Beef Heart Infusion Research Articles

Community-Associated Methicillin-Resistant Staphylococcus aureus Survival on Artificial Turf Substrates

Artificial turf has been suggested as a risk factor for community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). This is an experimental study looking at survival of CA-MRSA on artificial turf. MRSA strain USA-300-0114 was grown as either planktonic cells or biofilms in liquid cultures of beef heart infusion broth overnight at 37 °C. Beakers containing ProGrass (Pittsburgh, PA) turf were inoculated at the dirt interface with either ∼5 × 10 planktonic bacteria or with biofilms. The inoculum included varying nutrient conditions consisting of spent medium, saline, or 5% mucin. The beakers were incubated at 37 °C in ambient air. The main outcome measure was the number of surviving colony-forming units determined by plating on mannitol salt agar. Survival was biphasic with a colony-forming unit drop from ∼5 × 10 to ∼5 × 10 after the first week followed by survival of between 10 and 10 bacteria until termination of the experiment (20-50 d). Survival was dependent on nutrients, and washed cells survived less than 1 d. Mucin could serve as a nutrient source and slightly increased surviving numbers to 10-10 bacteria. Biofilm formation did not influence survival. CA-MRSA survivability on artificial turf surfaces is dependent on the availability of nutrients. These results suggest that CA-MRSA could survive on artificial turf in significant numbers for 1 wk, and lower numbers for at least 1 month, if supplied with appropriate nutrients. Outdoor environmental conditions may affect these findings.

Antimicrobial Properties of Milkfat Globule Membrane Fractions

Milkfat globule membranes (MFGMs) were prepared from bovine cream according to standard procedures. These membranes and peptide hydrolysates, which were generated by proteolysis with immobilized digestive enzymes, were screened for antibacterial activity against Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica Typhimurium, Pseudomonas fluorescens, Bacillus cereus, Lactobacillus acidophilus, and Lactobacillus gasseri. Assays were first performed on beef heart infusion (BHI) plates spotted with test protein-peptide fractions and then seeded with lawns of indicator cells to monitor the zone of growth inhibition. Under these experimental conditions, MFGMs were most active against Salmonella Typhimurium and P. fluorescens. However, antibacterial activity was not seen after plating on Luria-Bertani (LB) medium. We determined that the antimicrobial effects observed on BHI plates were due to the generation of H2O2 by xanthine oxidase, a major protein constituent of the MFGMs, as a result of purine catalysis. This substrate is present in BHI but lacking in LB medium. Evaluation of purified xanthine oxidase alone resulted in analogous data trends. The growth of probiotic Lactobacillus strains were affected only marginally when grown on lactobacilli deMan Rogosa Sharpe plates, suggesting the decreased sensitivity of these bacteria to H2O2. In this study, several MFGM hydrolysates exhibited variable antibacterial activity against test food pathogens on agar plates prepared with M9 minimal media, and this variation was not attributable to xanthine oxidase enzymatic activity. The probiotic microorganisms were mostly resilient to these antibacterial fractions. Bovine MFGM fractions may represent an excellent resource material from which to generate native, naturally occurring biodefensive proteins and/or peptides.

Menstrum for culture preservation and medium for seed preparation in a tetanus toxin production process containing no animal or dairy products

To completely eliminate animal and dairy products from the lyophilization menstrum and the seed medium used to produce tetanus toxin with Clostridium tetani. Tetanus toxin production in a recently developed fermentation medium lacking animal and dairy products was studied with different seed media. It was found that soy peptone could completely replace the beef heart infusion plus animal peptone previously used as seed medium. In addition, we found that cells lyophilized in soy milk could replace the usual type of cells lyophilized in cow's milk. We have now developed a complete tetanus toxin production process containing no animal and dairy products. Toxoid preparations made from toxin produced with animal and dairy products can contain undesirable contaminants such as the prion causing bovine spongiform encephalopathy (Mad Cow's Disease) or antigenic peptides that stimulate anaphylactic reactions and other undesirable immune reactions in immunized hosts. The new vegetable-based process described here avoids such unfortunate possibilities.

