**4.4 Opening the sample**

134 Salmonella – A Dangerous Foodborne Pathogen

Methods which is being used to detect pathogen microorganisms in the food are ineffective if such pathogenic microorganisms diffused rarely or the food is severely contaminated with other microorganisms. For these reasons, various indicator microorganisms are used for

To take samples from the foods for microbiological analysis and to bring this to the laboratory by "protecting all microbiological criteria at the moment of sampling" are quite difficult. Subjects such as how much and how many sample will be taken, bringing to the

Many national and international standards give the food quantity to be analyzed as 10 g (mL). This value is valid for quantitative analyses only. Pathogens are usually analyzed by

While 10 grams of sample is sufficient for a standard analysis, usually 25 grams of food is required for every additional pathogen test in accordance with special homogenization requirements as mentioned above. According to this, at least 60 grams of sample including10 grams for total bacteria, coliform group bacteria, yeast and mold and staphylococcus and 25 grams of each for *Salmonella* and Lysteria analysis should be brought

The requirement that how many items should be taken from a sample mostly causes

More samples should be taken from foods that have high pathogenic risk such as *Salmonella*,

The sample taken from the enterprise should be brought to the laboratory as cleared from all conditions that will increase or decrease the single microorganism count as soon as possible

In microbiologically stable products such as sterile, dry and humid resistant packages, no cooling is required during transportation. On the other hand, unprocessed, cooled, pasteurized, spoiled foods should be transported between 0°C and +4°C and frozen products should be transported at -18°C and those bulged (or having a risk of bulge) should

First, the sample should be accepted to be analyzed by the laboratory. For this acceptance, laboratory personnel should control whether the sample is brought to the laboratory under required conditions; if such personnel is sure that the sample was brought under standard conditions, she/he should accept the sample for the analysis; otherwise she/he should

For the sample which has come to the laboratory and accepted, date of acceptance, time of acceptance, all information related with the product (date of production, package features, batch number, shift number, time of sampling, temperature of sampling, temperature of arrival etc. if required) should be recorded according to the features of the laboratory. Frozen liquid products are not accepted for microbiological or somatic cell analysis. If chemical tests will be performed, sub-samples should be separated for microbiological analysis first.

be packaged separately against explosion and leakage and brought to the laboratory.

either reject or write all negativities related with this to the acceptance form.

confusion. In daily controls performed in food industry, only 1 sample is sufficient.

**4.3 Bringing the sample to the laboratory and acceptance** 

laboratory, opening and preparation for planting should be overemphasized.

various purposes.

**4.1 Sample taking** 

**4.2 Sample amount** 

to the laboratory.

Lysteria etc.

and analyzed.

present/absent test in 25 grams of food.

The sample which has come for analysis should be analyzed within the shortest period. If there is a necessity to wait for a while;


Frozen products should be thawed in +4°C refrigerator temperature. It should be considered that big particle products will thaw within a longer period than small particle products and psycrophile bacteria may develop within the thawing period, therefore the food should be frozen with portions not more than 50 grams within bounds of possibility. If the sample is frozen by weighing before, it may be thawed by transferring into homogenization solution directly.

Parallel of the sample which has come to the laboratory and accepted should be protected as witness of which features will not change until the termanitaion of the analysis.

Before opening the closed package, the place and its surroundings should be disinfected via 76% (v/v) alcohol or any appropriate chemical agent and if the package is appropriate, it should be singed. Packages that can not be singed (paper etc.) should be removed by cleaning with disinfecting sterile water after chemical disinfection and such disinfection should not be contacted with the food sample anyhow. Otherwise, negative result may be taken. Materials which will be used to open the sample such as scissors, tin opener, bottle opener etc. should have been disinfected or sterilized in the drying oven or autoclave by wrapping to an appropriate package (paper, kitchen type aluminum folio).

Liquid samples may be analyzed directly. Solid foods should be pre-processed such as weighing, homogenization etc.

Weighing to a certain weight (10 g, 25 g, etc.) in solid sample should be performed under aseptic conditions. To weight in vertical type planting cabinet is the most reliable method. The container that weighing will be performed should have been sterilized and should be in the size to take pre-enrichment medium like *Salmonella*.

If solid food consists of particles which may create a problem during weighing in terms of size and qualification, it should be divided into suitable sizes.

