which were increased to different extents when Ex-Cyte IV or FCS were added. The growth enhancing effect induced by Ex-Cyte IV was differently concentration dependent for each cell line. The clonogenicity of cells grown as spheroids in serum-free medium was lower than in spheroids grown in supplemented media. There was no difference in clonogenicity between the differently supplemented media. All four cell lines responded to growth in serum-free medium with a drop in the S-phase and G2M phase.The present study provides a novel approach to the study of human tumor cells in 3-dimensional culture under defined conditions. The human serum derived substance Ex-Cyte IV may provide a method to obtain information on specific biological parameters that could be masked in serum-supplemented media.