Introduction: Highly acid resistant Escherichia coli O157:H7 could survive the low pH of orange juice due to its ability to adapt to acidic conditions. It is also known that natural stress factors commonly encountered by bacteria during mild food production such as heat and cold can induce a “viable but non-culturable” (VBNC) state in E. coli. Flow cytometry (FCM) can be used to monitor changes in cellular viability and morphology, both in cells that are able to grow and in VBNC cells.
Purpose: The aims of this study were to investigate the viability and culturability of E. coli subjected to a novel Model Orange Juice (MOJ) using FCM and plate counting.
Methods: E. coli K-12 MG1665 in different growth phases (OD650 = 0.5, 0.75 and 1) were added to a MOJ (pH = 3.2, containing sucrose, fructose, glucose, citric and malic acids and potassium citrate). The behavior of E. coli in MOJ was investigated during 24 hours storage (4 ºC and 37 ºC) using traditional (recovery on nutrient agar plates) and FCM (propidium iodide/bis-oxonol viability staining) techniques. The results were statistically analyzed using the Mann-Whitney test.
Results: FCM results showed a significant decrease (P < 0.001) in the number of viable cells for each sample after three hours of incubation, followed by a modest increase (P < 0.05), regardless of the incubation temperature and/or growth stage. For instance, for mid-log phase cells (OD650 = 0.5) at 4 ºC, there was a 1.12 ± 0.04 log decrease and a 0.30 ± 0.07 log increase in cell count after 3 and 24 hours, respectively. Furthermore, compared to FCM, plate counting identified significantly fewer viable cells (4.26 ± 1.06 log, P < 0.001) after 24 hours, probably due to an increase in the proportion of VBNC cells.
Significance: These data suggest that plate counting grossly underestimated viable cells despite using the most common resuscitation and recovery technique. Considering the very low dose of infection of E. coli O157:H7, FCM could be a powerful technique for its identification in foods.