# Illegal Nitrite Treatment of Red Tuna and Prolonged Storage: What About Other Food Safety Risks?
## 2 Materials and Methods
### Microbiological Determinations  
The bacterial determinations were carried out according to standardized and reference methods as follow: ISO 4833-1:2013/Amd 1:2022 [28], ISO 21528-2:2017 [29], ISO 16649-2:2010 [30], ISO 6888-2:2021/Amd 1:2023 [31], ISO 21872-1:2017/Amd 1:2023 [32], ISO 6579-1:2017/Amd 1:2020 [33]. These protocols were followed for evaluating total microbial count, enumerating _Enterobacteriaceae_, beta-glucuronidase-positive _Escherichia coli_, and coagul-positive staphylococci (_Staphylococcus aureus_ and other species), and detecting _Vibrio_ spp. and _Salmonella_ spp., respectively. Twenty-five grams of tuna sample was placed in a Stomacher bag and 225 mL (1:10, \(w/v\)) of Buffered Peptone Water (BPW) was added and the mixture was blended using a BagMixer (Interscience, Milan, Italy). Then, serial dilutions (1:10 \(v/v\)) were plated on proper media in Petri dishes. More in depth, the following conditions and media were adopted: Violet Red Bile Glucose Agar (VRBGA) (Oxoid) incubated at 37 \({}^{\circ}\)C for 48 h for _Enterobacteriaceae_, plate count agar (PCA) (Oxoid, Hampshire, UK) incubated at 30 \({}^{\circ}\)C for 72 h for total microbial count, and Rabbit Plasma Fibrinogen Agar medium (RPFA medium) incubated at 37 \({}^{\circ}\)C for 24-48 h for staphylococci. Tuna dilutions in BPW were incubated at 37 \({}^{\circ}\)C for 18 h. Then, 0.1 mL of the culture was inoculated in 10 mL of Rapapport Vassiliadis Soy (RVS) broth (Microbiol, Cagliari, Italy) and incubated at 41.5 \({}^{\circ}\)C for 24 h. One milliliter of the same culture was transferred in 10 mL of Muller-Kauffmann Tetrathionate-Novobiocin (MKTTn) broth (Microbiol, Cagliari, Italy) and incubated at 37 \({}^{\circ}\)C for 24 h. At the end of the incubation, 10 mL of the culture from each tube was seeded using a sterile calibrated loop on Xylose Lysine Desoxycholate (XLD) (Microbiol, Cagliari, Italy) and _Salmonella_ Detection Agar (SDA) (Microbiol, Cagliari, Italy) for the detection of _Salmonella_ spp. _Vibrio_ spp. detection was performed by preparing a pre-enrichment of each sample (25 g) in 225 mL of alkaline peptone water (APW), which was then incubated at 37-41.5 \({}^{\circ}\)C overnight. Then, an inoculating loop of the culture broth was streaked on CHROMagar Vibrio (CHROMagar(tm), Paris, France) and thiosulfate citrate bile salt agar (TCBS) (Microbiol, Cagliari, Italy) plates and incubated for 24 h at 37 \({}^{\circ}\)C.  
All presumptive colonies isolated from selective media were identified at species level with the MALDI TOF-MS (Bruker Daltonics, Bremen, Germany) method. Each tuna sample was analyzed in duplicate, both with and without the nitrite treatment, within 24 h of collection (t\({}_{0}\)) and after 5 days of storage (t\({}_{5 gg}\)) in a refrigerator (5 \(\pm\) 3) \({}^{\circ}\)C.  
Enumeration of Selected Pathogens Added to Tuna  
Tuna samples, treated or not, were also contaminated with low and high levels of _Salmonella enteritidis_ ATCC(tm) 13076(tm) (10\({}^{2}\) and 10\({}^{4}\) CFU/mL) [34] to evaluate the survival and possible growth of this pathogen in raw tuna treated with nitrite during prolonged refrigerated storage. Slices of tuna samples were placed in a sterile container and mixed with each culture suspension of the _Salmonella_ strain. Subsequently, inoculated samples were subjected to nitrite treatment and analyzed immediately and after 5 days of storage in a refrigerator (5 \(\pm\) 3) \({}^{\circ}\)C to check the effect of nitrite treatment. The surface-plating method on selective media specific for _Salmonella_ spp. (XLD agar plates) (Microbiol, Cagliari, Italy) was used to determine the populations of target pathogen in inoculated tuna samples. The difference in the enumeration of _Enterobacteriaceae_ between the samples with and without the nitrite treatment and the blank sample was calculated and correlated with the presence of _Morganella morganii_, a typical example of histamine-producing bacteria (HPB). Briefly, tuna samples, treated or not, were contaminated with high levels of _M.