It should be remembered that this application is valid for weighing which is more than aimed weight and weighing over 5% should not be performed as far as possible. If microorganisms such as *Salmonella* was weighed as 26 grams instead of 25 grams in present/absent tests, to use a 234 (=225+9) mL medium instead of 225 mL of pre-enrichment medium is not a condition. Because, the process performed here by using 25 grams of food + 225 mL medium is not a dilution, but using 9 mL medium per 1 gram food. Tolerence of +/- 5% is always accepted. The deviation in this sample is only 4%.

Generally, it is the process to make solid and semi-solid foods homogenous in a homogenization solution. Liquid foods show a homogenous distribution in anyway. The purpose of homogeneity here is to distribute all microorganisms in the food to all mass to be analyzed. Homogenization process is performed as 1:9 in general whether it is used for counting or present/absent tests. According to this, 1 part food is homogenizated by 9 part solution. In present/absent tests, 1 part food is homogenizated by 9 parts of medium. If counting will be performed, 1:9 homogenization is also used as 10¹ dilution. Therefore, amount of the food and homogenization solution should be cared about.

Isolation and Identification of Salmonellas from Different Samples 137

To produce coliform bacteria, water is planted into Durham tubes including 2% peptone, 0,5% sodium taurocholate, 1% lactose and bromthymol blue (or bromeresol purple) as indicator. Amount of the water planted varies between 0,1 cm3 and 50 cm3. Concentrated medium is used if more water will be planted. The least water amount including coliform bacteria is detected by assays and contamination degree of the water is determined. Generally, 20 cm of 15 tubes included medium is taken. 0,1-1 cm3 water is put into 5 tubes; 1 cm3 water is put into 5 tubes; and 10 cm3 water is put into 5 tubes. Tubes are left at 37°C for 48 hours. After 24 and 48 hours, tubes are controlled and acid and gas formation is controlled. If gas is produced, it is examined that such gas has filled 1/10 of the small tube in Durham tube. Formation of gas which will occupy 1/10 of the tube after 24 hours shows possibility of Eschericihia coli reproduction. If no gas has formed within 24 hours or the gas occurred is less than 1/10 of the small tube, tubes are left at 37°C for 48 hours. Any gas formation is considered as suspicious.

To confirm whether reproduced bacteria are Eschericihia coli, planting is performed from tubes with least amount of water that reproduction was observed to eosine-methylene blue agar by decreasing method. Petri dishes are left at 37°C for 24 hours and examined and it

Same amount of water is added into tubes including selenite f medium which was prepared with one portion concentrate. After waiting at 37°C for 24 hours, planting is performed from every tube to SS agar. Furthermore, one cm3 each from tubes are taken and mixed with bismuth sulphite agar and poured into petri plates. When reproduction occurs, it is searched

Meat may include many germ types such as bacillus suptilis, Escherichia, proteus, *Salmonella* , staphylococcus species and fungus and especially anerobe spore forming bacteria show fundamental change. These bacteria cause formation of bad odor by making putrefaction in

To detect bacteria, one gram is weighed, it is meshed in mortar with sand. 1000, 10.000, 100.000 dilutions are prepared. Colony count is performed with these in the petri plate.

Fresh egg is not always sterile. Gran (+) coccus, gram (-) bacillus and some fungus species

The shell of the egg has a porous structure. Gases and microscopic particles may pass through these pores. Bacteria always exist on the shell of the egg. Escherichia coli is present almost on all shells. Humidity causes bacteria to pass through pores in humid and dirty

The egg which will be examined is cleaned well by washing with brush, water and soap. It is kept in 0,1% sublimated solution for 30 minutes. It is taken from here by a sterile spoon and put into 200 cm3 of sterile water. It is kept in the water for 10 minutes and put into 95% alcohol and left for 5 minutes. The egg is taken from the alcohol and left for drying and

may be present in the egg. Sometimes, egg may include *Salmonella* bacteria. (617, 618)

eggs. Therefore, eggs which sill be stored for a while must not be washed.

proteins. It is not very possible to decide on the status of the meat by bacteria on it.

No gas formation after 48 hours shows no reproduction of Eschericihia coli.

controlled whether the bacteria reproduced is Eschericihia coli.

**4.6.3 Searching for** *Salmonella* **bacteria** 

**4.7 Meat** 

**4.8 Egg** 

whether it is from *Salmonella* group. (551,602,611)

Diagnose of bacteria is performed if required.

**4.6.2 Approximate assay for coliform bacteria count** 
