COLI-AEROGENES BACTERIA IN FARM WATER SUPPLIES

SUMMARY: A series of 825 cultures of coli-aerogenes bacteria isolated at 30° and a series of 735 cultures isolated at 37° from 645 samples of farm water supplies were classified according to the recommendations of the Coliform Sub-Committee of the Society for Applied Bacteriology (Report, 1949). Klebsiella constituted 50% of the cultures isolated at 30°, whereas Escherichia coli I was the dominant type, forming 57%, among the cultures isolated at 37°. It would thus appear that isolation at 30° is as selective for Klebsiella as isolation at 37° is for Escherichia . Coli-aerogenes organisms, mainly 37° negative strains of Citrobacter freundii I and K. cloacae , were found in waters of high sanitary quality derived from protected springs and wells; but the coli-aerogenes microflora of polluted water was dominated by E. coli I, which formed 43% of the isolates at 30° and 76% of those at 37°. The results for a series of fortnightly samples from 11 farm water supplies showed a marked seasonal variation in the incidence of different types isolated at 30°; E. coli I formed a higher proportion in summer than in winter, while 37° negative strains of Klebsiella and Citrobacter formed a higher proportion in winter than in summer.     

Stability of plasmids in five strains of Salmonella maintained in stab culture at different temperatures

Four strains of Salmonella berta and one of Salm. enteritidis were stored as stab cultures in sugar-free agar at 5°, 22° and 30°C and in 15% glycerol at—80°C. The stability of the plasmid profiles in each of the strains was monitored over a period of 2·5 years.
Plasmid profiles were stable in all strains stored at—80°C, and only six of 450 colonies examined from strains kept in sugar-free agar at 5°C had lost plasmid molecules. Seventy of 440 colonies from stab cultures that were kept at 22°C, and 71 of 440 colonies at 30°C showed changed plasmid profiles. The total number of plasmids lost increased with time, and occasionally, more than one plasmid molecule was lost in the same strain.
The virulence associated plasmid of Salm. enteritidis was remarkably stable as it was maintained in all colonies examined at all temperatures investigated. Likewise, no change in Sma I restriction profile was observed in this plasmid molecule at any temperature.     

THE EFFECT OF TEMPERATURE ON INFECTION WITH STRAINS OF CUCUMBER MOSAIC VIRUS

Cucumber mosaic virus strains differed in their ability to multiply in plants at 37° C. Some strains multiplied in inoculated leaves and produced systemic symptoms in plants at this temperature; plants systemically infected with one such strain remained infected after prolonged treatment at 37° C. Other strains did not appear to multiply in inoculated leaves at 37° C. and heat treatment was successful in freeing plants from infection with these. Tests with one strain of each type showed both to be rapidly inactivated in expressed sap at 37° C.
Strains of cucumber mosaic virus forming small necrotic local lesions in leaves of french bean var. Canadian Wonder, produced many fewer lesions in plants kept after inoculation at 25° C. for 24 hr. and then at 15° C. than in plants kept continuously at the lower temperature.     

Nitrogenase activity (acetylene reduction) in root-associated, cold-climate species of Azospirillum, Enterobacter, Klebsiella and Pseudomonas growing at various temperatures

Abstract 23 Strains of diazotrophic root-associated bacteria isolated from various parts of Finland were tested for nitrogenase activity during growth at various temperatures. Nitrogenase activity was optimal at 20–37°C in cultures of Klebsiella pneumoniae , and at 14–20°C in cultures of Klebsiella terrigena and Enterobacter agglomerans . Strains of K. terrigena and E. agglomerans showed no activity at 37°C, and K. pneumoniae only minimal or no activity at 14°C. Azospirillum lipoferum exhibited high nitrogenase activity at both 28–37°C, but less than 25% of optimal activity at 20°C and no activity at 14°C. Pseudomonas sp. expressed nitrogenase activity at 14–28°C. None of the strains manifested nitrogenase activity at 4 or 42°C. There were only small local variations within a species between strains isolated at different locations.     

Life-Cycle of Trypanosoma conorhini. Influence of Temperature and Other Factors on Growth and Morphogenesis

SYNOPSIS. In continued observations on the in vitro growth and multiplication of the bloodstream trypanosome stage of Trypanosoma conorhini , a better medium was found for cultivating these forms at 37°C, but no subcultures could be obtained. The infectivity for mice of the blood type trypanosomes grown in vitro was comparable to that of the metacyclic trypanosomes. The only reproducing forms of T. conorhini found in the vertebrate were in the trypanosome stage.
It was also found that the in vitro reversion of the bloodstream trypanosome into crithidia, such as occurs in the invertebrate host and in the usual diphasic culture medium, is dependent on at least two factors: if incubated at 25–28° reversion did not occur in any of the liquid media tried (all containing blood serum and hematin or hemoglobin), unless total blood was part of the inoculum or washed red blood cells were added to the media; on the other hand, no reversion was seen, even in the presence of red blood cells if the cultures were incubated at 37°.     

