An improved selective medium, Tryptose-sulfite-cycloserine (TSC) agar, for the enumeration of Clostridium perfringens is described. It consists of the same basal medium as Shahidi-Ferguson-perfringens (SFP) agar, but with 400 p,g of D-cyclOserine per ml substituted for the kanamycin and polymyxin. Tolerance of C. perfringens for D-cycloserine, its production of lecithinase, and its ability to reduce sulfite were used as the basis for development of this medium. Comparisons were made between TSC and SFP agars for the recovery of vegetative cells of C. perfringens by using statistical methods. The results showed that TSC allowed virtually complete recovery of most of the C. perfringens strains while inhibiting practically all facultative anaerobes tested. SFP agar allowed a slightly higher rate of recovery of C. perfringens but was found to be much less selective.

The survival of four Salmonella strains in river water microcosms was monitored by culturing techniques, direct counts, whole-cell hybridization, scanning electron microscopy, and resuscitation techniques via the direct viable count method and flow cytometry. Plate counts of bacteria resuspended in filtered and untreated river water decreased several orders of magnitude within the first week of incubation, while they did not decrease as rapidly in autoclaved water. In situ hybridization studies suggested a rapid decrease in ribosomal content, as determined by the drastic decrease in the number of detectable cells after 72 h. In contrast, direct counts remained relatively constant during 45 days in all microcosoms. Although the culturable counts of two bacterial strains in filtered water after 31 days represented approximately 0.001% of the total counts, direct viable counts and resuscitation studies with a dilution series suggested that the number of viable bacteria was at least four orders of magnitude higher. Additionally, notable changes in forward scatter and in nucleic acid content were observed only after 4 h of nutrient amendments by flow cytometry. However, cells from the resuscitation experiments did not grow on solid media unless cell-free supernatant from viable cultures was added during the resuscitation period. The results in this study suggest the presence of a not immediately culturable status in Salmonella.

In an outbreak of gastrointestinal illness caused by consumption of home-grown raw vegetable sprouts contaminated by Bacillus cereus, victims developed symptoms after an incubation period of 6-15 hours. Four persons initially experienced nausea and vomiting, and this was followed in 3 cases by abdominal cramps and diarrhea. Bacteriologic investigation indicated that B. cereus on unsprouted seeds proliferated during germination in a commercially sold seed sprouting kit and reached levels in excess of 10(7) per gram. B. cereus isolated from the incriminated sprouts exhibited enterotoxigenic activity when tested by the ligated rabbit ileal loop technique, the dermal reaction in guinea pigs, and the rabbit skin capillary permeability test. The diversity of symptoms and incubation periods attributed to B. cereus requires analysis for this often overlooked organism whenever food-borne gastroenteritis is suspected.

A reversed passive latex agglutination (RPLA) test kit for Clostridium perfringens enterotoxin (CPE) marketed by the Denka-Seiken Co., Tokyo, Japan, was evaluated by using culture supernatant fluids and extracts from feces of food poisoning patients. Nanograms of CPE were detectable with the assay and the reaction was specific, as shown by parallel activity in a double antibody enzyme-linked immunosorbent assay (ELISA). Although less sensitive, the RPLA method is easier to perform than the ELISA and counterimmunoelectrophoresis, both of which require special test reagents and equipment not generally available.

Sprouting seeds (alfalfa, mung bean and wheat) were purchased at local health food stores and examined for Bacillus cereus by the official AOAC method. Of 98 units collected, 56 (57%) were positive for B. cereus at levels ranging from 3 to >500 per g. Population levels of B. cereus on sprouts grown from naturally contaminated seeds in a home sprouting kit ranged from a mean of log10 3.72 for alfalfa to 5.39 for wheat; the log10 mean for mung bean sprouts was 4.52. Washing contaminated sprouts for 10 min with warm tap water as recommended by the manufacturer of the sprouting kits reduced the B. cereus count for mung bean sprouts by approximately one log unit but was less effective for wheat sprouts. B. cereus populations large enough to cause food poisoning (>105/g) frequently remained on wheat sprouts even after three wash cycles, and significant numbers of viable B. cereus remained on wheat sprouts even after cooking for 20 min.