Quantitation of pH- and salt-tolerant subpopulations from Clostridium botulinum.

Plating efficiencies of Clostridium botulinum 62A spores on media with variable pH (7.0 to 5.5) and salt (0, 1, 2, and 3%) levels revealed that only a very small subpopulation could give rise to colonies. The relative size of this subpopulation decreased by orders of magnitude with decreasing pH and increasing salt concentrations. Strong interactions of pH with salt were noted. For example, on a medium containing 2% salt at pH 5.5, colonies could be formed from only 1 in 100,000 spores. Proper monitoring of medium anaerobiosis was critical in obtaining reproducible results.     

Characterization of a halo-acid-tolerant variant of Clostridium botulinum B-aphis.

Clostridium botulinum B-aphis spores plated on medium containing 4% salt at pH 6.0 yielded colonies at a frequency of ca. 1 in 10(6). A subculture of one of these colonies, designated strain Ba410, was compared with the parent strain, B-aphis, for a variety of traits. After 7 days of incubation at 37 degrees C, strain Ba410 grew in medium containing 7% NaCl, whereas strain B-aphis could not grow in salt concentrations greater than 5%. The strains also differed in cellular and colonial morphology. After exponential growth in the basal medium was completed, lysis of both strains was pH dependent; in media containing salt, lysis of Ba410 cells was pH independent. Strain Ba410 was more proteolytic than strain B-aphis in conditions of low pH and high salt, so that its toxin could be detected by the mouse assay. In a medium containing alanine and cysteine, the germination rate of B-aphis was 0.77% min-1, whereas that of Ba410 was 0.14% min-1; 2% salt inhibited the germination of Ba410 but not B-aphis.     

Characterization of a halo-acid-tolerant variant of Clostridium botulinum B-aphis

Clostridium botulinum B-aphis spores plated on medium containing 4% salt at pH 6.0 yielded colonies at a frequency of ca. 1 in 10(6). A subculture of one of these colonies, designated strain Ba410, was compared with the parent strain, B-aphis, for a variety of traits. After 7 days of incubation at 37 degrees C, strain Ba410 grew in medium containing 7% NaCl, whereas strain B-aphis could not grow in salt concentrations greater than 5%. The strains also differed in cellular and colonial morphology. After exponential growth in the basal medium was completed, lysis of both strains was pH dependent; in media containing salt, lysis of Ba410 cells was pH independent. Strain Ba410 was more proteolytic than strain B-aphis in conditions of low pH and high salt, so that its toxin could be detected by the mouse assay. In a medium containing alanine and cysteine, the germination rate of B-aphis was 0.77% min-1, whereas that of Ba410 was 0.14% min-1; 2% salt inhibited the germination of Ba410 but not B-aphis.     

Thermal inactivation and injury of Bacillus stearothermophilus spores.

Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55 degrees C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.04, 18.00, 8.00, 3.33, and 1.05 min at 112.8, 115.6, 118.3, 121.1, and 123.9 degrees C, respectively. Recovery on AAMS-S resulted in reduced decimal reduction time. The computed z value (the temperature change which will alter the D value by a factor of 10) for spores recovered on AAMS was 8.3 degrees C; for spores recovered on AAMS-S, it was 7.6 degrees C. The rates of inactivation and injury were similar. Injury (judged by salt sensitivity) was a linear function of the heating temperature. At a heating temperature of less than or equal to 118.3 degrees C, spore injury was indicated by the curvilinear portion of the survival curve (judged by salt sensitivity), showing that injury occurred early in the thermal treatment as well as during logarithmic inactivation (reduced decimal reduction time). Heat-injured spores showed an increased sensitivity not only to 0.9% NaCl but also to other postprocessing environmental factors such as incubation temperatures, a pH of 6.6 for the medium, and anaerobiosis during incubation.     

