Biomedical subjects
Publications and source records attributed to L R Beuchat.
Airborne Aspergillus flavus spores were rarely detected throughout the 1975 growing season by the agar plate method at cornfield sites in Georgia, Illinois, Maryland, South Carolina, and Virginia.
Metabolic inhibitors were added to a culture medium inoculated with theramlly stressed Vibrio parahaemolyticus to obtain information pertaining to biosynthetic processes required for recovery from heat damage. Ribonucleic acid and protein syntheses, in addition to membrane repair, were required during recovery of injured cells. Neither nucleic acid nor Mg2+ leakage was noted to occur during the time cells were subjected to heat stress. Studies revealed that Mg2+ was apparently taken up by cells of V. parahaemolyticus during the first 30 min after thermal treatment, indicating a possible increased requirement for Mg2+ for membrane and/or ribosome stability and repair.
Mid- to late logarithmic growth phase cells of Vibrio parahaemolyticus grown in tryptic soy broth (TSB) containing 0.5, 3.0, and 7.5% NaCl were heated for 8 min at 45 degrees C in 0.1 M phosphate buffer (pH 7.2) containing 3% NaCl. Colony formation on thiosulfate-citrate-bile salts-sucrose agar (TCBS) containing 2% NaCl was greatest for unheated cells that had been grown in 7.5% NaCl-TSB; cells grown in 0.5% NaCl-TSB formed a greater number of colonies on 1.0% NaCl-TCBS. Thermal injury was evident in heated cells, regardless of the NaCl concentration in TSB growth medium. The effects of Mg2+, K+, and Li+ added as chlorides to 0.5% NaCl-TSB on the growth of nonheated and heated V. parahaemolyticus were studied. Lower levels of Mg2+ and slightly higher levels of K+ were required to replace Na+ in TSB inoculated with thermally injured cells that had been originally grown in 3.0 and 7.5% NaCl-TSB. LiCl had an inhibitory effect on both nonheated and heated cells when present in the recovery medium (0.5% NaCl-TSB) at concentrations as low as 0.5%. Increased numbers of colonies were formed by heated cells plated in MgCl2-supplemented TCBS, regardless of the NaCl concentration in the original growth medium. Potassium had little, if any, effect on colony formation by nonheated V. parahaemolyticus recovered on TCBS and may have had a detrimental effect on heat-injured cells.
Ten strains of Byssochlamys fulva and three strains of B. nivea were cultured in a laboratory medium and tested for their ability to produce patulin. Two strains of B. fulva and all three strains of B. nivea produced the mycotoxin. One strain of B. fulva produced patulin in 11 of 13 processed fruit juices, with greatest amounts being produced in blueberry, red raspberry, and boysenberry juices, whereas no patulin was detected in prune or tomato juices. Grown in Concord grape juice at 18, 25, 30, and 38 degrees C, this strain produced the highest patulin concentration at 18 degrees C after 25 days, whereas biomass production was greatest at 25 and 30 degrees C after 20 and 25 days.
Vibrio parahaemolyticus cells were injured by chilling and heating, and their recovery was tested in glucose-salt-Teepol broth (GSTB), tryptic soy broth containing 7% NaCl (TSBS), Horie - arabinose - ethyl violet broth (HAEB), and water blue - alizarin yellow broth (WBAY). Exponential phase cells were more sensitive to cold shock than were stationary phase cells. Exposure of chill-injured V. parahaemolyticus to GSTB and TSBS resulted in 70 to 80% death; about 70% lethality was noted for heat-injured cells inoculated into TSBS. Neither HAEB nor WBAY enrichment media were lethal to stressed cells, although rates of growth were retarded. The 3% NaCl in 0.1 M potassium phosphate (pH 7.0) diluent proved to be most suitable for protecting against inactivation of cold- and heat-injured cells.
Potassium sorbate, sodium benzoate, sulfur dioxide, and diethylpyrocarbonate (DEPC) were tested for their effectiveness in preventing the outgrowth of Byssochlamys nivea Westling ascospores. Sulfur dioxide was the most inhibitory of the test antimycotics, complete inhibition of colony formation occuring in acidified (pH 3.5) potato dextrose agar containing 50 ppm of the preservative. Complete inhibition of B. nivea ascospore outgrowth in grape juice stored for 60 days was noted in the presence of 300 ppm sulfur dioxide, 400 ppm potassium sorbate, and 600 ppm DEPC. Growth was observed in grape juice containing 1000 ppm sodium benzoate. The presence of up to 100 ppm potassium sorbate in grape juice during heat activation appears to have a stimulatory effect on breaking dormancy, while the other test preservatives at this concentration decrease the heat resistance of B. nivea ascospores. The time elapsed between heat shock and exposure to DEPC or sodium benzoate is critical with respect to the sensitivity of ascospores to these preservatives.
