**2. Taking samples for tests**

The first stage of microbiological analysis of food consists in taking and preparing a sample for analyses. Incorrect sampling can lead to obtaining false negative or false positive results. When talking about taking samples, the term "representative sample" is often used. The sample should reflect the image of the product from which it originates as precisely as possible. It is quite easy to take a representative sample from liquid products, e.g. milk, if the milk has been sufficiently mixed before taking the sample. On the other hand, when the subject of examination is a product of high viscosity, with slow flow or of a heterogeneous structure, then it is very difficult to assess the microbiological quality of the entire batch (e.g. a barrel or a

Detection of *Salmonella* spp. Presence in Food 395

and brilliant green in order to inhibit the growth of Gram-positive bacteria. In the case of acid and soured food products, peptone water should be used with double concentration of components, while for meat and food of high fat content, pre-enrichment should be

25 g food in 225ml of 10% buffered pepton water 37°C, 24 h

 0.1 ml in 10 ml Rappaport-Vassiliadis Soy Broth 37°C, 24 h 1 ml in 10 ml Tetrathionate broth (Müller-Kauffman) 41.5°C, 24 h

**Streaking on nutrient agar** 37°C, 24 h

**Biochemical confirmation** 37°C, 24 h

37°C, 24 h

performed in lactose broth with the addition of Triton X-100.

TSI

 LDC ONPG VP Indole

 O-antigens H-antigens

Fig. 1. Flow diagram for detection of *Salmonella*.

**Non-selective pre-enrichment** 

**Selective enrichment** 

**Isolation**  XLD with an inoculation loop BGA or Hektoen or other selective agar plates with an inoculation loop

Urea broth

**Serotyping** 

green, which, in turn, inhibits the growth of Gram-positive bacteria.

After the non-selective pre-enrichment stage, a 0.1cm3 sample is taken from the culture and inoculated on 10cm3 of selective medium, Rappaport-Vassiliadis with soya, and on **Muller-Kauffmann** medium in the amount of 1 cm3. **Rappaport-Vassiliadis (RVS)** medium is solid, strongly selective and contains malachite green and sodium chloride (inhibiting the growth of accompanying microflora). Soya peptone, pH 5.2, and increased temperature of incubation (41.5°C) favour the growth of *Salmonella* sp. strains. The medium is dark blue and clear. *Salmonella* sp. strains grow on this medium in the form of milky residue, while the colour of the medium itself does not change. The other selective medium, **Muller-Kauffmann broth (MKTTn)**, contains sodium thiosulphate and potassium iodide, which react to form a compound known as sodium tetrathionate, inhibiting the growth of the coliforms. *Salmonella* sp. are able to reduce this compound. The broth also contains brilliant

truckload) by examining only one 25-gram sample. The answer to the question concerning the required number of single samples is extremely difficult. In view of the high costs of microbiological tests, the number of samples is generally limited. In a microbiological laboratory, samples are taken with the use of sterile tools, e.g. spoons, scalpels, knives, spatulas and pipettes. Frozen products should be first thawed at below 5°C (for not longer than 12 hours). In the case of deeply frozen samples, sterile drills are used for sampling.

Determination of *Salmonella* sp. in food products always consists in detecting the presence of those bacteria in a specified amount of the product (generally 25g/ml, very rarely 10g/ml), but the number of those microorganisms in food is not determined. Both in the classical method and in its modifications, the first stage of detection is non-selective enrichment. This is crucial, since food production involves its technological treatment, e.g. heating, which can cause the death of most cells or cause sub-lethal injured. Omission of the stage of preenrichment of the sample and inoculating the material directly on the solid medium can give false negative results. If the examined material includes a very low number of living cells, or the cells have been sub-lethally damaged during the technological processes, we may not receive macroscopically-visible colonies on the solid medium. In such a case there is a risk of releasing the product to market although it does not satisfy safety criteria. During the storage of such a product, damaged cells can be repaired and bacteria can proliferate to a level that would be hazardous for the consumers.

There are many methods to determine *Salmonella sp.* in food and, for this reason, the present study focuses on the classical culture method – the application of a Vidas device – as the only fully automated one. Additionally, the PCR method (a commonly-applied alternative to the plate method) and the FISH method (which is still not popular, although work on its optimization is ongoing) are also described.
