Characterization of dysgonic, heterotrophic bacteria from drinking water.

Only a small percentage of the heterotrophic bacteria encountered in water distribution systems are identifiable, because of these organisms fail to grow on the conventional media used for biochemical characterization. Organisms that would not subculture from the same standard plate count agar used for initial isolation were successfully subcultured on a low-nutrient medium, R3A. These cultures were then inoculated to a modified O/F base medium containing specific substrates. This, combined with a lower incubation temperature (30 degrees C), increased the enzymatic activity of many of the organisms. These reactions established a groundwork for tentative taxonomy.     

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.     

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.     

Cholesterol-reducing bacterium from human feces.

An anaerobic, gram-positive diplobacillus that reduces cholesterol to coprostanol was isolated from human feces and rat cecal contents. The isolates closely resemble a cholesterol-reducing organism isolated by Eyssen et al. (H. Eyssen et al., Eur. J. Biochem. 36:412-421, 1973) from a rat's cecum. These organisms would not form colonies and were isolated and cultivated in an anaerobic medium containing homogenized pork brains (naturally high in cholesterol). These organisms require free or esterified cholesterol for growth. They were isolated by serially diluting feces or cecal contents and inoculating brain medium. Colony-forming organisms, which did not reduce cholesterol, were eliminated by addition of inhibitory agents to the brain medium cultures. This serial dilution procedure was performed until a pure culture of a cholesterol-reducing organism was obtained.     

Detection and Enumeration of a Tagged Pseudomonas fluorescens Strain by Using Soil with Markers Associated with an Engineered Catabolic Pathway

Previously we described a novel gene tagging method, using the moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955, to identify microorganisms destined for release into the environment. Here, we used the engineered strain Pseudomonas fluorescens PF5MT12 carrying the moc region integrated into the bacterial chromosome to demonstrate the usefulness of the markers for detection and direct selection of marked organisms present in soil samples. Using this system, we routinely detected population levels as low as 10(sup2) CFU per g of soil sampled. In addition to direct selection, we developed an immunologically based assay using MOP cyclase, a unique enzyme associated with moc, as the epitope for detecting the tagged organism. The colony immunoblot assay proved to be highly specific and without any false-positive signals when used to identify organisms cultured from soil on nonselective medium. The numbers of colonies that were immunoreactive with the anti-MOP cyclase antibody were essentially equal to those that grew out on selection plates. This indicates that MOP cyclase can be used as a marker and that we can use nonselective medium to retrieve the marked genetically engineered microorganisms and then identify them by using colony immunoblot assays. These direct selection and colony immunoblot methods provide a sensitive and accurate strategy for identifying and enumerating marked organisms recovered from soil samples. We also developed a rapid assay for MOP cyclase that does not require cell permeabilization with toluene. This assay can be used to verify tagged organisms isolated by other methods or to screen large numbers of colonies for the tag following nonselective isolation.     

Cholesterol-reducing bacterium from human feces.

An anaerobic, gram-positive diplobacillus that reduces cholesterol to coprostanol was isolated from human feces and rat cecal contents. The isolates closely resemble a cholesterol-reducing organism isolated by Eyssen et al. (H. Eyssen et al., Eur. J. Biochem. 36:412-421, 1973) from a rat's cecum. These organisms would not form colonies and were isolated and cultivated in an anaerobic medium containing homogenized pork brains (naturally high in cholesterol). These organisms require free or esterified cholesterol for growth. They were isolated by serially diluting feces or cecal contents and inoculating brain medium. Colony-forming organisms, which did not reduce cholesterol, were eliminated by addition of inhibitory agents to the brain medium cultures. This serial dilution procedure was performed until a pure culture of a cholesterol-reducing organism was obtained.     

Evaluation of different malachite green brands in the performance of rappaport's medium

Summary Three malachite green dyes, Merck's concentrated for microscopy and bacteriology (old), Merck's malachite green oxalate (oxal), and Difco's malachite green, were used for preparation of 3 batches of Rappaport's medium. These media were tested for growth of 40 Salmonella serotypes and for inhibition of competing organisms. All Rappaport's media behaved similarly and supported excellent growth of 38 serotypes. When Salmonella cultures were diluted in fecal-saline suspensions and inoculated in all Rappaport media the competing organisms in these suspensions were efficiently inhibited. However, the inhibition of competing organisms was less efficient when Rappaport's media were inoculated with preenrichment cultures of 20 samples of minced meat in buffered peptone water. In this respect, Difco's malachite green was clearly inferior to the other two dyes. It is concluded that Merck's malachite green oxalate is as efficient as the old dye of the same brand which is no longer produced, and can replace it in the preparation of Rappaport's medium.     

