RU2848543C1 - Method for detecting salmonella spp. on work surfaces - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a method for detecting Salmonella spp. on work surfaces. The essence of the invention: a liquid culture medium containing per 1 litre: 5.00 g of animal tissue peptic hydrolysate, 4.00 g of mannitol, 10.00 g of sodium phosphate, 4.80 g of bile, 4.00 g of sodium hydroselenite, 0.0085 g of methyl yellow, and water. Selectivity is ensured by bile and hydroselenite, which inhibit the growth of non-target microorganisms. Methyl yellow induces a visual colour change in the medium from yellow to orange in the presence of Salmonella. Method of application: using a sterile swab moistened with NaCl solution, wipe the surface (area ≥ 10 cm²), place the swab in the medium and incubate for 24 hours at 37°C. A colour change indicates the presence of bacteria. The method is applicable to plastic, glass and stainless steel.

EFFECT: increased selectivity, sensitivity (CFU≤ 5) and simplification of the detection method.

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Description

Field of technology to which the invention relates

The invention relates to microbiology, veterinary science, hygiene and sanitation and can be used to detect bacterial contamination on work surfaces during quality control of their processing.

State of the art

Salmonella bacteria cause salmonellosis, an acute foodborne infection that affects the gastrointestinal tract, leading to intoxication and dehydration. Individuals with weakened immune systems, pregnant women, children, and the elderly are at risk for more serious complications. Human infection occurs through foodborne exposure, most commonly through the consumption of beef, pork, poultry, and, less commonly, seafood, vegetables, and fruits. Food contamination can be caused by improper temperature control and poor cooking. Using culture media to detect Salmonella bacteria on work surfaces can help prevent salmonellosis outbreaks in food processing and catering establishments.

A nutrient medium for salmonella is known, containing sodium dibasic phosphate, potassium monobasic phosphate, bentonite clay, and distilled water [RU Patent for Invention No. 2139344, Application 97122257/13, 12/23/1997, Published: 10/10/1999].

A disadvantage of this medium is its low selectivity, which means that a positive result may occur when non-target microorganisms other than Salmonella are isolated. This medium also has low sensitivity, as the infective dose is 1000 microbial cells per 1 ml of medium. In addition to the above-mentioned disadvantages, this medium requires more time for preparation, significantly increasing the time required for the work.

A differential diagnostic medium for isolating Salmonella spp. is known, which includes pancreatic hydrolysate of fish, feed yeast extract, sodium chloride, lactose, sucrose, brilliant green, phenyl red, microbiological agar, tris (hydroxymethyl) aminomethane [RU Patent for Invention No. 2233884, Application 2002116391/13, 17.06.2002, Published: 10.08.2004, Bulletin No. 22].

A disadvantage of this medium is the presence of an expensive component, tris(hydroxymethyl)aminomethane, which increases the cost of the medium and limits its practical use in enterprises and clinical diagnostic laboratories. Also, when using this medium, the isolation of a widespread and clinically significant microorganism, Escherichia coli, is observed, indicating the medium's low selectivity.

A known nutrient medium for salmonella contains buffered peptone water and a component for producing acid by salmonella. Propylene glycol is used as the component for producing acid by salmonella [Russian Federation Patent for Invention No. 2570386, Application 2014136524/15, September 8, 2014, Published: December 10, 2015, Bulletin No. 34].

A disadvantage of this medium is the need for a selective enrichment step, which increases the time required for testing. This method allows the proposed culture medium to be used for the detection of Salmonella in food products only, which limits its use to food production facilities. A disadvantage of this medium is the lack of information on the number of isolated microorganisms, making it impossible to assess the sensitivity of the culture medium.

Disclosure of invention

The objective of the invention is to improve quality control of work surface treatment at various enterprises where it is necessary to control bacterial contamination.

The technical result of the invention is the development of a method and environment for detecting Salmonella spp. on work surfaces made of various types of materials, with an area of at least 10 ^cm2 , with a CFU count of ≤5, characterized by high selectivity, ease of detection of the result, and general ease of use.

