RU2508399C1 - DRY DIFFERENTIAL DIAGNOSTICS FEED MEDIUM FOR DETECTION AND CONSIDERATION OF E.coli AND COLIFORM BACTERIA - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention can be used for detection and considering of E.coli and coliform bacteria in water, food products, clinic material, etc. Feed medium contains pancreatic hydrolysate of fish flour dried with Tergitol 7 on the bases of 0.1 g of Tergitol 7 per 6 g of dry pancreatic hydrolysate of fish flour, yeast extract, 1-water D (+) lactose, bromthymol blue, sodium dodecyl sulphate, 2,3,5-triphenyltetrazolium chloride, sodium carbonate and microbiological agar in the specified ratio.

EFFECT: invention allows preserving sterility of feed medium during 10 days, increasing accuracy of differentiation of coliform bacteria and Ecoli and simplifying a feed medium preparation method.

2 tbl, 4 ex

Description

The invention relates to sanitary and clinical microbiology and can be used to detect and account for E. coli and coliform bacteria in water, food products, clinical material, etc.

Leading foreign companies produce lactose agar with tergitol 7, or Tergitol 7 agar based on peptone or a mixture of pancreatic hydrolysates of animal tissues and casein, yeast extract, meat extract and lactose, as well as contain Tergitol 7, agar and indicator - Merck blue bromothymol blue Microbiology , 2004/2005, p. 168), HiMedia (HiMedia Laboratories Pvt. Limited (India), 1993, p. 233), Fluka (Scientific research 2003/2004, p. 1483), GEM LLC (Bacteriological media. Catalog 2009-2010, p. 88).

The disadvantage of commercial media is the difficulty of preparation: separate sterilization of the base medium by autoclaving and sterilization of a solution of 2,3,5-triphenyltetrazolium chloride (TTX) by membrane filtration are required, because TTX is destroyed at elevated temperatures, which leads to a lack of differentiation with respect to lactose-negative but reducing TTX bacteria.

In Russia there is no commercial dry nutrient medium for the detection and recording of E. coli and coliform bacteria with tergitol 7. GOST R 52426-2005 (ISO 9308-1: 2000) “Drinking water”, part 1, “Membrane filtration method”, regulates the detection and quantification of E. coli and coliform bacteria; use of lactose TTX agar with sodium heptadecyl sulfate (medium with tergitol 7) - laboratory preparation. The component content in g / l is presented in table 1.

The disadvantage of lactose TTX agar with sodium heptadecyl sulfate (tergitol 7 medium) of laboratory manufacture is the difficulty in preparation, which includes separate preparation of the base medium, sodium heptadecyl sulfate solutions and 2,3,5-triphenyltetrazolium chloride (TTX), followed by separate sterilization and preparation of a complete medium with compliance with aseptic rules, non-standard.

Closest to the proposed differential diagnostic nutrient medium for the detection and recording of E. coli and coliform bacteria (lactose agar with tergitol 7) is a selective differential medium for the isolation and recording of E. coli and coliform bacteria in water by membrane filtration lactose agar with tergitol 7 and TTX by Merck, which consists of the following components, g / l:

Lactose 20,0 Peptone 10.0 Yeast extract 6.0 Meat extract 5,0 Bromothymol blue 0.05 Tergitol 7 0.1 Agar 12.7

Additionally, a sterile solution of 2,3,5-triphenyltetrazolium chloride (TTX) is prepared, which is then introduced into the base sterile medium.

The disadvantage of this environment is the difficulty of preparation and the availability of special equipment for sterilization by membrane filtration.

To date, in Russia, due to the lack of a domestic commercial environment with tergitol 7, water control by membrane filtration for the detection and recording of E. coli and coliform bacteria is carried out on Endo medium, the use of which is planned until 2010, for the period required for accumulation of statistical data on the use of tergitol 7. (3)

The technical result of the invention is the creation of a domestic dry nutrient medium, ease of preparation while maintaining sterility for at least 10 days, the stability of the results in terms of growth and differentiating properties.

The technical result is achieved by balancing the component composition of the medium, which contains pancreatic hydrolyzate of fish meal, dried with a calculated amount of tergitol 7, yeast extract, lactose, 2,3,5-triphenyltetrazolium chloride (TTX), bromothymol blue, sodium carbonate, microbiological agar and additional sodium dodecyl sulfate in the following ratio, g / l:

Pancreatic hydrolyzate of fish meal with tergitol 7 (PGRM with tergitol 7) 5.5-6.7 Yeast extract 4.0-6.0 D (+) - lactose, 1-water 8.0-12.0 Bromothymol blue 0.07-0.09 Sodium dodecyl sulfate 0.08-0.12 2,3,5-triphenyltetrazolium chloride 0,023-0,026 Sodium carbonate 0.14-0.16 Microbiological agar 8.0-14.0 pH 7.4 ± 0.2

The difference between the proposed environment and the prototype is the use as a protein base of pancreatic hydrolyzate of fish meal with tergitol 7 (PGRM with tergitol 7), additional sodium dodecyl sulfate and the absence of meat extract. The quantitative ratio of the components of the nutrient medium, its pH, ensures that the medium remains sterile for at least 10 days, has an inhibitory effect on gram-positive microorganisms, good growth and primary differentiation of coliform bacteria and E. coli.

