# A Review on Food Safety: The Case of _Citrobacter_ sp., Fish and Fish Products
## 5 Isolation and Detection of Enterobacteria in Food Case: _Citrobacter_ sp.
in food samples (including fish), a method has been developed by preparing decimal dilutions of the sample and using a culture medium consisting of 0.5% tryptone, 0.5% yeast extract, 2.7% L-histidine-2 HCl, 0.5% NaCl, 0.1% CaCO\({}_{3}\), 2.0% agar, and 0.006% bromoresol purple (pH 5.3), incubating the plates at 37 \({}^{\circ}\)C/72 h. After these procedures, the presence of histamine-forming colonies make the medium color vary from yellow to purple, indicating an increase in pH and a positive reaction due to histamine accumulation [23; 56; 57]. _Determination of Biogenic Amines (Histamine) in Fish and Fish Products_  
Histamine, once generated in food, cannot be eliminated by any heat treatment, so to guarantee the safety of fish and fish products, in relation to histamine content, various colorimetric, immunoassay, and chromatographic methods have been developed and proposed, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), and thin-layer chromatography (TLC) of qualitative, semiquantitative, and quantitative natures [62; 78; 79; 80; 81]. The choice of method to be used considers its efficiency, sensitivity, simplicity, speed, cost of materials, and used reagents [82]. Among the different methods are fluorometric-AOAC 977.13, which is the most used procedure for fish samples. It is sensitive and reproducible, but it is also complex and consumes a lot of analysis time; this methodology presents extraction with methanol and passage through an ion exchange column, as well as derivatization and quantification with external standards [55; 68]. In the spectrophotometric method, the sample is subjected to processes of homogenization, centrifugation, and extraction with saline and butanol solutions, measuring the absorbance of extracts at 496 nm. The histamine concentration in the sample is calculated by means of a calibration curve using a histamine standard [23]. Immuno enzymatic methods are useful for the detection of histamine in fish and are used for studies in case of surveillance of conditions and adequate hygienic practices for product preservation, suspected intoxication, and applications in HACCP systems; the method can be qualitative or semiquantitative [80; 83]. The basic principle of an immunoassay catalytic study, such as the enzyme-linked immunosorbent assay (ELISA), is to use an enzyme to detect the binding of antigen to antibody. The enzyme converts a colorless (chromogenic) substrate to a colored product, indicating the presence of an antigen-binding antibody. Therefore, the ELISA can be used to detect the presence of antigens or antibodies in the sample, depending on its design [84]. The commercial kits available to detect histamine in fresh fish products and meals can only discriminate products with less than 50 ppm, but for health regulation purposes, such as the European Union Regulation 2073/2005 that considers several detection levels (100, 200, and 400 mg/kg) of histamine content in fishery products, further confirmation of the positive sample is needed with higher-precision analytical techniques based on HPLC [80]. In recent years, various methods based on high-performance liquid chromatography (HPLC) and/or gas chromatography (GC) have been developed for the determination of histamine in fish, which require chemical derivatization (dansyl chloride) or O-phthalaldehyde (OPA) to improve the detection of biogenic amine by UV-vis absorption or fluorescence, and where the derivatization step can be pre-column, column, or post-column, and quantification can be represented by a standard curve. These methods are fast, sensitive, and reproducible for the determination of one or more biogenic amines [55; 56; 80; 85; 86; 87]. Regardless of the method carried out for the determination of histamine in fish and fish products, when they are sent to the laboratory, both the suspicious fish and/or residues of the same batch must be transported in a cold chain (frozen) to avoid the new formation of histamine during the transfer process for analysis.