This is achieved by the fact that, unlike known methods for detecting bacterial contamination on surfaces, it is proposed to use a liquid nutrient medium containing (per 1 liter of the prepared medium):

Peptic hydrolysate of animal tissues 5.00 g Mannitol 4.00 g Sodium phosphate 10.00 g Bile 4.80 g Sodium hydroselenite 4.00 g Methyl yellow (analytical grade)
Distilled water 0.0085 g
Up to 1 l

The medium is prepared using a standard method: the components of the nutrient medium, except methyl yellow, are added to approximately 900 ml of distilled water and mixed thoroughly. The nutrient medium is heated to boiling to completely dissolve the particles. After the medium has cooled to 45-50°C, 0.0085 g of methyl yellow, previously dissolved in 10 ml of sterile distilled water, is added under aseptic conditions. The volume of the medium is brought to 1 liter under aseptic conditions with sterile distilled water. The medium is thoroughly mixed and poured into sterile test tubes. The prepared medium is yellow in color.

The work surface is swabbed using a sterile swab soaked in 100 µl of sterile sodium chloride solution. The swab is then placed in a test tube containing the recommended culture medium, which is then incubated at 37°C for 24 hours. If Salmonella bacteria are present, the culture medium changes color from yellow to orange after incubation. If Salmonella bacteria are absent, the culture medium remains yellow after incubation.

The quantitative ratio of the components of the nutrient medium in the form of peptic hydrolysate of animal tissues, mannitol, sodium phosphate provides the cultured microorganisms with the necessary nutrients - nitrogen, amino acids, carbohydrates, phosphates, electrolytes, lipids.

The selectivity of the medium is ensured by the addition of bile and sodium hydroselenite, which helps suppress the growth of non-target microorganisms.

The addition of the indicator component methyl yellow makes it possible to easily visualize the change in color of the nutrient medium during the growth of microorganisms of the genus Salmonella .

The proposed method does not involve complex manipulations, is based on the use of a nutrient medium from available components, the result is identified visually with the naked eye, the analytical characteristic - the color of the nutrient medium - has a high differentiation of results, which overall ensures the ease of use of the developed method for determining the presence of Salmonella bacteria on work surfaces.

The specified method makes it possible to determine Salmonella spp. on surfaces of various types, including plastic, glass and steel, with a CFU count of ≤5 per at least 10 cm ² .

The proposed method was tested on various surface types: plastic, glass, and stainless steel. Solutions with varying protein concentrations were applied to a surface area of 10 ^cm2 .

When testing on plastic surfaces, Petri dishes were used (TU 9398-003-55058819-2011).

When testing on glass-type surfaces, laboratory glassware made of borosilicate glass was used (GOST EN 1748-1-1-2016).

When testing on stainless steel type surface, stainless steel plates (SS316) were used.

Example 1. Using a sterile viscose swab moistened with 100 μl of sterile sodium chloride solution, a swab was taken from the plastic surface of a Petri dish (TU 9398-003-55058819-2011) with an area of 10 ^cm2 containing CFU≤5 Salmonella typhimurium. The swab was placed in a test tube with a nutrient medium with the following composition:

Peptic hydrolysate of animal tissues 5.00 g

Mannitol 4.00 g;

Sodium phosphate 10.00 g;

Bile 4.80 g;

Sodium hydroselenite 4.00 g;

Methyl yellow (analytical grade) 0.0085 g;

Distilled water up to 1 liter.

The tube was then incubated at 37°C for 24 hours. At the end of the incubation, an orange coloration of the nutrient medium was observed, confirming the presence of Salmonella typhimurium on the surface.