Effective levels of tetanus toxin can be made in a production medium totally lacking both animal (e.g., brain heart infusion) and dairy proteins or digests (e.g., casein hydrolysates)

We have developed a fermentation medium for Clostridium tetani that results in the formation of tetanus toxin and contains no meat (e.g., beef heart infusion) or dairy (e.g., casein digest) products, thus obviating the problem of possible prion diseases. Particular preparations of hydrolyzed soy proteins, especially Quest Hy-Soy ®, have been found to replace both the meat extract and casein digest components of traditional tetanus toxin production media and to yield even higher toxin titers. The comparison of the traditional versus the new medium has been carried out repeatedly by us and the superiority of our medium has been consistently observed. To our knowledge, this is the first time that such a medium has been devised.

Method for sampling bacteria of human dental enamel lesions

A method is described for the isolation, identification and frequency evaluation of bacteria inside primary enamel lesions (PEL) of freshly extracted human molars. PEL were localized by transillumination. Molar surfaces were sterilized by ultraviolet light. The PEL and the sound enamel controls were aseptically excavated, cultivated in beef-heart infusion medium and the resulting bacteria were identified by biochemical and gas-chromatographic methods. One hundred and four PEL were studied. Four bacterial genera and eight species were found. The genus Streptococcus displayed the highest frequency, being present in 56.2% of PEL. Actinomyces naeslundii was the most frequent species being present in 25% of PEL followed by S. sanguis 21.9% and S. mitis 18.7%.

Binding of Todd-Hewitt broth antigens by Streptococcus mutans

Streptococcus mutans 10449, grown in chemically defined culture medium, was tested for its ability to bind 3H-labeled Todd-Hewitt broth components (greater than 12,000 Mr). Maximum adsorption of radioactivity occurred within 5 min at room temperature, and cell-bound material was not completely removed by extended washing with buffer. Heat-killed, arsenate-inhibited, and viable bacteria bound similar quantities. Only 0.09% of the radioactivity in the preparation of high Mr Todd-Hewitt broth components was removed by absorption with excess numbers of S. mutans 10449 cells. Binding followed saturation kinetics and was competitively inhibited by unlabeled medium components, both the dialyzable and nondialyzable fractions. Other oral streptococci were also found to bind these complex medium components. Rabbit antiserum elicited to the high-molecular-weight Todd-Hewitt broth components reacted with monkey cardiac muscle and with S. mutans coated with medium components. Absorption of the anti-Todd-Hewitt broth serum with homogenized heart removed antibodies that reacted with Todd-Hewitt broth-coated S. mutans. Therefore, the tissue-specific antigens of this beef heart infusion medium that adsorb to S. mutans can interfere with the detection and characterization of antigens shared by these bacteria and animal tissues.

GROWING SPORES OF P.A. 3679 IN FORMULATIONS OF BEEF HEART INFUSION BROTH

ABSTRACTSpores of the putrefactive anaerobe (P.A.) 3679 are widely used in inoculated pack studies designed to evaluate the adequacy of heat processes for low acid foods. For this purpose, it is essential to have the ability to repeatedly grow large amounts (> 106) of test spores with comparable heat resistance. Beef heart infusion broth fortified with 1.0% tryptone, 1.0% gelatin, 0.05% glucose, 0.4% dibasic Potassium phosphate, 0.3% sodium citrate, and either 0.5% isoelectric casein or 1.5% skim milk powder, in the presence of beef heart particles was found to be dependable for producing > 109 heat resistant spores/liter medium when incubated 2 wk at 30°C followed by 1 wk at 20°C. With the beef heart debris either remaining or absent after two washing, the heat resistance of the spores did not change during 1 yr of storage at 4°C.

The influence of acetylsalicylic acid, phenylbutazone, indomethacin, and flufenamic acid on the kinetics of leucocytes during acute inflammation.

Five groups of ten male Wistar rats each were used for the purpose of this experiment. All subjects suffered from sterile acute inflammation in the peritoneal cavity. A sterile beef heart infusion broth reinforced with proteose-peptone was injected to cause the inflammation. The following substances mixed with tragacanth gum were given orally at in 12 h intervals: --Acetalsalicylic acid 150 mg/kg body weight to the first group; --Phenylbutazone 100 mg/kg body weight to the second group; --Indomethacin 15 mg/kg body weight to the third group; --Flufenamic acid 100 mg/kg body weight to the fourth group. Finally the fifth group received only tragacanth gum. The rats were sacrificed after 48 hours and leucocytes were collected from the peritoneal cavity by washing with 5 ml 0.9% NaCl solution. The total leucocyte number and the percent of each type were estimated for each group individually. A statistically significant decrease in the number of leucocytes migrated to the peritoneal cavity due to the inflammation was observed in rats that received one of the drugs (group I to IV) when compared to rats that received only the excipient (group V). Likewise, significant differences were noted in the composition of each type.