Thermal inactivation and injury of Bacillus stearothermophilus spores

Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55 degrees C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.04, 18.00, 8.00, 3.33, and 1.05 min at 112.8, 115.6, 118.3, 121.1, and 123.9 degrees C, respectively. Recovery on AAMS-S resulted in reduced decimal reduction time. The computed z value (the temperature change which will alter the D value by a factor of 10) for spores recovered on AAMS was 8.3 degrees C; for spores recovered on AAMS-S, it was 7.6 degrees C. The rates of inactivation and injury were similar. Injury (judged by salt sensitivity) was a linear function of the heating temperature. At a heating temperature of less than or equal to 118.3 degrees C, spore injury was indicated by the curvilinear portion of the survival curve (judged by salt sensitivity), showing that injury occurred early in the thermal treatment as well as during logarithmic inactivation (reduced decimal reduction time). Heat-injured spores showed an increased sensitivity not only to 0.9% NaCl but also to other postprocessing environmental factors such as incubation temperatures, a pH of 6.6 for the medium, and anaerobiosis during incubation.     

Effect of Sodium Chloride and pH on the Outgrowth of Spores of Type E Clostridium botulinum at Optimal and Suboptimal Temperatures

The sodium chloride inhibition of spore outgrowth of four strains of type E Clostridium bolulinum was determined in a Trypticase-peptone-glucose (TPG) medium. At 16, 21, and 30 C, spores of three strains required 5.0% and one strain 4.5% salt for complete inhibition during 1 year of incubation. At 8 and 10 C, spores of the four strains required 4.5% salt for definite inhibition. Salt concentrations slightly lower than those providing inhibition tended to extend spore outgrowth time at low temperatures. The minimal pH permitting outgrowth of type E spore inocula was affected by the concentration of reducing compound present in the system. When either 0.02% sodium thioglycolate or 0.05% L-cysteine hydrochloride was used, outgrowth at 30 and 8 C occurred at much lower pH levels than when 0.2% thioglycolate was added. At 30 C, spores of one strain showed outgrowth in TPG medium as low as pH 5.21 with an inoculum of 2 million spores per replicate tube. At a 10-fold higher inoculum, the same strain showed outgrowth at pH 5.03 in one of five replicate tubes. At 8 C, spore outgrowth of the four strains occurred at pH 5.9, but not at pH 5.7, in TPG medium containing L-cysteine hydrochloride.     

Role of Curing Agents in the Preservation of Shelf-stable Canned Meat Products

Experiments were conducted to gain a better understanding of the mechanism by which sodium chloride, sodium nitrate, and sodium nitrite supplement the action of heat in preserving canned cured meat products. Heated spores of putrefactive anaerobe 3679h were less tolerant of all three curing agents in the outgrowth medium than were unheated spores. When the curing agents were added to the heating menstruum, but not to the outgrowth medium, sodium chloride and sodium nitrate tended to protect the spores against heat injury, but sodium nitrite did not. When the spores were both heated and cultured in the presence of the curing agents: (i) nitrate and salt increased the apparent heat resistance at low concentrations (0.5 to 1%) but decreased it at concentrations of 2 to 4%; (ii) nitrite was markedly inhibitory, especially at pH 6.0. At the normal pH of canned luncheon meats (approximately 6.0), nitrite appears to be the chief preservative agent against spoilage by putrefactive anaerobes.     

A method for isolating milky disease, Bacillus popilliae var. rhopaea, spores from the soil

A method based on the tyndallization procedure is described for isolation of Bacillus popilliae var. rhopaea spores from the soil. A soil suspension is diluted with a germinating medium, which promotes the germination of most spores except B. popilliae var. rhopaea, and is treated with a series of seven heat shocks (70°C for 20 min) at hourly intervals. This treatment reduced the number of contaminant spores by over 95%. The suspension is then plated out onto “J” medium which allows the germination and growth of all surviving spores including the milky disease spores. The plates are incubated anaerobically at 28°C for 7 days before the characteristic small transparent colonies of B. popilliae var. rhopaea are counted. In testing the method it was revealed that about 15% of the milky disease spores in the soil produced visible colonies, and that a spore concentration of over 1.2 × 10⁵ spores/g dry wt of soil could be quantified. This concentration of spores produces only 3% infection in Rhopaea verreauxi larvae. The method may be applicable to other varieties of B. popilliae which will grow on “J” medium.     

Separate isolation of Clostridium difficile spores and vegetative cells from the feces of newborn infants.