A control study was conducted in parallel. Using a sterile viscose swab moistened with 100 μl of sterile sodium chloride solution, a 10 ^cm2 area of a non-sterile plastic Petri dish (TU 9398-003-55058819-2011) containing no Salmonella typhimurium was swabbed. The swab was placed in a test tube containing a nutrient medium with the following composition:

Mannitol 4.00 g;

Sodium phosphate 10.00 g;

Bile 4.80 g;

Sodium hydroselenite 4.00 g;

Methyl yellow (analytical grade) 0.0085 g;

Distilled water up to 1 liter.

The tube was then incubated at 37°C for 24 hours. At the end of the incubation, the color of the medium remained yellow, indicating the absence of Salmonella typhimurium on the surface.

Example 2. Using a sterile cotton swab moistened with 100 μl of sterile sodium chloride solution, a sample was swabbed from the surface of a borosilicate glass laboratory flask (GOST EN 1748-1-1-2016) with an area of 10 ^cm2 containing ≤5 CFU Salmonella typhimurium . The swab was placed in a test tube containing a nutrient medium with the following composition:

Peptic hydrolysate of animal tissues 5.00;

Mannitol 4.00 g;

Sodium phosphate 10.00 g;

Bile 4.80 g;

Sodium hydroselenite 4.00 g;

Methyl yellow (analytical grade) 0.0085 g;

Distilled water up to 1 liter.

The tube was then incubated at 37°C for 24 hours. At the end of the incubation, an orange coloration of the nutrient medium was observed, confirming the presence of Salmonella typhimurium on the surface.

A control study was conducted in parallel. Using a sterile cotton swab moistened with 100 μl of sterile sodium chloride solution, a 10 ^cm2 sample containing ≤5 CFU of Escherichia coli was swabbed from the surface of a borosilicate glass laboratory flask (GOST EN 1748-1-1-2016). The swab was placed in a test tube containing a nutrient medium with the following composition:

Peptic hydrolysate of animal tissues 5.00 g;

Mannitol 4.00 g;

Sodium phosphate 10.00 g;

Bile 4.80 g;

Sodium hydroselenite 4.00 g;

Methyl yellow (analytical grade) 0.0085 g;

Distilled water up to 1 liter.

The tube was then incubated at 37°C for 24 hours. At the end of the incubation, the color of the medium remained yellow, indicating the absence of Salmonella spp. on the surface.

Example 3. Using a sterile microfiber swab moistened with 100 μl of sterile sodium chloride solution, a 10 ^cm2 sample containing ≤5 CFU of Salmonella typhimurium was swabbed from the surface of a stainless steel plate (SS316). The swab was placed in a test tube containing a nutrient medium with the following composition:

Peptic hydrolysate of animal tissues 5.00 g;

Mannitol 4.00 g;

Sodium phosphate 10.00 g;

Bile 4.80 g;

Sodium hydroselenite 4.00 g;

Methyl yellow (analytical grade) 0.0085 g;

Distilled water up to 1 liter.

The tube was then incubated at 37°C for 24 hours. At the end of the incubation, an orange coloration of the nutrient medium was observed, confirming the presence of Salmonella typhimurium on the surface.

A control study was conducted in parallel. Using a sterile microfiber swab moistened with 100 μl of sterile sodium chloride solution, a 10 ^cm2 surface of a stainless steel plate (SS316) containing ≤5 CFU of Staphylococcus aureus was swabbed. The swab was placed in a test tube containing a nutrient medium with the following composition:

Peptic hydrolysate of animal tissues 5.00 g;

Mannitol 4.00 g;

Sodium phosphate 10.00 g;

Bile 4.80 g;

Sodium hydroselenite 4.00 g;

Methyl yellow (analytical grade) 0.0085 g;

Distilled water up to 1 liter.

The tube was then incubated at 37°C for 24 hours. At the end of the incubation, the color of the medium remained yellow, indicating the absence of Salmonella spp. on the surface.

