# 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.
Various generalized methods have been developed in liquid and/or solid media for the isolation and enumeration of enterobacteria in food, which are generally used as indicators of food contamination, inadequate hygiene practices during and after processing, and health risks from the consumption of contaminated food [38], as well as the identification of where _Citrobacter_ species can be found and identified through the use of several differential and selective media. Among the existing methods are the ISO 21528-1:2017 method for detection and enumeration by the most probable number (NMP) technique, and ISO 21528-2:2017 for detection and enumeration by colony count. [PERSON] et al. [74] proposed a method for detection and enumeration by colony count which involves the dilution of the sample in peptone water and subsequent inoculation in a selective culture medium (Red Violet Bile agar) and incubation at 35 \({}^{\circ}\)C/48 h and 44.5 \({}^{\circ}\)C/24 h for subsequent quantification of total coliforms and fecal coliforms, respectively, as well as subsequent identification of colonies through different biochemical tests. In addition, the methods proposed by [PERSON] and [PERSON] [38] present the phases of pre-enrichment, enrichment, and sample dilution in different nutritive culture media, such as peptone water or tryptone soy broth (TSB), Mossel EE broth for enterobacteria and subsequent growth and identification in differential culture medium, such as Violet Red Bile Glucose Agar, and confirmation through various biochemical tests for enterobacteria, such as the oxidase test and Klieger's Iron Agar (KIA) test. On the other hand, sometimes the absence of a relationship between the phenotypic characteristics of the isolate under study and those corresponding to bacterial strains of the type species cause the phenotypic methods to make the most probable but not definitive identification. To face these drawbacks, modern or genotypic and/or proteomic identification methods have been developed and implemented as complementary or alternative procedures that are faster and more sensitive in obtaining results [69; 73]. Therefore, modern technologies for the detection and identification of _Citrobacter_ species in laboratory samples have been developed, which through molecular analysis of the subunit ribosomal RNA gene (16S rRNA) by polymerase chain reaction (PCR) in its different variants, have reported good utility and are even being used for epidemiological studies [21; 35; 40; 42; 70]. Meanwhile, proteome analysis using matrix-assisted laser-desorption ionization and time-of-flight mass spectroscopy (MALDI-TOF MS) to identify strains in culture is also an alternative to traditional methods for greater sensitivity, specificity, and speed in obtaining results [40; 75; 76; 77]. Finally, for the microbiological analysis of a phenotypic nature to determine the presence and quantification of histidine-decarboxylalting and histamine-producing bacteria \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Test** & _S. enterica_ **subsp.** & _P. mirabilis_ & _C. freundii_ & _C. koseri_ \\  & _ enterica_ & & & \\ \hline H\({}_{2}\)S & +a & + & + & – \\ Phenylalanine & – & + & – & – \\ deaminase & \(+\) & – & – & – \\ Lysine & \(+\) & – & – & – \\ decarboxylalse & \(-\) & – & + & + \\ \hline \hline \end{tabular} a: 90% or more of positive results. +: positive reaction; \(-\): negative reaction.  
\end{table} 
Table 3: Biochemical identification tests for the differentiation of _Citrobacter freundii_ and _C. koseri_ with other enterobacteria [41; 72].