Variable Pressure Scanning Electron Microscopy (VP-SEM) with a cooling stage allows the observation of water-containing wet samples with minimum shrinkage or deformation without conventional sample preparations such as fixation, dehydration, drying, and metal-coating. The VP-SEM with a cooling stage also reduces water vaporization and electron beam damage, so it will enable us to make an observation for a long time.
We studied white radish sprouts using the VP-SEM with the cooling stage and a Yttrium Aluminum Garnet (YAG) backscattered electron detector for better image quality at various accelerating voltages. In addition to non-cleaned samples, the following three cleaning techniques for the samples were examined: 1) Cleaning by running tap water; 2) Dipping in strongly acidic electrolyzed water and cleaning by running tap water; 3) Dipping in formula consisting mainly of fumaric acid and cleaning by running tap water.
A low accelerating voltage of 5 kV made it easy to recognize bacteria on the samples. The fairly large tissue surfaces of non-cleaned samples were covered by bacteria and the bacteria were lined up along the grooves of cell boundaries. Running tap water did not markedly remove bacteria from the surface of the samples. On the other hand, strongly acidic electrolyzed water or formula consisting mainly of fumaric acid showed a marked cleaning effect. Moreover, the bacteria along the grooves of cell boundaries of those stuck to the surface of cells remained in spite of cleaning.

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Fresh vegetables were examined for bacterial contamination by standard plate count (SPC), Escherichia coli, enterohaemorrhagicE. coliO157 andSalmonellabetween August and November, 1998. A total of 299 vegetable samples including 37 white radishes, 29 carrots, 28 cabbages, 28 Japanese leeks, 32 lettuces, 29 cucumbers, 40 tomatoes, 28 onions, 32 white radish sprouts, 13 alfalfa sprouts, 1 salad lettuce, 1 spinach, and 1 bean sprout were got at some markets.
The SPC of the vegetables were widely distributed from <10 to 10⁸cfu/g . In Particular, the SPC of alfalfa and white radish sprouts were high at 10⁷cfu/g . AlthoughE. coliwas detected in a Japanese leek, enterohemorrhagicE. coliO157 was negative for all samples.SalmonellaO13, serotype Cubana was detected in one sample of alfalfa sprouts.
SalmonellaO13, serotype Cubana isolates of alfalfa sprouts were compared with 2 isolates of human carrier that were isolated during the same period in Tokyo . Antibiotic resistance patterns, plasmid profile and PFGE pattern after digestion with restriction enzymeXbaIandSpeIwere studied for epidemiological makers, but no significant relationship was found.

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Fluorescence polarization technique can specifically detect nucleotide sequences by hybridization without separation procedures. We applied the technique to the detection of the Shiga toxin genes (stx1andstx2) in enterohemorragicEscherichia coli(STEC).
STEC possessesstx1 and/orstx2 and using MK primers, the amplification ofstx1 and/orstx2 can be performed universally, but the band sizes for both genes are almost identical; thus the genotype is hard to determine using electrophoresis. To identify stx genotype, conventional PCR combined with electrophoresis requires a second amplification process with more than 25 temperature cycles using other genotype-specific primer pairs. However, using the newly developed method only a few temperature cycles of the asymmetric PCR using the same MK primers and two different probes forstx1andstx2 were needed. The incubation time for probe hybridization was 10 minutes, and the detection time for fluorescence polarization was within about 1 minute per sample. Thus, we considered that the detection method using fluorescence polarization technique was a rapid and reliable method for detectingstx1and/orstx2.

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We investigated the usefulness of a rapid screening method for the detection of VTEC andSalmonellafrom swab specimens of flayed carcasses by detecting the Vero toxin (VT) andSalmonella invAgenes by PCR using multiplex primer sets (MK and SIN primer sets) for the meat inspection at slaughterhouse. Thirteen strains of VTEC and 3 strains ofSalmonellawere examined in the presence of other bacteria (non-VTEC and non-Salmonella) concomitantly present in the swab specimens. The multiplex PCR could detect VT andinuAgenes at a concentration of 2.0×10⁴cfu/ml and 2.1×10³cfu/ml respective1y, even in the presence of other bacteria at a concentration of 10⁹cfu/ml in the broth suspension. Brief shaking-incubation of the swab specimens at 36°Cfor 8 hrs in Tryptic Soy Broth (TSB), during which period both VTEC andSalmonella, if present, grew to 10^6.6-10^8.4cfu/ml, enhanced the detection rate of the multiplex PCR test. Cultivation of the swab specimens in either EEM or N-mEC media showed restricted growth, and subsequent lower detection rates compared with that in TSB. With the combination of brief shaking-culture in TSB and the multiplex PCR, we could detect as little as 1-10cfu/ml of VTEC andSalmonellapresent in the swab specimens. This method can shorten the time and reduce the number of staff needed to perform meat inspection.

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