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Two peptones were extracted from raw shrimp waste after autolytic digestion. Digests were derived from shrimp heads and shrimp hulls, both of which are by-products of the shrimp processing industry. Digests were evaluated for suitability in supporting growth of microorganisms by measuring the total cell mass produced by five genera of bacteria and five genera of fungi in broths formulated from the peptones. Comparison was made to five commercially available medium preparations and a catfish peptone. A 0.5% solution of the lyophilized shrimp head digest, heated at 121 C for 15 min, resulted in a cloudy suspension. However, the digest supported excellent growth of fungi and good growth of bacteria. A heated 0.5% solution of the hull digest was clear and supported good growth of both bacteria and fungi.
Vibrio parahaemolyticus was grown in tryptic soy broth (TSB) containing NaCl levels of 0.5, 3.0, and 7.5% (wt/vol). Cultures incubated at 21, 29, and 37 C were harvested in late exponential phases and thermal death times at 47 C (D47 c; time at 47 C required to reduce the viable population by 90%) were determined in phosphate buffer containing 0.5, 3.0, and 7.5% NaCl. At a given NaCl concentration in the growth medium, D47 c values increased with elevated incubation temperatures and with elevated levels of NaCl in the heating menstrua. Differences in thermal resistance of cells cultured at a particular temperature were greater between those grown in TSB containing 0.5 and 3.0% NaCl than between those grown in TSB containing 3.0 and 7.5% NaCl. D47c values ranged from 0.8 min (grown at 21 C in TSB with 0.5% NaCl) to 6.5 min (grown at 37 C in TSB with 7.5%, heated in 7.5% NaCl buffer). Methyl esters of major phospholipid fatty acids extracted from cells were quantitated. The ratio of saturated to unsaturated fatty acids in cells grown at a given NaCl concentration increased with elevated incubation temperature. At a particular growth temperature, however, saturated to unsaturated fatty acids ratios were lowest for cells grown in TSB containing 3.0% NaCl.
Survival of Salmonella senftenberg 775W, S. anatum, and S. typhimurium during exposure to currently practiced, as well as abusive, pecan processing and storage conditions was studied. Thermal treatments normally carried out during the processing of pecans are inadequate to consistently destroy salmonellae in highly contaminated inshell nuts. Pecan nut packing tissue was toxic to salmonellae, thus affording some protection against high initial contamination and subsequent survival of the organisms. Examinations of inoculated inshell pecans stored at -18, -7, 5, and 21 C for up to 32 weeks revealed that the extent of survival was inversely correlated to the storage temperature. S. senftenberg 775W and S. anatum were not detectable on inshell nuts after 16 weeks of storage at 21 C. Little decrease in viable population of the three species was noted on inoculated pecan halves stored at -18, -7, and 5 C for 32 weeks. Due to organoleptic quality deterioration in pecan nutmeats at elevated temperatures, sterilization methods other than thermal treatment appear to be required for the elimination of viable salmonellae from pecan nuts.
Pecan nuts were selected at various points during routine harvesting, and nutmeats were analyzed for gross and internal fungal contamination and for the presence of Aspergillus flavus and A. parasiticus. Fungi were isolated from a large percentage of nutmeats at all points of examination. No correlations could be made between increased incidence of fungi and particular harvesting procedures.
