Problems encountered with the capillary tube immunodiffusion method for detection of botulinal toxin.

By using antitoxin specific for the neurotoxin molecule, the capillary tube immunodiffusion method did not detect low levels of crystalline toxin. Reactions described earlier with crude toxin and less specific antitoxin were probably due to nontoxigenic botulinal antigens.

Development of culture vessel for sterility testing of bandages and other medical devices.

A cylindrical glass culture vessel equipped with a vented nylon closure was developed and evaluated for sterility testing. The sterility testing cylinder (STC), made in 2 sizes, accommodates 250 or 400 mL culture broth. A tight-fitting, skirted nylon cap protects the upper part of the cylinder, and a membrane filter in a recessed opening allows venting during autoclaving and ensures sterility during removal and replacement of the cap. The configuration of the vessel in terms of the opening size and ratio of the horizontal to vertical cross section provides depths desirable for testing many shapes of medical devices, including elongated and narrow ones, without excessive waste of medium. They are easy to charge with medium, and to autoclave, store, inoculate, and observe for growth.

Detection and growth of enteropathogenic Escherichia coli in soft ripened cheese.

The organism most frequently encountered during the 1971 outbreak of enteropathogenic Escherichia coli (EPEC) in soft ripened cheese was a strain that failed to ferment lactose broth within 48 h. Since existing methods for E. coli are dependent upon fermentation of this sugar, such strains can remain undetected, particularly when present in low numbers. Therefore a cultural testing procedure was developed to insure isolation of both lactose-positive and -negative strains. This method used GN broth, modified by substituting lactose and arabinose for glucose and D-mannitol, as an enrichment medium. MacConkey agar, used as a plating medium, was modified by substituting arabinose for half the lactose. The cultural procedure was used in conjunction with a fluorescent antibody method to screen cheese for the presence of presumptive enteropathogenic E. coli. Suspected isolates were subjected to further biochemical and serological testing and identified as members of specific serogroups. These methods were used for the analysis of over 2,000 wheels of cheese; over 10% of the samples tested were found to contain strains belonging to six different serogroups associated with diarrheal diseases. No attempt was made to confirm pathogenicity by in vivo tests. Enumeration of E. coli in cheese showed that numbers increased during storage. Cheese with less than 10 organisms/g initially increased to over 10-5 at room temperature and over 10-3 at 4 C within 10 days. With higher initial counts, levels up to 10-9 were found at 4 C. These studies showed that the high levels of E. coli encountered in these products cannot be used as a direct indicator of post-processing contamination.

Rapid detection of Clostridium botulinum toxin by capillary tube diffusion.

A micro capillary agar-gel diffusion system for the detection of botulinal toxin in foods and cultures was developed and evaluated. Toxins types A, B, and E, produced in culture broth with and without added trypsin, and type E toxin, produced in inoculated canned clams, were tested with this system and with the mouse bioassay procedure. With nontrypsinized toxin, the capillary diffusion system detected as little as 100 minimal lethal doses (MLD) per ml but was effective only at higher levels, 10(6) to 1.5 x 10(7) MLD/ml, when used with trypsinized toxin. The inability to detect lower levels of trypsinized toxin was due to thioglycolate present in the medium used to produce toxin. Evidently, trypsinization of toxin produces polypeptides still held together by disulfide bonds. Cleavage of these bonds by reduction with thioglycolate reduces the sensitivity of the capillary method. Trypsinized toxin produced in broth without thioglycolate was detected as readily as nontrypsinized toxin. Toxin was detected in canned clams containing as low as 100 MLD/ml. No cross-reactions were observed with type E toxin and types A and B antitoxins. Extensive studies using the capillary method for detecting types A and B toxins were not performed; however, a suspected sample of commercially canned mushrooms gave a positive type B reaction but not a type A reaction. This typing was confirmed later by the mouse bioassay. Toxin was present at a level of 100 MLD/ml. The procedure developed may prove useful as a rapid screening method for the detection of botulinal toxin in foods, with final identification made by using the mouse bioassay.