Example 4. Using a sterile cotton swab moistened with 100 μl of sterile sodium chloride solution, a sample was collected from the surface of a borosilicate glass laboratory flask (GOST EN 1748-1-1-2016) with an area of 10 ^cm2 containing ≥10 CFU Salmonella enterica . The swab was placed in a test tube containing a nutrient medium with the following composition:

Peptic hydrolysate of animal tissues 5.00 g;

Mannitol 4.00 g;

Sodium phosphate 10.00 g;

Bile 4.80 g;

Sodium hydroselenite 4.00 g;

Methyl yellow (analytical grade) 0.0085 g;

Distilled water up to 1 l.

The tube was then incubated at 37°C for 24 hours. At the end of the incubation, an orange coloration of the nutrient medium was observed, confirming the presence of Salmonella enterica on the surface.

A control study was conducted in parallel. Using a sterile cotton swab moistened with 100 μl of sterile sodium chloride solution, a 10 ^cm2 surface swab was taken from a non-sterile borosilicate glass laboratory flask (GOST EN 1748-1-1-2016) free of Salmonella spp. The swab was placed in a test tube containing a nutrient medium with the following composition:

Peptic hydrolysate of animal tissues 5.00 g;

Mannitol 4.00 g;

Sodium phosphate 10.00 g;

Bile 4.80 g;

Sodium hydroselenite 4.00 g;

Methyl yellow (analytical grade) 0.0085 g;

Distilled water up to 1 l.

The tube was then incubated at 37°C for 24 hours. At the end of the incubation, the color of the medium remained yellow, indicating the absence of Salmonella spp. on the surface.

Example 5. Using a sterile viscose swab moistened with 100 μl of sterile sodium chloride solution, a 10 ^cm2 sample containing ≤5 CFU of Salmonella typhimurium was swabbed from the plastic surface of a Petri dish (TU 9398-003-55058819-2011). The swab was placed in a test tube containing a nutrient medium with the following composition:

Peptic hydrolysate of animal tissues 5.00 g;

Mannitol 4.00 g;

Sodium phosphate 10.00 g;

Bile 4.80 g;

Sodium hydroselenite 4.00 g;

Methyl yellow (analytical grade) 0.0085 g;

Distilled water up to 1 l.

The tube was then incubated at 37°C for 24 hours. At the end of the incubation, an orange coloration of the nutrient medium was observed, confirming the presence of Salmonella typhimurium on the surface.

A control study was conducted in parallel. Using a sterile viscose swab moistened with 100 μl of sterile sodium chloride solution, a 10 ^cm2 sample containing ≤5 CFU of Pseudomonas aeruginosa was swabbed from the plastic surface of a Petri dish (TU 9398-003-55058819-2011). The swab was placed in a test tube containing a nutrient medium with the following composition:

Peptic hydrolysate of animal tissues 5.00 g;

Mannitol 4.00 g;

Sodium phosphate 10.00 g;

Bile 4.80 g;

Sodium hydroselenite 4.00 g;

Methyl yellow (analytical grade) 0.0085 g;

Distilled water up to 1 l.

The tube was then incubated at 37°C for 24 hours. At the end of the incubation, the color of the medium remained yellow, indicating the absence of Salmonella spp. on the surface.

The results of selectivity and sensitivity tests of the proposed medium with different CFU and non-target microorganisms are presented in Tables 1-3.

Table 1. Results of tests of the nutrient medium for detection of Salmonella spp. at different numbers of CFU Salmonella typhimurium (in 10 repetitions).