A modified taurocholate-cefoxitin-cycloserine-fructose agar medium, pH 5.5, on which vegetative cells alone could grow, was newly devised for separate isolation of Clostridium difficile vegetative cells and spores from feces. The ratio of C. difficile-positive feces from healthy newborn infants younger than 10 days of the age was 30.8%, and 93.3% of feces from healthy infants older than 20 days were positive for C. difficile. C. difficile spores alone were detected in twenty-one samples (75%) of C. difficile-positive Twenty-eight specimens. Only 10.7% (3/28) C. difficile vegetative cells alone were detected. C. difficile spores alone were detected in one of nine healthy adults. These collective results offer potential explanations for high frequent isolations of C. difficile from newborn infants without occurrence of pseudomembranous colitis.     

The effect of nisin-sodium chloride interactions on the outgrowth of Bacillus licheniformis spores

The effect of salt (NaCl) on the efficacy of nisin in preventing outgrowth of Bacillus licheniformis spores was determined in Plate Count Agar (PCA). An equivalent liquid medium was used for heat activation. Nisin and salt were added to the heat-activation medium, the PCA, or both. The spores were extremely sensitive to nisin; outgrowth were completely inhibited in salt-free media when 10 iu/ml of nisin was present in both the heat-activation and the growth media or when 100 iu/ml nisin was present in either the heat-activation and the growth medium. In media supplemented with 1% salt, outgrowth occurred from 1% of spores exposed to 100 iu/ml nisin in either the heat-activation or the growth medium. A 3% salt supplement was necessary before detectable outgrowth occurred when both the heat-activation and the growth media contained 100 iu/ml nisin. Salt appears to antagonize the sporicidal action of nisin by interfering with nisin adsorption onto the spore.     

Effect of electrical fields on polar growth of Phycomyces blakesleeanus

Abstract When Phycomyces blakesleeanus spores germinated in an electrical field of 10–20 V · cm⁻¹, the hyphal tips showed a strong tendency to grow towards the cathode in a medium with pH 7. A similar response was found during growth at pH 5 but a maximal response was only obtained at higher field strength. Also the emergence of the germ tubes from the spores could be polarized by such fields but the degree of polarization was much smaller at pH 7 and almost nonexistent at pH 5. Ionic strength of the medium did not play an important role since only minor differences were found in media with 0, 1 or 10 mM KH₂ PO₄ as the only salts. The results suggest that a voltage-dependent rearrangement of (membrane) proteins affects the polar growth of the fungus.     

Spontaneous mutants of Microsporon gypseum resistant to griseofulvin

Summary 1) The spores of the microconidial mutant I–18 of the dermatophyteMicrosporon gypseum in agar medium with GF germinated and formed germ tubes deformated in a characteristic way. From 1µg GF/ml up with an increasing antibiotic concentration (expressed in logarithms) the munber of colonies grown (expressed in probits) decreased linearly.2) As a sensitivity measure of the spores the median efficient dose ED 50 was used which was determined by means of a graphic probit analysis. For the strain used this value was determined in the range between 1.35–1.95µg GF/ml in three independent experiments.3) From the smears of a thickened spore suspension (1.6–14.2 × 10⁷ viable spores) in medium containing a high GF concentration a very small, but as for the order a stable number of colonies grew, as found in eight independent experiments. On the medium containing 20µg GF/ml in average 61 colonies grew, on 40µg GF/ml 20 colonies, on 80µg GF/ml 3 colonies and on 160µg GF/ml 0.3 colony (expressed in 10⁷ viable spores tested).4) A part of these colonies were isolated and transferred 29 times on a medium without the antibiotic. Two isolates only show a permanently increased resistance to GF, viz. the strain D-29 which is 50 × more resistant and the strain N-53 which is 3.5 × more resistant than the wild strain I-18.     

The effect of recovery medium on the estimated heat-inactivation of spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic strains of Clostridium botulinum at 85°C, followed by enumeration of survivors on a highly nutrient medium indicated a 5 decimal kill in less than 2 min. The inclusion of lysozyme or egg yolk emulsion in the recovery medium substantially increased apparent spore heat-resistance, with as little as 0.1 μg lysozyme/ml sufficient to give an increase in the number of survivors. After heating at 85°C for 2 min between 0.1% and 1% of the spores of 11 strains (5 type B, 4 type E, 2 type F) formed colonies on medium containing 10 μg lysozyme/ml. Enumeration of survivors on a medium containing lysozyme showed that heating at 85°C for 5 min resulted in an estimated 2.6 decimal kill of spores of strain 17B (type B). These findings are important in the assessment of heat-treatments required to ensure the safety with respect to non-proteolytic Clostridium botulinum of processed (pasteurized) refrigerated foods for extended storage such as sous-vide foods.     

