Journal: mSphere
Article Title: Comparative pathogenomic analysis reveals a highly tetanus toxin-producing clade of Clostridium tetani isolates in Japan
doi: 10.1128/msphere.00369-23
Figure Lengend Snippet: Isolation and identification process of C. tetani . The soil samples were initially added to cooked meat medium (CMM), a widely used culture medium for Clostridium spp., and subjected to incubation at 37°C for 2–3 days. This was performed after heated and unheated treatments to facilitate subsequent bacteriological, biochemical, genetic, and immunological testing. To isolate and identify C. tetani , a CM culture solution was inoculated dropwise onto the blood agar medium, followed by anaerobic incubation at 37°C for 18–24 h. C. tetani exhibited swarming behavior on the medium (arrow). Gram staining of the swarming tips on the medium revealed filamentous long rods. The swarm tip was introduced into fresh CMM; C. tetani was cultured in pure culture; and the spores were confirmed using Gram staining. The presence of the tetanus toxin gene was verified through PCR. M, DNA ladder; 1 and 5 indicate positive control; 2 and 6 indicate negative control; 3, 4, 7, and 8 denote isolates. If the tetanus toxin gene was present, PCR products were detected at 331 bp for GAT1/GAT2 and 229 bp for GAT5/GAT6 using the two sets of primers. For biochemical characterization, a pure culture of C. tetani from CMM was anaerobically incubated at 37°C for 18–24 h on a blood agar medium. The bacillus solution was then prepared and analyzed using API kit. The presence of tetanus toxin in the mice was confirmed by assessing its biological activity. The tetanus culture supernatant solution was subsequently injected into the inner left thigh of two mice, and their condition was observed for 4 days. The mice exhibited symptoms of tetanus, including characteristic left hind limb protrusion, tonic paralysis, limping, and gradual decline in walking ability.
Article Snippet: The horseradish peroxidase (Shigma, Tokyo, Japan)-labeled anti-tetanus toxin mouse monoclonal antibody (TH-11; Fujifilm, Tokyo, Japan) was diluted 1,000-fold using a conjugate diluent solution.
Techniques: Isolation, Incubation, Staining, Cell Culture, Positive Control, Negative Control, Activity Assay, Injection
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