Combined effects of ionizing-irradiation and different environments on Clostridium botulinum type E spores.

We examined the combined effects of gamma-radiation (24 degrees C) on spores of Clostridium botulinum-type Eklund strain suspended in different gas-saturated Na-phosphate buffer in absence or presence of protectors or sensitizers. Response surface methodology (RSM) was also used to ascertain the effects of radiation on the recovery of spores using a medium containing various levels of NaCl or Na-thioglycollate. The former (< 0.5%) decreased viable spore counts, but the latter (0.15%) did not. Irradiation inactivation of Eklund spores was most effective in air-saturated buffers compared to N2O and N2 gas. The Na2-EDTA (0.01 M) was the most efficient radioprotector of spores due to its reactivity toward hydroxy radicals, followed by t-butanol (0.1 M) in NO2 or N(2)-saturated buffers, respectively. Catalase (10.0 mg ml(-1)) and DL-cysteine (0.1 mM) sensitized the spores during irradiated N2O or N(2)-saturated buffers, and NaCl (0.01 M) only sensitized spores in N2 environment. Spores frozen at -75 degrees C for 30 days and thawed prior to use were more sensitive to radiation damage compared to freshly prepared spores. Glycerol (15%), in Na-phosphate buffer (pH 7.0, 0.06 M), protected Eklund spores and increased the number of spores from 10(6) to 10(11) colony forming unit (CFU) ml(-1), and enhanced their radiosensitivities. Seven strains of C. botulinum type E were screened for plasmids and strain BL764 had two plasmids (15.8 and 46.8 mDa), BL4028 also had two (4.4 and 13.2 mDa), BL4850 contained only one (4.9 mDa), whereas EQA, BL211, Eklund, and Beluga had none. Gamma-Radiation (10 kGy, absorbed dose) cured the 15.8-mDa plasmid in strain BL764, but its absence yielded no changes in toxigenicity.

Bacterial amelioration of bauxite residue waste of industrial alumina plants.

The high alkali content of bauxite residue deposits from alumina production plants in industrial nations poses a challenge to reestablish flora and fauna at the deposit sites. The present study demonstrated that low levels of injured bacterial cells in the bauxite residue actively grew using various added nutrients and/or hay. The organisms grew from less than 10 to more than 10(9) cells g(-1) bauxite residue and formed organic acids that lowered the pH from 13 to about 7.0. A total of 150 cultures was isolated from treated bauxite residue and included species of Bacillus, Lactobacillus, Leuconostoc, Micrococcus, Staphylococcus, Pseudomonas, Flavobacterium and Enterobacter. Scanning electron micrographs demonstrated that untreated particles (control) of the bauxite residue were clumped together, and in treated bauxite residue these particles were highly dispersed with microcolonial structures. Furthermore, the treated bauxite residue supported growth of several plants and earthworms that survived for over 300 days. In a test plot bioremediation on a residue deposit at Alcoa Point Comfort, TX, the Bermuda grass hay used was effective mulch material and encouraged water filtration, leading to establishment and growth of salt-tolerant vegetative species.

Detection of Clostridium botulinum type F using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify a portion of the Clostridium botulinum type F toxin gene. An 1137-bp fragment was amplified from 11 different strains of type F C. botulinum with primers derived from the published sequence of type F strain no. 202. This fragment was not amplified from the DNA of C. botulinum types A, B and E, or from other clostridial organisms examined. When used as a hybridization probe, the 1137-bp PCR-generated fragment generated from one of the type F strains (the proteolytic strain type F Langeland) hybridized to the PCR products from all other type F toxin-producing strains tested. Portions of fragments amplified from the type F Langeland strain were sequenced. The sequence of this strain was found to exhibit approximately 3% variation from the published sequence of the non-proteolytic type F strain no. 202. Primers designed to pair with the regions of maximum sequence variation between strain 202 and the Langeland strain gave amplification products only with DNA from type F strains that exhibited the same proteolytic properties as the strain from which the primer sequences were derived. These findings underscore the need to consider variations in sequence when designing oligonucleotide probes and PCR primers in order to avoid false negative results.

Monoclonal antibody to type F Clostridium botulinum toxin.

Hybridomas synthesizing monoclonal antibodies (MAbs) against type F Clostridium botulinum toxin were developed. MAb from one stable hybridoma, hybridoma 223, consisted of kappa light chains and an immunoglobulin G subclass 2a heavy chain. This MAb was used in a double-sandwich enzyme-linked immunosorbent assay to detect type F toxin in foods, culture fluids, and purified toxin preparations. The sensitivity of the double-sandwich enzyme-linked immunosorbent assay was approximately 10 mouse lethal doses of toxin per ml of toxic fluid.

Monoclonal antibody for the detection of Clostridium botulinum type A toxin.