Influence of growth temperature on the production of extracellular virulence factors and pathogenicity of environmental and human strains of Aeromonas hydrophila

The biochemical properties, virulence for mice and trout, and the extracellular virulence factors at 28° and 37°C of 11 environmental and nine human strains of Aeromonas hydrophila were compared. All the environmental isolates and four of the human group were virulent for trout at 3 x 10⁷ cfu, but only human strains were able to cause death or lesions in mice by the intramuscular route. Extracellular virulence factors such as haemolysins, cytotoxins and proteases were also investigated in supernatant fluids of cultures grown at 28°C and 37°C. The production of haemolysins, caseinases, elastases and growth yields of environmental strains decreased sharply during cultivation at 37°C but cytotoxins were produced to the same extent, or slightly less, than at 28°C. The human strains differed from the environmental strains in response to growth temperatures: protease activity decreased at 37°C, although growth yield was not affected, but more haemolysins and cytotoxins were produced by the virulent strains at this temperature than at 28°C. Sodium caseinate SDS-PAGE of culture supernatant fluids of selected human strains revealed that temperature selectively inhibited the production of certain proteases.     

Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating

H umphrey , T.J. 1990. Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating. Journal of Applied Bacteriology 69 493–497.
Storage of cultures of Salmonella enteritidis PT4 at either 4° or 8°C before heating significantly increased heat sensitivity. The differences between fresh and stored cultures, which became apparent after 4–7 h, were more pronounced with cultures stored at the lower temperature and in those heated at 60° rather than 55°C. Incubation of the stored cultures in either egg or Lemco broth for 30 min at 37°C prior to heating enabled the organisms to recover heat resistance.     

Growth, Fine Structure and Cyst Formation of a Microbial Mat Ciliate: Pseudocohnilembus pusillus (Ciliophora, Scuticociliatida)

ABSTRACT Pseudocohnilembus species exhibit a polymorphic life cycle consisting of trophic cells, theronts, and cysts. Pseudocohnilembus pusillus isolated from the intertidal mats of Laguna Figueroa, Baja California Norte, Mexico, forms desiccation-resistant cysts in response to bacterial food depletion. This isolate is a euryhaline organism, able to grow at salinities from freshwater to 96 ppt total salinity and from pH 6–9. Electron micrographs show that oral and somatic cilia and kinetids are retained inside young cysts. Cyst walls are composed of a single layer (0.1 μm) of granular material. Under all conditions, as bacterial food was depleted, P. pusillus cells formed cysts, except for a small proportion (1–5%) that continued to swim. Changes in pH and salinity did not directly induce cyst formation. Salinity did greatly affect growth rate. Doubling times were shortest at 16 ppt salinity and at pH 7–8. Cyst formation occurred later in the growth cycle as more food bacteria were added. Additionally, ciliates grown in small culture volumes (10 ml) formed cysts sooner than cultures in larger volumes (100 ml), suggesting that crowding may influence cyst formation. Mature cysts may survive desiccation at least as long as one month at 37° C and for as long as one year at 20 ± 3° C. Although trophic cells did not survive desiccation or anoxia, encysted ciliates from liquid stationary phase cultures kept in anoxic seawater for one month excysted into swimming cells within 2.5 h after exposure to air. The adaptability of P. pusillus to extremes of salinity, pH, desiccation, and anoxia permits survival in its environmentally variable, microbial mat habitat.     

The protective effect of some food ingredients on Staphylococcus aureus MF31

The upper limiting temperature of growth of Staphylococcus aureus MF31 in heart infusion broth (HI) was about 44°C but addition of monosodium glutamate (MSG) and soy sauce permitted the organism to grow above this temperature. This effect is similar to that of NaCl. Tomato ketchup, Worcestershire and HP sauces added to HI did not allow growth at the non-permissive temperature of 46°C but death was delayed. Staphylococcus aureus died in unsupplemented chicken meat slurry at 46°C but grew at 48°C in slurry supplemented with 5.8% NaCl and survived incubation for 18 h at 50°C in slurry supplemented with 5.8% NaCl and 5% MSG. Cultures grown at 37°C had a D ₆₀ value of 2 min in 50 mmol/l Tris (pH 7.2) buffer. Cultures grown at 46°C in HI containing 5.8% NaCl had a D ₆₀ value of 8 min in Tris buffer. Addition of 5.8% NaCl plus 5% MSG to the buffer increased the D ₆₀ by a factor of about 7 for both cultures. In storage experiments at room temperature, the culture grown at 37°C and at 46°C plus 5.8% NaCl died at about the same rate in salami. In milk powder, however, the count of 37°C culture decreased from 10⁹/g to 10⁶/g in 5 weeks while the count of 46°C culture remained unchanged. In cottage cheese, freeze-dried rice and macaroni, the 37°C cultures also died more rapidly. It is suggested that cultures grown at 46°C plus 5.8% NaCl may be suitable for experiments with artificially contaminated foods.     