Ultrastructural changes observed in Rickettsia rickettsii incubated at different temperatures in cell-free medium

When cells of Rickettsia rickettsii were suspended at different temperatures in growth medium free of host cells, ultrastructural changes were observed in some of these organisms. Depending upon the temperature and length of incubation, loosely organized cells developed into compact, intensely stained, rod-shaped organisms. These compact cells closely resembled the morphology of the original culture of Rickettsia used for inoculation. Morphological changes were primarily noted in cells maintained at 21 degrees C. The viability of the cells was also affected by the temperature of incubation.     

Defined minimal medium for a thermophilic Bacillus sp. developed by a chemostat pulse and shift technique

Summary A new method for the design and optimization of a minimal medium is described. A chemostat pulse technique was used to identify growth limiting nutrients. By combining this pulse technique with medium shifts, the essential medium components were determined and the composition quantified. The described technique has useful applications for fastidious organisms and organisms which cannot be easily cultivated in shake flasks. A minimal medium is given for thermophilic Bacillus caldotenax, which exhibits methionine and biotin auxotrophy.     

Performance of Pseudomonas CFC-selective medium in the fish storage ecosystems

Pseudomonas agar base supplemented with cephaloridine, fucidin, and cetrimide (CFC) was used to count Pseudomonas populations on fish. Both Enterobacteriaceae and Shewanella putrefaciens were able to grow on the CFC medium. Evaluation of the performance of CFC-selective for pseudomonads medium, on fish samples stored aerobically and under a modified atmosphere at 0, 10 and 20 degrees C was tested.The selectivity of the medium was affected by storage temperatures and the type of packaging of the fish samples. The selectivity of the medium diminished as the population increased and for samples stored at high temperature (20 degrees C) or under modified atmospheres.When designing adequate selectivity of a medium, interfering organisms should be taken into account, especially when the background flora tends to be more robust than the organisms to be counted or detected.     

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.     

Rapid Technique for the Enumeration of Clostridium perfringens

A new medium, Tryptone-sulfite-neomycin (TSN) agar, and an incubation procedure for the enumeration of Clostridium perfringens are described. Tolerance to neomycin, optimal growth at 46 C, and sulfite-reducing properties of C. perfringens were used as a basis for development of the medium. Comparisons were made between sulfite-polymyxin-sulfadiazine (SPS) agar and TSN agar at 37 and 46 C with C. perfringens and other organisms. These studies indicate the quantitative and selective superiority of TSN agar, incubated at 46 C, over SPS agar.     

Trace elements incorporated into the culture medium of Mycobacterium tuberculosis promote the presence of tuberculoprotein C in the preparation of purified protein derivatives

Two purified protein derivatives (PPDs) were prepared from Mycobacterium tuberculosis, H37Ra. One of the PPDs was prepared from the culture filtrate of organisms grown on Proskauer-Beck medium to which trace elements had been added. The other PPD was prepared from the culture filtrate of organisms grown on the same medium but without trace elements, and was used as the control. Comparative skin reactions in sensitized rabbits showed that the PPD prepared from organisms grown in the presence of trace elements was less potent than the control. Since it has long been recognized that of the many tuberculoproteins present in PPD, the C protein (a 44 to 66 kDa protein) was always the least potent fraction when tested in equivalent concentrations in both serological and skin test assays, the possibility existed that the PPD obtained from organisms grown in trace element medium had more of the C protein complex than the control. Comparative studies of these two PPDs in terms of their chemical composition, skin test responses, and electrophoretic profiles obtained by SDS-polyacrylamide gel electrophoresis provide support for this assumption.     

Suppressive Activity of Streptomycin on the Growth of Mycobacterium lepraemurium in Macrophage Cultures

The effect of streptomycin on the growth of an obligate intracellular bacterium was studied in a new host-parasite cell system. The system consisted of Mycobacterium lepraemurium grown in cultures of mouse peritoneal macrophages. Since these organisms do not grow in bacteriological media, the influence of extracellular bacterial growth can be ruled out. The suppressive activity of streptomycin was observed in a total of five experiments. At the end of 4 weeks, the average number of organisms per macrophage for the controls was 65.7; for cultures with streptomycin at concentrations of 0.5, 1, 5, 10, 50, and 100 mug/ml of medium, it was 45.4, 38.3, 28.7, 22.7, 13.4, and 8.2, respectively. A good dose-response relationship was evident. M. lepraemurium which had been treated in macrophage cultures with various concentrations of the antibiotic for 6 to 8 weeks was used to infect fresh macrophages. These cultures were in turn treated with streptomycin. Resistance of the organisms to streptomycin did not occur.     