Hybridomas producing monoclonal antibodies against type A Hall strain Clostridium botulinum toxin were generated by fusing mouse myeloma cell line P3-X63-Ag8.653 with spleen cells of Balb/c mice immunized with C. botulinum type A toxoid. The monoclonal antibody from one hybridoma, identified as No. 424, was selected from 61 others for its high antibody titre. This monoclonal antibody was used in a double-sandwich enzyme-linked immunosorbent assay (ELISA) system to detect type A toxin in culture fluids and in foods. The monoclonal antibody did not react with either C. botulinum toxin types B, C, D, E and F or with other clostridial species tested. This particular monoclonal antibody (No. 424) did not neutralize type A toxin in the mouse bioassay procedure but detected approximately 10 mouse lethal doses of type A toxin/ml culture fluid. Monoclonal antibody and rabbit antitoxin to type A C. botulinum toxin were useful in a double-sandwich ELISA for the rapid and specific detection of type A toxic fluids in culture and in food samples.

Physicochemical factors affecting ethanol adsorption by activated carbon.

Powder and granular activated charcoal were evaluated for ethanol adsorptivity from aqueous mixtures using an adsorption isotherm. Ethanol adsorption capacity was more pronounced at 25 degrees C as compared to 5, 15, and 40 degrees C. When pH of the ethanol-buffer mixture (0.09 ionic strength) was changed from acidic (2.3) to neutral and then to alkaline (11.2), ethanol adsorption was decreased. Increasing ionic strength of the ethanol-buffer mixtures from 0.05 to 0.09 enhanced ethanol adsorption but a further increase to 0.14 showed no significant effect. Ethanol adsorption was more efficient from an aqueous ethanol mixture as compared to semidefined and nondefined fermentation worts, respectively. Heating granular charcoal to 400 degrees C for 1 h and 600 degrees C for 3 h in N(2) increased ethanol adsorptivity and heating to 1000 degrees C (1 h) in CO(2) decreased it when ethanol was removed from dilute solutions by simple pass adsorption in a carbon packed column. Granular charcoal was superior to powdered charcoal and an inverse relationship was noted between the weight of the granular carbon bed in the column and ethanol adsorbed/g carbon. Decreasing the column feed flow rate from 7.5 to 2.0 L aqueous ethanol/min increased the adsorption rate.

Transfer of Firmly Attached 32P- Salmonella typhimurium from Raw Poultry Skin to Other Surfaces.

Salmonellae adhere firmly to poultry skin during processing. Loosely attached bacteria cross-contaminate work surfaces. This study was undertaken to determine if firmly attached bacteria present a health hazard through transfer to work surfaces. Attached 32P-labeled S. typhimurium cells were serially rinsed with 2 to 4 L of Salmonella -free potable tap water or with sterile 0.85% NaCl. Rinsing removed 61 to 89% of attached labeled cells. However, after rinsing, 11 to 39% of cells remained attached, and of these, 3 to 10% were able to detach and transfer from skin to stainless steel surfaces. It was concluded that large rinse volumes may not remove all attached salmonellae from poultry skin surfaces and the potential for cross-contamination does exist.

Selective and differential medium for detecting Clostridium botulinum.

A selective and differential growth medium was developed for detection of Clostridium botulinum types A, B, and F. The medium consisted of peptone-glucose-yeast extract agar supplemented with cycloserine, 250 micrograms/ml; sulfamethoxazole, 76 micrograms/ml; and trimethoprim, 4 micrograms/ml as selective inhibitors and various types and levels of botulinal antibodies for type differentiation in the immunodiffusion reaction. Growth of proteolytic types of C. botulinum were not affected by the incorporation of the selective agents, but some nonproteolytic types were suppressed. Cross-reactions between types A and B were visually distinguishable, whereas cross-reactions between type F and Clostridium sporogenes did not occur at the optimum antibody titer. Optimum antibody titer varied with toxin type. The proposed selective differential medium should be valuable in isolating and typing of proteolytic C. botulinum types A, B, and F from samples containing mixed microbial populations.

Acid hydrolysis of sweet potato for ethanol production.

Studies were conducted to establish optimal conditions for the acid hydrolysis of sweet potato for maximal ethanol yield. The starch contents of two sweet potato cultivars (Georgia Red and TG-4), based on fresh weight, were 21.1 +/- 0.6% and 27.5 +/- 1.6%, respectively. The results of acid hydrolysis experiments showed the following: (1) both hydrolysis rate and hydroxymethylfurfural (HMF) concentration were a function of HCL concentration, temperature, and time; (2) the reducing sugars were rapidly formed with elevated concentrations of HCl and temperature, but also destroyed quickly; and (3) HMF concentration increased significantly with the concentration of HCl, temperature, and hydrolysis time. Maximum reducing sugar value of 84.2 DE and 0.056% HMF (based on wet weight) was achieved after heating 8% SPS for 15 min in 1N HCl at 110 degrees C. Degraded 8% SPS (1N HCl, 97 degrees C for 20 min or 110 degrees C for 10 min) was utilized as substrate for ethanol fermentation and 3.8% ethanol (v/v) was produced from 1400 mL fermented wort. This is equal to 41.6 g ethanol (200 proof) from 400 g of fresh sweet potato tuber (Georgia Red) or an ethanol yield potential of 431 gal of 200-proof ethanol/acre (from 500 bushel tubers/acre).

Immunodiffusion method for detection of type A Clostridium botulinum.

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.