Growth of parasitic Mycoplasma without serum of serum fraction.

Lipid-free beef heart infusion would support the growth of six strains of parasitic Mycoplasma when supplemented with a combination of cholesterol, lecithin, and cardiolipin.

Recovery patterns of spores of putrefactive anaerobe 3679 in various subculture media after heat treatment.

A comparative study was made of the heat resistance of spores of putrefactive anaerobe 3679 grown in two different sporulation media and of the recovery pattern of these spores in several subculturing media after treatment with moist and dry heat. The heat resistance of the spores was characterized in the form of D and z values. The D values were determined by the modified Schmidt method. The z values were established by the graphic method. The results revealed significant differences in D and z values, depending on the type of heat and sporulation and subculture media. Spores grown in beef heart infusion showed higher heat resistance than those grown in Trypticase. Among the seven subculture media used, the largest number of spores was recovered in beef infusion. The magnitude of the D values at 121.1 C obtained with spores heated in moist heat decreased, depending on the subculture medium used, in the following order: beef infusion, pea infusion, yeast extract, liver infusion, Eugonbroth, Trypticase, synthetic medium. With spores subjected to dry heat, D values at 148.9 C decreased with the subculture medium in the following order: beef infusion, yeast extract, pea infusion and liver infusion, Trypticase, Eugonbroth, synthetic medium. The z values obtained with spores subjected to dry heat were approximately double those obtained with moist heat. Their relative magnitude varied slightly, depending on the type of subculture medium used. However, the relative magnitudes of the D values and z values with reference to the subculture media used were different with moist heat from those obtained with dry heat. Two theories are discussed as possible explanations for the logarithmic order of death of bacterial spores. The results obtained in these experiments, together with the findings of other workers, are most compatible with the theory that heat treatment of spores results in an increased rate of random injury to the genetic material of the spores.

Recovery Patterns of Spores of Putrefactive Anaerobe 3679 in Various Subculture Media after Heat Treatment1

A comparative study was made of the heat resistance of spores of putrefactive anaerobe 3679 grown in two different sporulation media and of the recovery pattern of these spores in several subculturing media after treatment with moist and dry heat. The heat resistance of the spores was characterized in the form of D and z values. The D values were determined by the modified Schmidt method. The z values were established by the graphic method. The results revealed significant differences in D and z values, depending on the type of heat and sporulation and subculture media. Spores grown in beef heart infusion showed higher heat resistance than those grown in Trypticase. Among the seven subculture media used, the largest number of spores was recovered in beef infusion. The magnitude of the D values at 121.1 C obtained with spores heated in moist heat decreased, depending on the subculture medium used, in the following order: beef infusion, pea infusion, yeast extract, liver infusion, Eugonbroth, Trypticase, synthetic medium. With spores subjected to dry heat, D values at 148.9 C decreased with the subculture medium in the following order: beef infusion, yeast extract, pea infusion and liver infusion, Trypticase, Eugonbroth, synthetic medium. The z values obtained with spores subjected to dry heat were approximately double those obtained with moist heat. Their relative magnitude varied slightly, depending on the type of subculture medium used. However, the relative magnitudes of the D values and z values with reference to the subculture media used were different with moist heat from those obtained with dry heat. Two theories are discussed as possible explanations for the logarithmic order of death of bacterial spores. The results obtained in these experiments, together with the findings of other workers, are most compatible with the theory that heat treatment of spores results in an increased rate of random injury to the genetic material of the spores.

Cellular proteolytic enzymes of Penicillium cyaneo-fulvum.

Soluble cellular fractions of surface and submerged cultures of Penicillium cyaneo-fulvum, grown in beef heart infusion (BHI) medium supplemented with glucose, showed well-defined fluctuations in proteolytic activity. Four proteases were shown to occur in the cells, and fluctuations in protease specific activity were considered to be related to the appearance and disappearance of individual enzymes. Synthesis of these enzymes seemed to be regulated by the pH of the external medium.Several proteases also occurred in filtrates of cultures grown in BHI–glucose medium. A comparison of intra- and extra-cellular alkaline proteases (optima, pH 10) showed that, although they were similar in many respects, the enzymes could be distinguished by their action on polyamino acids. Inability to detect the extracellular alkaline protease in cell preparations suggested that this enzyme was released into the medium as rapidly as it was produced.

Production and characteristics of hemolysins of Escherichia coli.

