The problem here is that gas gangrene may have settled in dead tissue. The most likely causative agent of gas gangrene is Clostridium perfringens, a gram-positive bacterium that forms endospores. It is an obligate anaerobic that develops in tissues without oxygen. Since dead tissue is no longer supplied with oxygen from the circulatory system, dead tissue provides pockets with an ideal environment for the growth of C. perfringens. Examination of mandatory anaerobes requires special equipment. Mandatory anaerobic bacteria should be grown under oxygen-free conditions. The most common approach is culture in anaerobic glass (Figure 9.30). Anaerobic glasses contain chemical packs that remove oxygen and release carbon dioxide (CO2). An anaerobic chamber is a closed box from which all oxygen is removed. Gloves sealed at the box openings allow crops to be handled without exposing the crop to air (Figure 9.30). The enzyme catalase converts hydrogen peroxide into water and oxygen, as shown in reaction 3.

According to standard methods, 10 g of each sample of ham, chicken or sliced minced meat were mixed with 90 ml of sterile peptone water solution (0.1%) for 1 min in a sterile gastric pouch (Stomacher Lab System Model 400; Colworth, London, UK) with a Stomacher (Colworth Stomacher 400; Colworth, London, United Kingdom). Subsequent dilutions were obtained by mixing 1 ml of aliquot homogenate with 9 ml of peptonic water solution (0.1%). These dilutions were then analyzed in parallel by aerobic colony count on standard nutrient agar plates and counted visually after an incubation period of 48 h at 30 °C and by fluorescence-based dosing, where they were then delivered with the standard working oxygen sensor in microwaves and monitored as before on a plate reader to determine the times required to achieve threshold intensities. The CFU/ml of the agar plates were then compared to those calculated using calibration curves to convert threshold times to CFU/ml. Aerobic respiration explains the need for oxygen and higher growth rates under aerobic conditions. But where there is O2, there are also by-products of oxygen or reactive oxygen species (ROS) that need to be detoxified. Even organisms that don`t use aerobic respiration need a way to break down some of the ROS that can form from molecular oxygen. Three main enzymes break down these toxic by-products: superoxide dismutase, peroxidase and catalase. Each catalyzes a different reaction.

Reactions of the type in reaction 1 are catalyzed by peroxidases. Staphylococci and enterobacteriaceae are examples of facultative anaerobes. Staphylococci are located on the skin and upper respiratory tract. Enterobacteriaceae are mainly found in the intestines and upper respiratory tract, but can sometimes spread to the urinary tract where they can cause infection. It is not uncommon to see mixed bacterial infections in which facultative anaerobes deplete oxygen and create an environment in which obligate anaerobes can thrive. We can easily observe different molecular oxygen requirements by growing bacteria in thioglycolate tube cultures. A test-tube culture begins with an autoclaved thioglycolate medium, which contains a small amount of agar-agar, to allow mobile bacteria to move through the medium. Thioglycolate has strong reducing properties and autoclaving removes most of the oxygen. The tubes are inoculated with the bacterial cultures to be tested and incubated at the appropriate temperature. Over time, oxygen slowly diffuses from above through the culture of the thioglycolate tube.

Bacterial density increases in the area where oxygen concentration is most appropriate for the growth of that particular organism. For active growing and rapidly breathing cells, such as those used in this study, sealing samples to exclude oxygen in ambient air, which would otherwise interfere with the destruction of oxygen gradients associated with cellular respiration, seems unnecessary and superfluous. In this case, a mineral oil seal applied to each sample was found to have little effect on signal profiles (Table 2).2). At the same time, sealing the entire plate with an adhesive film (Mylar) proved desirable to minimize evaporation and cross-contamination of samples in long-term experiments and avoid accidental spills. Other clostridia responsible for serious infections include C. tetani, the causative agent of tetanus, and C. perfringens, which causes gas gangrene. In both cases, the infection begins in the necrotic tissue (dead tissue that is not oxygenated by the bloodstream). This is why deep puncture wounds are associated with tetanus.

If tissue death is accompanied by a lack of blood circulation, gangrene is still a danger.

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