Repeat number CFU Incubation temperature Color of the nutrient medium before incubation Time of color change of the environment (in hours) Color of the nutrient medium after incubation Result Negative control 1 ≤5 37°C Yellow 20 Orange Positively Yellow 2 ≤5 37°C Yellow 22 Orange Positively Yellow 3 ≤5 37°C Yellow 24 Orange Positively Yellow 4 ≤5 37°C Yellow 20 Orange Positively Yellow 5 ≤5 37°C Yellow 19 Orange Positively Yellow 6 ≤5 37°C Yellow 22 Orange Positively Yellow 7 ≤5 37°C Yellow 24 Orange Positively Yellow 8 ≤5 37°C Yellow 20 Orange Positively Yellow 9 ≤5 37°C Yellow 22 Orange Positively Yellow 1 From 6 to 10 37°C Yellow 23 Orange Positively Yellow 2 From 6 to 10 37°C Yellow 20 Orange Positively Yellow 3 From 6 to 10 37°C Yellow 19 Orange Positively Yellow 4 From 6 to 10 37°C Yellow 20 Orange Positively Yellow 5 From 6 to 10 37°C Yellow 22 Orange Positively Yellow 6 From 6 to 10 37°C Yellow 24 Orange Positively Yellow 7 From 6 to 10 37°C Yellow 20 Orange Positively Yellow 8 From 6 to 10 37°C Yellow 23 Orange Positively Yellow 9 From 6 to 10 35°C Yellow 19 Orange Positively Yellow 10 From 6 to 10 37°C Yellow 24 Orange Positively Yellow 1 ≥10 37°C Yellow 20 Orange Positively Yellow 2 ≥10 37°C Yellow 19 Orange Positively Yellow 3 ≥10 37°C Yellow 24 Orange Positively Yellow 4 ≥10 37°C Yellow 22 Orange Positively Yellow 5 ≥10 37°C Yellow 20 Orange Positively Yellow 6 ≥10 37°C Yellow 18 Orange Positively Yellow 7 ≥10 37°C Yellow 20 Orange Positively Yellow 8 ≥10 37°C Yellow 24 Orange Positively Yellow 9 ≥10 37°C Yellow 21 Orange Positively Yellow 10 ≥10 37°C Yellow 23 Orange Positively Yellow

Table 2. Results of tests of the nutrient medium for detection of Salmonella spp. with different numbers of CFU of the non-target microorganism Escherichia coli (in 10 repetitions).

Repeat number CFU Incubation temperature Color of the nutrient medium before incubation Time of color change of the environment (in hours) Color of the nutrient medium after incubation Result Negative control 1 ≤5 37°C Yellow - Yellow Negative Yellow 2 ≤5 37°C Yellow - Yellow Negative Yellow 3 ≤5 37°C Yellow - Yellow Negative Yellow 4 ≤5 37°C Yellow - Yellow Negative Yellow 5 ≤5 37°C Yellow - Yellow Negative Yellow 6 ≤5 37°C Yellow - Yellow Negative Yellow 7 ≤5 37°C Yellow - Yellow Negative Yellow 8 ≤5 37°C Yellow - Yellow Negative Yellow 9 ≤5 37°C Yellow - Yellow Negative Yellow 1 From 6 to 10 37°C Yellow - Yellow Negative Yellow 2 From 6 to 10 37°C Yellow - Yellow Negative Yellow 3 From 6 to 10 37°C Yellow - Yellow Negative Yellow 4 From 6 to 10 37°C Yellow - Yellow Negative Yellow 5 From 6 to 10 37°C Yellow - Yellow Negative Yellow 6 From 6 to 10 37°C Yellow - Yellow Negative Yellow 7 From 6 to 10 37°C Yellow - Yellow Negative Yellow 8 From 6 to 10 37°C Yellow - Yellow Negative Yellow 9 From 6 to 10 37°C Yellow - Yellow Negative Yellow 10 From 6 to 10 37°C Yellow - Yellow Negative Yellow 1 ≥10 37°C Yellow - Yellow Negative Yellow 2 ≥10 37°C Yellow - Yellow Negative Yellow 3 ≥10 37°C Yellow - Yellow Negative Yellow 4 ≥10 37°C Yellow - Yellow Negative Yellow 5 ≥10 37°C Yellow - Yellow Negative Yellow 6 ≥10 37°C Yellow - Yellow Negative Yellow 7 ≥10 37°C Yellow - Yellow Negative Yellow 8 ≥10 37°C Yellow - Yellow Negative Yellow 9 ≥10 37°C Yellow - Yellow Negative Yellow 10 ≥10 37°C Yellow - Yellow Negative Yellow

Table 3. Results of tests of the nutrient medium for detection of Salmonella spp. with different numbers of CFU of the non-target microorganism Staphylococcus aureus (in 10 repetitions).