Improved Solid Medium for the Detection and Enumeration of Pectolytic Bacteria

An improved solid agar medium (MP medium) has been developed which allows detection of pectolytic activity in bacteria. Organisms tested exhibited a variety of regulatory controls governing pectate lyase synthesis. The medium contains mineral salts, pectin, and yeast extract. After growth of the organisms, the agar plate is flooded with a polysaccharide precipitant, and pectolytic activity is shown by clear zones around active colonies. High concentrations of phosphate are shown to be necessary for pectic enzyme formation on solid media. The medium has successfully been used to detect pectolytic organisms in soil, forest litter, and rotting vegetable samples.     

Rich culture medium for the radiochemical labeling of proteins and nucleic acids.

Yeast extract was treated with tyrosine decarboxylase and used to prepare a rich, complex medium virtually free of tyrosine. The medium supported maximal growth rates for Escherichia coli prototrophs, as well as for defined and undefined auxotrophs. It has made possible the efficient radiochemical labeling of cells growing optimally in complex medium and the characterization of mutants with undefined requirements. Similarly prepared media may be useful for the study of fastidious organisms and organisms for which no defined medium has been described.     

Accumulation of iron by yersiniae.

Escherichia coli, Bacillus megaterium, and three species of yersiniae grew rapidly without significant production of soluble siderophores in a defined iron-sufficient medium (20 microM Fe3+). In iron-deficient medium (0.1 to 0.3 microM Fe3+) all organisms showed reduced growth, and there was extensive production of siderophores by E. coli and B. megaterium. Release of soluble siderophores by Yersinia pestis, Y. pseudotuberculosis, or Y. enterocolitica in this medium was not detected. Citrate (1 mM) inhibited growth of yersiniae in iron-deficient medium, indicating that the organisms lack an inducible Fe3+-citrate transport mechanism. Uptake of 59Fe3+ by all yersiniae was an energy-dependent saturable process, showing increased accumulation after adaptation to iron-deficient medium. Growth of Y. pseudotuberculosis and Y. enterocolitica but not Y. pestis on iron-limited solid medium was enhanced to varying degrees by exogenous siderophores (desferal, schizokinen, aerobactin, and enterochelin). Only hemin (0.1 pmol) or a combination of inorganic iron plus protoporphyrin IX promoted growth of Y. pestis on agar rendered highly iron deficient with egg white conalbumin (10 microM). Growth of Y. pseudotuberculosis and Y. enterocolitica was stimulated on this medium by Fe3+ or hemin. These results indicate that hemin can serve as a sole source of iron for yersiniae and that the organisms possess an efficient cell-bound transport system for Fe3+.     

Effect of sodium chloride on growth of heterotrophic marine bacteria

The effect of NaCl on the growth rates and yields of 31 gram-negative, heterotrophic, marine bacteria was determined. The strains used were representative of aerobic genera (Alteromonas, Pseudomonas, Alcaligenes, Bdellovibrio) as well as genera comprised of facultative anaerobes (Beneckea, Photobacterium). Two media were used-the first, a medium designed for the cultivation of marine bacteria and, the second, a medium used for the cultivation of terrestrial strains. These two media differed in the concentrations of divalent cations; the terrestrial medium (TM) contained 2 mM Mg⁺⁺ and 0.55 mM Ca⁺⁺ while the marine medium (MM) contained 50 mM Mg⁺⁺ and 10 mM Ca⁺⁺. The amount of NaCl necessary for optimal growth varied in different strains and was usually considerably higher in TM (100 to 460 mM) than in MM (70 to 300 mM). Many strains which grew in MM and TM had a shorter generation time in the former than in the latter medium. In addition, four strains which grew well in MM usually failed to grow in TM. These results show that higher levels of divalent cations are either essential for growth or stimulate growth rate, indicating that for many marine strains a terrestrial medium modified by the addition of NaCl cannot support optimal growth. Fourteen terrestrial strains of the genera Pseudomonas, Alcaligenes, Acinetobacter, Salmonella, Aeromonas, and Vibrio did not have ionic requirements comparable to those of the marine strains. All of the terrestrial organisms grew in TM without added NaCl (0.068 mM Na⁺ was present as a contaminant). In some terrestrial organisms, growth was stimulated by the addition of NaCl, the highest stimulation being found in Vibrio cholerae. The optimal growth rates and yields for four strains of this species were observed at 2.5 to 5.0 mM NaCl while the growth rates and yields in TM with no added NaCl were 40 to 50% of the optimum.     

Bacillus protein secretion: an unfolding story

Bacillus subtilis and its close relatives are widely used for the production of enzymes for the detergent, food and beverage industries. These organisms not only produce an appropriate range of enzymes but also have the capacity to secrete them into the culture medium at high concentrations. Purification from the culture medium rather than from the cytoplasm considerably reduces downstream processing costs. In recent years, considerable effort has been aimed at developing B. subtilis as a host for the production of heterologous proteins. The folded state of the target protein at various stages of the secretion pathway has proved to be important.