Snyder, Irvin S. (University of Iowa, Iowa City), and Nancy A. Koch. Production and characteristics of hemolysins of Escherichia coli. J. Bacteriol. 91:763-767. 1996.-Filterable and nonfilterable hemolysins were produced by Escherichia coli in beef-heart infusion, casein hydrolysate, and chemically defined media. Differences were found among the three media in the time of appearance and in the relationship between production of these two hemolysins. The nonfilterable hemolysin produced in the three media, as well as the filterable hemolysin produced in the beef-heart infusion medium, were destroyed in 1 hr at 56 C. The filterable hemolysin produced in the casein hydrolysate and chemically defined media was unaffected by exposure to 56 C for 1 hr. Formalin inactivated the hemolysins produced in all three media. The optimal pH for activity of the nonfilterable hemolysin varied with time of production, whereas the optimal pH for the filterable hemolysin was constant. Certain carbohydrates were required for the production of filterable hemolysin by E. coli grown in casein hydrolysate and chemically defined media.

Comparative effects of beta-propiolactone on mice, mouse-derived cell cultures, and Venezuelan equine encephalomyelitis virus.

Studies were made comparing the toxicity of beta-propiolactone (BPL) for mammalian (mouse) cells in vitro and for mice and for Venezuelan equine encephalomyelitis (VEE) virus which is highly cytopathogenic for each. The mammalian cells grown in tissue culture were found to be adversely affected by BPL in concentrations ranging from 0.001 to 0.1 mg/ml of supernatant fluid. The difference in response was influenced by the menstruum in which the BPL was suspended and the difference in cell types tested. Tenfold less BPL appeared to be required to destroy the cells when it was suspended in a balanced salt solution than when it was suspended in protein-containing solutions such as beef heart infusion broth or medium 199 plus 20% horse serum. Secondary embryonic mouse lung cells seemed slightly more adversely affected by BPL than the established embryonic lung or L cells. BPL given to mice by intranasal instillation and by intracerebral injection was lethal to half of the animals within 2 days at doses of 0.31 and 0.39 mg, respectively. Higher concentrations of BPL were required to rapidly inactivate the virus in vitro than were required to kill mice or to cause a toxic effect on cells in culture. It required 10 mg/ml of BPL to completely inactivate a high-titered VEE virus preparation in 5 min and 1 mg/ml to inactivate most, but not all, of the virus in 15 min. A concentration of 0.1 mg/ml of BPL had only a slight effect on the virus after a period as long as 60 min. Evidence is presented indicating that simultaneous inactivation of all of the properties of the VEE virus particles by BPL aerosols did not occur at the same time but that, after treatment, the virus possessed a limited ability to immunize mice despite a loss in infectivity.

Comparative Effects of β-Propiolactone on Mice, Mouse-derived Cell Cultures, and Venezuelan Equine Encephalomyelitis Virus

Studies were made comparing the toxicity of beta-propiolactone (BPL) for mammalian (mouse) cells in vitro and for mice and for Venezuelan equine encephalomyelitis (VEE) virus which is highly cytopathogenic for each. The mammalian cells grown in tissue culture were found to be adversely affected by BPL in concentrations ranging from 0.001 to 0.1 mg/ml of supernatant fluid. The difference in response was influenced by the menstruum in which the BPL was suspended and the difference in cell types tested. Tenfold less BPL appeared to be required to destroy the cells when it was suspended in a balanced salt solution than when it was suspended in protein-containing solutions such as beef heart infusion broth or medium 199 plus 20% horse serum. Secondary embryonic mouse lung cells seemed slightly more adversely affected by BPL than the established embryonic lung or L cells. BPL given to mice by intranasal instillation and by intracerebral injection was lethal to half of the animals within 2 days at doses of 0.31 and 0.39 mg, respectively. Higher concentrations of BPL were required to rapidly inactivate the virus in vitro than were required to kill mice or to cause a toxic effect on cells in culture. It required 10 mg/ml of BPL to completely inactivate a high-titered VEE virus preparation in 5 min and 1 mg/ml to inactivate most, but not all, of the virus in 15 min. A concentration of 0.1 mg/ml of BPL had only a slight effect on the virus after a period as long as 60 min. Evidence is presented indicating that simultaneous inactivation of all of the properties of the VEE virus particles by BPL aerosols did not occur at the same time but that, after treatment, the virus possessed a limited ability to immunize mice despite a loss in infectivity.

