A sample preparation method for microbiological analysis of seeds

Abstract: There have been several outbreaks of food poisoning linked to the consumption of raw sprouts. The two most common pathogens associated with sprout consumption are Salmo-nella spp and verocyto-toxin producing Escherichia coli. During sprouting the warm, humid and nutrient rich conditions are ideal for bacterial growth. Pathogenic bacteria can infect seeds at very low numbers only to grow to dangerous levels during sprouting. Mi-crobiological analysis of dry seeds is difficult because of the low amount of pathogens and because hard seeds are difficult to handle. In this study a safe and reliable sample preparation method for seeds used for sprouting have been developed. The developed method starts with soaking the seeds in water and germinating them for one day. Soaking and germination softens seeds, make them easier to crush and allows bacterial growth, which increases chances of detection. After germination, the seeds are crushed with a mortar and pestle and placed in double stomacher bags. Buffered peptone water is added to samples, which are then pummeled in a stomacher. After enrichment at 37 ˚C, samples are analyzed according to validated methods. In order to get faster test result it is possible to analyze soaking water in addition to seeds. The soaking water is blended with equal amounts of double strength buffered peptone water and bacteria are enriched by incubation. Samples of alfalfa (Medicago sativa), fenugreek (Trigonella foenum-graecum) and mung bean (Vigna radiata) were used in the seed processing trials. Detection trials were performed on seeds inoculated with Salmonella Enteritidis and E. coli O157 and detection was performed with both cultivation- and PCR-based methods. Using cultivation based methods, Salmonella Enteritidis could be detected in all samples of soaking water, seeds before germination and after two days of germination, when inoculated in levels of 8-12 CFU/g dried seed. E. coli O157 was detected using real-time PCR detection of an 88 bp section of the rfbE-gene. At inoculation levels of approximately 40 CFU/g dried seeds, E. coli O157 was not detected in all samples. Detection rate was higher when using soaking water and after at least one day of germination. To determine the optimal germination time, bacterial growth on sprouting seeds was studied by following the growth of inherent aerobic bacteria for five days and inoculated E. coli O157 during three days of germination. The amount of aerobic reached its maximum after two to three days of germination. In seeds inoculated with E. coli O157, the bacteria were only detected in fenugreek seeds where most of the growth occurred during the first two days of germination.