航天诱变对昆虫病原真菌的生物学效应

8个昆虫病原真菌菌株经过航天搭载后,有5个菌株的孢子在航天搭载后全部死亡,菌株M2029和MR8的存活率仅约10-7,菌株M189的存活率可达到20%以上。分离得到航天诱变的单菌落菌株,检测表明航天诱变使昆虫病原真菌的形态、生长速度、产孢量、致病力等多项生物学特性发生了不同程度的变异,变异率高,变异趋向有正负双向性。测定结果表明航天诱变菌株HM189-68s、HM189-32c、HM189-127和HM189-17的生长特性和致病力优于原始菌株。航天诱变为选育生物防治优良菌株提供了新途径。     

Effects of pH shifts, bile salts, and glucose on sporulation of Clostridium perfringens NCTC 8798.

The sporulation of Clostridium perfringens NCTC 8798 was studied after exposing vegetative cells to: pH values of 1.5 to 8.0 in fluid thioglycolate broth (for 2h) and then transferring them to Duncan-Strong (DS) sporulation medium; sodium cholate or sodium deoxycholate (0.3 to 6.5 mM) in DS medium; or Rhia-Solberg medium with 0.4% (wt/wt) starch, glucose, or both added at 0 to 55 mM. At pH 1.5, no culturable heat-resistant spores were formed. For cells exposed to pH 3.0, 4.0, 5.0, or 6.0, increases in heat-resistant spores were not seen until after a lag of 12 to 13 h, whereas the lag was only 2 to 3 h for cells exposed to pH 7.0 or 8.0. Maximal spore crops were produced after only 6 to 8 h for cells exposed to pH 7 or 8, but 16 to 18 h was required for production of maximal spore crops by cells exposed to the lower-pH media. The addition of sodium cholate (3.5 to 6.5 mM) to DS medium only slightly reduced the culturable heat-resistant spore count from 1.9 X 10(7) to 3 X 10(6)/ml. The addition of 1.8 mM or more sodium deoxycholate reduced the culturable heat-resistant spore count to less than 10/ ml. When either starch or glucose alone was added to Rhia-Solberg medium there was no production of culturable heat-resistant spores, but a combination of 0.4% (wt/wt) starch and 4.4 mM glucose yielded 6 X 10(5) spores/ml. The spore production remained at this level for glucose concentrations of 6 to 22 mM, but then declined to about 3 X 10(3) spores per ml at higher concentrations.     

Effects of pH shifts, bile salts, and glucose on sporulation of Clostridium perfringens NCTC 8798

The sporulation of Clostridium perfringens NCTC 8798 was studied after exposing vegetative cells to: pH values of 1.5 to 8.0 in fluid thioglycolate broth (for 2h) and then transferring them to Duncan-Strong (DS) sporulation medium; sodium cholate or sodium deoxycholate (0.3 to 6.5 mM) in DS medium; or Rhia-Solberg medium with 0.4% (wt/wt) starch, glucose, or both added at 0 to 55 mM. At pH 1.5, no culturable heat-resistant spores were formed. For cells exposed to pH 3.0, 4.0, 5.0, or 6.0, increases in heat-resistant spores were not seen until after a lag of 12 to 13 h, whereas the lag was only 2 to 3 h for cells exposed to pH 7.0 or 8.0. Maximal spore crops were produced after only 6 to 8 h for cells exposed to pH 7 or 8, but 16 to 18 h was required for production of maximal spore crops by cells exposed to the lower-pH media. The addition of sodium cholate (3.5 to 6.5 mM) to DS medium only slightly reduced the culturable heat-resistant spore count from 1.9 X 10(7) to 3 X 10(6)/ml. The addition of 1.8 mM or more sodium deoxycholate reduced the culturable heat-resistant spore count to less than 10/ ml. When either starch or glucose alone was added to Rhia-Solberg medium there was no production of culturable heat-resistant spores, but a combination of 0.4% (wt/wt) starch and 4.4 mM glucose yielded 6 X 10(5) spores/ml. The spore production remained at this level for glucose concentrations of 6 to 22 mM, but then declined to about 3 X 10(3) spores per ml at higher concentrations.     