Cultivation of Mycoplasma of Avian Origin

N GENERAL, members of the genus Mycoplasma of animal origin have strict growth requirements. The basal media used for growth of these organisms usually are heart infusions and 1 percent peptone enriched with either serum, ascitic fluid, or serum fraction. The growth of many strains has been improved by cholesterol, yeast products, desoxyribonucleic acid, and various sugars. These factors have been adequately reviewed by Edward.4 The growth requirements of Mycoplasma of avian origin, in contrast, appear to be less strictly defined. A number of different media have been used to isolate these organisms. Markham and Wong9 used a modified Edward's3 medium. This medium is quite complex and contains a beef-heart infusion medium enriched with yeast extract and equine serum.

Studies on a toxin-inactivating substance excreted by Penicillium cyaneo-fulvum.

SUMMARY: The reaction between staphylococcal α-toxin and a toxin-inactivating substance (TIS) excreted by Penicillium cyaneo-fulvum was found to proceed optimally at about pH 7·8, and in the temperature range 36–44°. A modified assay method, based on these findings, was used to follow TIS excretion by the mould. Appearance of the active principle in the culture filtrate followed closely upon the period of active sugar utilization by the mould. TIS was excreted in quiescent cultures, but not in shake cultures. The presence of certain nutritional factors found in beef heart infusion appeared to be necessary for TIS excretion, since detoxifying activity could not be detected in filtrates from cultures of the mould grown in the absence of this nitrogen source. The marked correlation between detoxifying and protease activities of culture filtrates during growth of the mould under various conditions, as well as during purification of TIS, and its inactivation by heat and by DFP, suggested that both activities were manifestations of the same enzymic reaction.

An investigation of "A" substance in T.A.B. vaccine diluted with saline or toxoid.

"A" substance, comparable to that found in human red blood cells, has been reported to be present in toxoid and vaccine made on media containing tryptic or peptic digests of animal tissue. Our tests were carried out with T.A.B. vaccine produced on (1) beef leg infusion containing 1% Bacto peptone and 3% Bacto agar, and (2) on beef heart infusion containing 1% N-Z case (tryptic digest of casein) and 3% agar. When either type of vaccine was diluted with saline, for human use, no trace of "A" substance was found. When T.A.B. vaccine was diluted with tetanus toxoid (T.A.B.T.) or with tetanus and diphtheria toxoids (T.A.B.T.D.), considerable "A" substance was present if the toxoids were produced on hog stomach autolyzate medium (peptic digest of hog stomach tissue). However, when the vaccines were diluted with toxoids produced on N-Z case medium, no "A" substance was found.

Clostridium botulinum type E toxin and toxoid.

A strain of Clostridium botulinum Type E in a beef heart infusion – peptone broth yields moderate amounts of toxin when grown at 30 °C. but fails to produce toxin at 37°. When grown in a cellophane sac suspended in a similar medium, at 30 °C, there is a 5- to 10-fold increase in the yield of toxin. Toxoid has been prepared from the high potency cellophane produced toxin by clarification with charcoal, Mandler filtration, and detoxification with 0.3% formalin at 30 °C. for 20 days. Three doses of the fluid toxoid protects mice against 500 M.L.D. of homologous toxin. One dose of the same toxoid adsorbed on alum affords the same level of protection as three doses of the fluid toxoid. Mixing Type E toxoid with equal amounts of Types A and B in vitro augments the antigenic action of the Type E toxoid.

The antimycobacterial activity of a peptide preparation derived from calf thymus.

A stable, water-soluble substance which possesses potent antimycobacterial activity under certain conditions in vitro has been prepared from calf thymus. This substance has been tentatively named thymus peptide. In final concentrations of 1 to 10 microg. per ml. of an albumin medium it inhibits the growth of various strains of mammalian mycobacteria, but manifests only little or no inhibitory activity against a variety of other microbial species. The ability of thymus peptide to inhibit the multiplication of tubercle bacilli diminishes when the inoculum is large, or when the medium is acidic. It is also markedly antagonized by addition of enzymatic hydrolysate of casein or beef heart infusion broth to the culture medium. Thymus peptide does not exert a rapid bactericidal action on tubercle bacilli, but organisms exposed to this compound for longer than 2 weeks could not be made to multiply in ordinary culture media. Substances similar or identical to the thymus peptide preparation could be extracted from calf spleen, sheep thymus, beef lymph nodes, and calf pancreas, but not from calf lung or calf liver.