The enumeration of sublethally injured populations of Staphylococcus aureus in foods

Substantiating earlier investigations, pure cultures of Staphylococcus aureus were found to be equally well recovered on Baird-Parker agar at 37°C as at 42°C, whereas Micrococcus spp. are suppressed at the latter temperature to an extent exceeding 5 log₁₀ cycles. It was also established that egg yolk dissimilation by Staph. aureus is intensified at 42°C. Heat treated (60°C) populations of Staph aureus were quantitatively recovered on Baird-Parker agar at 42°C, though acid-injured populations were not. Acid-injury (2% lactic acid at 37°C) could be completely restored by solid medium repaiar during at least 6 h at 23°C on tryptone soya peptone yeast extract egg yolk pyruvate agar. Pure culture studies were confirmed in surveys on trade samples of foods.     

Effect of temperature in the regulation of DNA synthesis in synchronous cultures of Chlorella

The influence of temperature on DNA replication of Chlorella pyrenoidosa grown at 30 °C was studied in synchronous cultures. Final DNA content and cell counts/ml were 30 to 40% less than controls after a 4 h exposure to 15 °C, but reached normal levels after being kept temporarily at 10 °C. The rates of DNA synthesis at these two temperatures were, respectively, one-sixth and one-tenth of the rate at 30 °C, but during the 15 °C treatment the cells lost the ability to enter a further replication cycle more rapidly than at 10 °C. This loss of capacity to initiate further replication cycles was prevented by inhibiting protein synthesis with cycloheximide.     

Factors influencing alkali-induced heat resistance in Salmonella enteritidis phage type 4

The culture of cells of Salmonella enteritidis PT4 at either 25, 30 or 37°C in media at pH values between 8.0 and 9.75 resulted in significant increases in heat resistance. At 37°C, induction was rapid and was dependent on protein synthesis, being inhibited by chloramphenicol. Thermotolerance was stable when cells were transferred from pH 9.2 to pH 7.0 and cultures only became heat sensitive again following significant multiplication at the lower pH.     

DNA replication in Escherichia coli 15T- growing at 20 degrees C

Escherichia coli 15T⁻ grows slowly in succinate or aspartate-M9 media. In both media, a gap in DNA replication is observed at 37 °C which is either not present at 20 °C or of very much shorter duration than at 37 °C. However, dichotomous replication is not observed in glucose M9 at 20 °C. The results suggest that initiation of replication in glucose is different from that in aspartate or succinate cultures.     

The effect of temperature, pH and medium in a surface adhesion immunofluorescent technique for detection of Listeria monocytogenes

A rapid surface adhesion-based immunofluorescence technique was used to detect Listeria monocytogenes from inoculated culture systems. The effect of culture type (pure, mixed and meat), pH (7·00, 6·40, 4·76 and 3·13), acids (citric and HCl) and temperature (25°, 30° and 37°C) on the adhesion of Listeria to the polycarbonate membrane used in this technique was determined. It was found that pH had a significant effect ( P < 0·05) with higher numbers of Listeria adhering at low pH values (4·76). Culture type was also important with significantly higher numbers of Listeria ( P < 0·05) adhering to membranes immersed in meat cultures than in pure or mixed cultures. This effect was seen at 30°C but not at 25° or 37°C. The total viable count (TVC) on the membrane was unaffected by pH but temperature had an influence with optimum adhesion occurring at 25°C. The reasons for observed differences and their implications for the surface adhesion immunofluorescent rapid method are discussed.     