Repeat number CFU Incubation temperature Color of the nutrient medium before incubation Time of color change of the environment (in hours) Color of the nutrient medium after incubation Result Negative control 1 ≤5 37°C Yellow - Yellow Negative Yellow 2 ≤5 37°C Yellow - Yellow Negative Yellow 3 ≤5 37°C Yellow - Yellow Negative Yellow 4 ≤5 37°C Yellow - Yellow Negative Yellow 5 ≤5 37°C Yellow - Yellow Negative Yellow 6 ≤5 37°C Yellow - Yellow Negative Yellow 7 ≤5 37°C Yellow - Yellow Negative Yellow 8 ≤5 37°C Yellow - Yellow Negative Yellow 9 ≤5 37°C Yellow - Yellow Negative Yellow 1 From 6 to 10 37°C Yellow - Yellow Negative Yellow 2 From 6 to 10 37°C Yellow - Yellow Negative Yellow 3 From 6 to 10 37°C Yellow - Yellow Negative Yellow 4 From 6 to 10 37°C Yellow - Yellow Negative Yellow 5 From 6 to 10 37°C Yellow - Yellow Negative Yellow 6 From 6 to 10 37°C Yellow - Yellow Negative Yellow 7 From 6 to 10 37°C Yellow - Yellow Negative Yellow 8 From 6 to 10 37°C Yellow - Yellow Negative Yellow 9 From 6 to 10 37°C Yellow - Yellow Negative Yellow 10 From 6 to 10 37°C Yellow - Yellow Negative Yellow 1 ≥10 37°C Yellow - Yellow Negative Yellow 2 ≥10 37°C Yellow - Yellow Negative Yellow 3 ≥10 37°C Yellow - Yellow Negative Yellow 4 ≥10 37°C Yellow - Yellow Negative Yellow 5 ≥10 37°C Yellow - Yellow Negative Yellow 6 ≥10 37°C Yellow - Yellow Negative Yellow 7 ≥10 37°C Yellow - Yellow Negative Yellow 8 ≥10 37°C Yellow - Yellow Negative Yellow 9 ≥10 37°C Yellow - Yellow Negative Yellow 10 ≥10 37°C Yellow - Yellow Negative Yellow

Thus, the conducted studies demonstrate positive results when using the stated composition of the nutrient medium components. High sensitivity of the proposed medium (positive result at CFU≤5) and specificity (absence of growth of non-target microorganisms) are demonstrated. Escherichia coli , Staphylococcus aureus, Pseudomonas aeruginosa ). The contrasting color transition of the nutrient medium from yellow to orange upon a positive result, as well as its ease of use, allow this medium to be used for the detection of Salmonella spp. not only in microbiology laboratories but also for quality control of work surface treatment in various enterprises where bacterial contamination must be monitored.

Claims (10)

A method for detecting Salmonella spp. on work surfaces, which consists in the fact that The surface is washed using a swab soaked in 100 µl of sterile sodium chloride solution, after which the swab is placed in a test tube with a nutrient medium having the following composition per 1 liter of the prepared medium: Peptic hydrolysate of animal tissues 5.00 g; Mannitol 4.00 g; Sodium phosphate 10.00 g; Bile 4.80 g; Sodium hydroselenite 4.00 g; Methyl yellow 0.0085 g; Distilled water up to 1 l, After this, the test tube is placed in a thermostat, where it is incubated at a temperature of 37°C for 24 hours, after which a change in the color of the nutrient medium in the test tube is observed: if the color of the medium changes from yellow to orange, a conclusion is made about the presence of Salmonella spp. on the working surface; if there is no change in the color of the medium and the original yellow color is maintained, a conclusion is made about the absence of Salmonella spp. on the working surface.