Purification of glucose oxidase from complex fermentation medium using tandem chromatography

A fast and efficient purification method for recombinant glucose oxidase (rGOx) for flask fermentation scale (up to 2L) was designed for the purposes of characterization of rGOx mutants during directed protein evolution. The Aspergillus niger GOx was cloned into a pYES2-alphaMF-GOx construct and expressed extracellularly in yeast Saccharomyces cerevisiae. Hydrophobic interaction (HIC)/size exclusion (SEC)-tandem chromatographic system was designed for direct purification of rGOx from a conditioned complex expression medium with minimum preceding sample preparation (only adjustments to conductivity, pH and coarse filtering). HIC on Butyl 650s (50 mM ammonium acetate pH 5.5 and 1.5 M ammonium sulphate) absorbs GOx from the medium and later it is eluted by 100% stepwise gradient with salt free buffer directly into SEC column (Sephadex 200) for desalting and final polishing separation. The electrophoretic and UV-vis spectrophotometric analyses have proven enzyme purity after purification.     

Colony-forming capacity of porcine liver epithelial cells in culture

Previously, we reported the presence of certain nonparenchymal epithelial cells (NPECs) in adult porcine livers that demonstrate differentiation patterns including an emergence of duct-like structures (DLSs) in the colonies. In the present study, we examined the effect of supplements to the NAIR-1 medium (Dulbecco modified Eagle medium [DMEM]-F12 containing 5% fetal bovine serum [FBS] and 11 supplements) used in these cultures on formation of DLSs-emerged colonies (type I colonies). No type I colonies were observed in the cultures of the nonparenchymal cell fraction when Roswell Park Memorial Institute-1640 medium or DMEM-F12 (1:1) supplemented with 5% FBS was used as the culture medium. NAIR-1 medium without each component did not produce any significant results. No type I colonies were formed when epidermal growth factor, and hydrocortisone and insulin mixture (A) or nicotinamide and l-ascorbic acid phosphate magnesium salt (Asc2P) mixture (B) was added to the DMEM-F12 medium supplemented with 5% FBS. However, when a combination of A and B was added, colonies were formed at a significant level. Together, the number of type I colonies was increased in the combination of A and B containing a higher concentration of Asc2P. We conclude that NPECs need a mixture of Asc2P and other components as supplements for type 1 colony formation.     

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

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

Studies on the mechanism of the osmoresistance of spores of Bacillus subtilis

AIMS: To determine the reason that spores of Bacillus species, in particular Bacillus subtilis, are able to form colonies with high efficiency on media with very high salt concentrations. METHODS AND RESULTS: Spores of various Bacillus species have a significantly higher plating efficiency on media with high salt concentration (termed osmoresistance) than do log or stationary phase cells. This spore osmoresistance is higher on richer media. Bacillus subtilis spores lacking various small, acid-soluble spore proteins (SASP) were generally significantly less osmoresistant than were wild-type spores, as shown previously (Ruzal et al. 1994). Other results included: (a) spore osmoresistance varied significantly between species; (b) the osmoresistance of spores lacking SASP was not restored well by amino acid osmolytes added to plating media, but was completely restored by glucose; (c) the osmoresistance of spores lacking SASP was restored upon brief germination in the absence of salt in a process that did not require protein synthesis; (d) significant amounts of amino acids generated by SASP degradation were retained within spores upon germination in a medium with high but not low salt; (e) slowing but not abolishing SASP degradation by loss of the SASP-specific germination protease (GPR) did not affect spore osmoresistance; (f) sporulation at higher temperatures produced less osmoresistant spores; and (g) spore osmoresistance was not decreased markedly by the absence of the stress sigma factor for RNA polymerase, sigmaB. CONCLUSIONS: Spore osmoresistance appears as a result of three major factors: (1) specific characteristics of spores and cells of individual species; (2) the precise sporulation conditions that produce the spores; and (3) sufficient energy generation by the germinating and outgrowing spore to allow the spore to adapt to conditions of high osmotic strength; the substrates for this energy generation can come from either the endogenous generation of amino acids by SASP degradation or from the spore's environment, in the form of a readily taken up and metabolized energy source such as glucose. SIGNFICANCE AND IMPACT OF STUDY: These results provide information on the mechanisms of spore osmoresistance, a spore property that can be of major applied significance given the use of high osmotic strength with or without high salt as a means of food preservation.