A rapid method for identifying and quantifying soft rot erwinias directly from plant material based on their temperature tolerances and sensitivity to erythromycin

A method is described for identifying and quantifying three soft rot erwinias directly from plant tissue and from other sources that is particularly useful in epidemiological studies. Colonies of these bacteria form characteristic deep cavities on selective-diagnostic crystal violet pectate (CVP) medium. Bacteria from individual presumptive erwinia colonies on CVP plates spot inoculated on plates of CVP medium with or without erythromycin (35 μg/ml) added and incubated at 27, 33°5 and 37°C can be identified according to the pattern of cavity formation. Erwinia carotovora pv. atroseptica forms the characteristic cavities only at 27°C and E. carotovora pv. carotovora at 27 and 33.5°C but not at 37°C on CVP with or without erythromycin. Erwinia chrysanthemi forms cavities at all temperatures and can also be identified by failure to grow at 27°C on CVP with erythromycin. Similarly, erwinias in mixed populations can be quantified by dilution plating on CVP with or without erythromycin and incubating at the different temperatures. Using this method, ca 80% of 183 erwinia strains in a culture collection were correctly identified, the precision increasing to over 95% when recently isolated erwinia strains were examined.     

A study of the growth kinetics of Yersinia enterocolitica serotype O:3 in pure and meat culture systems

The growth kinetics of a virulence plasmid-bearing (P+) and a plasmid-cured (P−) strain of Yersinia enterocolitica serotype O:3 in pure and meat culture were investigated. Growth studies were carried out at 25 and 37 °C in supplemented phosphate-buffered saline, buffered peptone water , cefsulodin-irgasan-novobiocin broth base or supplemented broth base (CIN). The lag phase durations and growth rates under these conditions were determined by linear regression analysis. In pure culture, under most sets of equivalent conditions, P+ and P− strains had similar lag phase durations. However, under one set of conditions, i.e. CIN broth at 37 °C, the lag phase duration of the P+ strain was significantly longer than P−. In all but the most selective medium, P+ strains had slower growth rates than P− strains at 37 °C, probably due to the increased metabolic burden entailed in the maintenance of the virulence plasmid. In the most selective medium, i.e. CIN broth, P+ strains grew significantly faster than P−. This finding suggests that possession of virulence plasmid confers an enhanced ability to grow in the presence of selective agents. In meat cultures, both strains had longer lag phases than in equivalent pure cultures, with longer lag phases noted at 37 than at 25 °C. No significant differences were observed between the length of lag phases of P+ and P− strains in meat culture. Both strains of Y. enterocolitica displayed faster growth rates in meat cultures than in pure cultures, indicating that one or more components of meat enhanced the growth of this organism. The effects and interaction of incubation temperature, enrichment broth and meat on the growth kinetics of plasmid-bearing and plasmid-cured Y. enterocolitica strains are discussed.     

The enumeration of thermotrophic types amongst the Enterobacteriaceae colonizing perishable foods

A total of 41 pure cultures of Enterobacteriaceae, comprising 32 thermotrophic and nine psychrotrophic strains, pathogens or marker organisms, were examined for numbers of colony forming units obtained at 37° and 42°5°C (thermotrophs) and 30°C (psychrotrophs), when surface-plated on a rich infusion agar and violet red bile agar. In addition 42 food and water samples, collected in a rural area of the Philippines, were examined by surface inoculating violet red bile AIPC (agar immersion plating and contact; 'dip') slides and incubating at 37° and 42°5°C. At 42°5°C there was almost total recovery of the thermotrophic Enterobacteriaceae, whereas the psychrotrophic strains were completely suppressed. At 37°C the psychrotrophs were only slightly inhibited. The Philippine foods, predominantly cooked meals, milk and drinking water, appeared to be significantly colonized by thermotrophic Enterobacteriaceae. It is concluded that incubation at 42°5°C satisfactorily selects enteropathogenic and other enteric Enterobacteriaceae while suppressing the psychrotrophic types which are mainly of vegetable origin. It is emphasized that, regardless of the temperature used, a resuscitation procedure for Enterobacteriaceae populations that have incurred sublethal injury in food has to precede counts on or in the usual selective media.     

Trypanosoma brucei brucei: In Vitro Production of Metacyclic Forms

ABSTRACT An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei . Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27° C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27° C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.     

Comparison of selective agars for the isolation and identification of Klebsiella oxytoca and Escherichia coli from environmental drinking water samples

Various selective media were assessed for their ability to detect and differentiate Klebsiella oxytoca and Escherichia coli in environmental water samples. Only two, Membrane Lauryl Sulphate agar and Deoxycholate Agar, could differentiate the two coliforms from each other and from the 'background' heterotrophs in water and this was a consequence of E. coli's ability to grow at 44°C and 37°C whereas Kl. oxytoca could only grow at 37°C. Modified M-FC medium effectively differentiated Kl. oxytoca but not E. coli in environmental samples. Other media characterized the different coliforms in pure culture but failed to do likewise in environmental samples. For example, pure cultures of E. coli fluoresced when MUG was added to the medium but single colonies on a mixed species plate failed to do so. MT7 agar distinguished the two coliforms from water heterotrophs but not from each other.