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Vibrio parahaemolyticus was grown at 36 C in tryptic soy broth (pH 7.8) containing added levels of NaCl ranging from 0.5 to 7.9% (wt/wt). The fastest generation time was 16.4 min in tryptic soy broth containing 2.9% NaCl (TSBS) which corresponded to a water activity (a(w)) of 0.992 (+/-0.005). Tryptic soy broth containing lower or higher levels of NaCl resulted in higher or lower a(w), respectively, and slower generation times. Growth was measured turbidimetrically at 36 C in TSBS containing added amounts of NaCl, KCl, glucose, sucrose, glycerol, or propylene glycol. The solutes used to reduce a(w) to comparable levels resulted in extended lag times of varied magnitude, dissimilar growth rates, and different cell numbers. Reduction of a(w) with glycerol was less inhibitory to growth than similar a(w) reductions with NaCl and KCl. Sucrose, glucose, and propylene glycol generally had the greatest effect on extending the lag times of V. parahaemolyticus when the addition of these solutes was made to establish similar a(w) levels lower than 0.992. Minimal a(w) for growth at 15, 21, 29, and 36 +/- 0.2 C for each of four strains of V. parahaemolyticus was tested in TSBS containing added solutes. Reduced a(w) was generally most tolerable at 29 C, whereas higher minimal a(w) for growth was required at 15 C. Solutes added to TSBS to achieve reduction in a(w), minimal a(w) for growth after 20 days, and incubation temperatures were as follows: glycerol, 0.937, 29 C; KCl, 0.945, 29 C; NaCl, 0.948, 29 C; sucrose, 0.957, 29 and 36 C; glucose, 0.983, 21 C; and propylene glycol, 0.986, 29 C. Each of the four strains tested responded similarly to investigative conditions. It appears that minimal a(w) for growth of V. parahaemolyticus depends upon the solute used to control a(w).
Thermal resistance and minimal pH and temperature conditions for growth of Vibrio parahaemolyticus in artificial media containing 3 and 7% sodium chloride were studied. Growth was observed at pH 4.8 and at 5 C.
Bacillus megaterium NRRL B-1368 cells and spores were produced on Trypticase Soy Broth (TSB) and Agar (TSA) containing 3.8 mug of aflatoxin B(1) per ml, analyzed for selected chemical constituents, and compared to cells and spores of B. megaterium produced on nontoxic Trypticase Soy Media. There was an initial 30% kill of cells after inoculation into toxic TSB and during the first 3.5 hr of incubation followed by a logarithmic growth phase in which the generation time was 75 min as compared to 20 min for the control culture. Chemical analyses revealed an increase in protein, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) on both a per cell basis and a per cent dry weight basis when B. megaterium was grown in toxic TSB. There was a concurrent decrease in the total amounts of cellular protein, DNA, and RNA synthesized in toxic TSB. Amino acid analyses of control and test cell walls showed little, if any, difference in cell wall composition. About 97% sporulation of B. megaterium occurred after 3 days on nontoxic TSA although 6 days were required to attain 65% sporulation on toxic TSA. Germination of spores was not inhibited by 4.0 mug of aflatoxin per ml but outgrowth was. No significant differences were observed in the heat resistance, protein, DNA, RNA, or dipicolinic acid content of spores formed on toxic TSA and nontoxic TSA.
Morphologically abnormal cells were produced by Bacillus megaterium NRRL B-1368 in response to aflatoxin B(1). Filamentous forms were characterized by early granulation and unusually large and numerous deposits of poly-beta-hydroxybutyric acid within the cells. Pantoyl lactone was without effect as a reversing agent for the observed inhibition of cell septum formation. B. megaterium cells and spores produced on toxic (3.8 mug of aflatoxin B(1) per ml) and nontoxic Trypticase Soy Broth and Trypticase Soy Agar (TSA) were observed by using phase contrast and electron microscopy. Transfer of aberrant forms to nontoxic TSA yielded macrocolonies with daughter cells morphologically indistinguishable from untreated cells. Agar slide cultures of filamentous cells transferred to nontoxic TSA indicated that normal cells were formed. Electron photomicrographs showed a decreased number of mesosomes in filamentous cells as compared to control cells. There were no observable morphological differences in spores formed on toxic or nontoxic TSA.
Modifications of a commercial 2,450-megahertz microwave oven were made so that 6 ml of microbial suspension could be exposed to the microwave field for various periods of time. The microorganisms were contained in the central tube of a modified Liebig condenser positioned in the approximate geometric center of the oven cavity. Kerosene at -25 C was circulated through the jacket of the condenser during microwave exposure permitting microwaves to reach the microbial suspension. Flow rates of the kerosene were varied to permit the temperature of the suspension to range from 25 to 55 C during microwave exposure. Conductive heating experiments using similar temperatures were also conducted. A thermocouple-relay system was employed to measure the suspension temperature immediately after the magnetron shutoff. Continuous application of microwaves to suspensions of 10(8) to 10(9)Streptococcus faecalis or Saccharomyces cerevisiae per ml appeared to produce no lethal effects other than those produced by heat. Respiration rates of microwave-exposed Scerevisiae were directly related to decreases in viable count produced by increased microwave exposure times.