Example 1. To prepare the medium at the stage of production of liquid PGRM, tergitol 7 is added to the clarified pancreatic hydrolyzate of fishmeal at the rate of 0.1 g of tergitol 7 per 6.0 g of PGRM in terms of dry matter and dried on a drying unit with a A1- vibro-boiling layer PMU. Take samples of the following ingredients, g / l:

Pancreatic hydrolyzate of fish meal with tergitol 7 (PGRM with tergitol 7) 5.5 Yeast extract 4.0 D (+) - lactose, 1-water 8.0 Bromothymol blue 0,07 Sodium dodecyl sulfate 0.08 2,3,5-triphenyltetrazolium chloride 0,023 Sodium carbonate 0.14 Microbiological agar 8.0 pH 7.4 ± 0.2

Samples are stirred in 1 liter of distilled water, heated to boiling and boiled with constant stirring for 3-4 minutes on low heat until the agar is completely melted. Cool to a temperature of 40-50 ° C and pour into Petri dishes. Before sowing, the plates are dried at room temperature, observing aseptic rules for 40-60 minutes.

The proposed environment provides the growth of the following test strains when sowing 0.1 ml of microbial suspension from a dilution of 10 ^-6 :

Escherichia coli 3912/41 (055: K59),

Escherichia coli ATCC 25922,

Klebsiella pneumoniae 418,

Shigella sonnei "S-form,

Salmonella typhimurium 79

and inhibition of S.aureus Wood-46 from a 10 ^-3 dilution after 20-24 hours of incubation at a temperature of (37 ± 1) ° C.

In this colony E. coli 3912/41 (055: K59), E. coli ATCC 25922 - round yellow-orange in color with a yellow zone, with a diameter of 2.0-3.0 mm; colonies of K. pneumoniae 418 - round, mucous, orange, with a diameter of 2.5-4.0 mm; colonies of S.sonnei “S-form” - round, red-brown in color with a blue zone, with a diameter of 1.5-2.5 mm; colonies of S. typhimurium 79 - red-brown in color with an uneven edge and with a blue zone, with a diameter of 2.0-3.5 mm.

The differential diagnostic properties are checked by sowing a mixture of equal volumes of test strains of E. coli 3912/41 (055: K59) and S. typhimurium 79 from a 10 ^-6 dilution.

The inhibitory properties of the medium are checked by sowing 0.1 ml of the microbial suspension of the S. aureus Wood-46 test strain from a 10 ^-3 dilution.

The results of biological control are presented in table.2

Example 2. The environment is prepared in accordance with example 1.

Pancreatic hydrolyzate of fish meal with tergitol 7 (PGRM with tergitol 7) 6.1 Yeast extract 5,0 D (+) - lactose, 1-water 10.0 Bromothymol blue 0.08 Sodium dodecyl sulfate 0.1 2,3,5-triphenyltetrazolium chloride 0,025 Sodium carbonate 0.15 Microbiological agar 11.0 pH 7.4 ± 0.2

The results of biological control are presented in table 2 and are similar to the results of the verification in example 1.

Example 3. The environment is prepared in accordance with example 1.

Pancreatic hydrolyzate of fish meal with tergitol 7 (PGRM with tergitol 7) 6.7 Yeast extract 6.0 D (+) - lactose, 1-water 12.0 Bromothymol blue 0.09 Sodium dodecyl sulfate 0.12 2,3,5-triphenyltetrazolium chloride 0,026 Sodium carbonate 0.16 Microbiological agar 14.0 pH 7.4 ± 0.2

The results of biological control are presented in table 2 and are similar to the results of the verification in example 1.

Example 4. The environment is prepared in accordance with example 1.

Pancreatic hydrolyzate of fish meal with tergitol 7 (PGRM with tergitol 7) 4.9 Yeast extract 3.0 D (+) - lactose, 1-water 6.0 Bromothymol blue 0.06 Sodium dodecyl sulfate 0.06 2,3,5-triphenyltetrazolium chloride 0,023 Sodium carbonate 0.13 Microbiological agar 5,0 pH 7.4 ± 0.2

Violation of the quantitative ratio of the ingredients of the medium leads to a sharp deterioration in growth and, consequently, differentiating properties of the medium.

The results of biological control are presented in table 2.

Example 5. The environment is prepared in accordance with example 1.

Pancreatic hydrolyzate of fish meal with tergitol 7 (PGRM with tergitol 7) 7.3 Yeast extract 7.0 D (+) - lactose, 1-water 14.0 Bromothymol blue 0.1 Sodium dodecyl sulfate 0.14 2,3,5-triphenyltetrazolium chloride 0,027 Sodium carbonate 0.17 Microbiological agar 17.0 pH 7.4 ± 0.2

The results of biological control are presented in table 2 and show the deterioration of growth and differentiating properties of the medium.

The table shows that the proposed nutrient medium prepared in accordance with examples 1, 2, 3, has good growth properties, high accuracy of differentiation of E. coli and coliform bacteria from other enterobacteria, inhibits the growth of staphylococcus.

A change in the quantitative ratio of the components of the environment leads to a violation of the biological quality indicators of the proposed environment.

So, in the case of preparation of the medium in accordance with examples 4, 5, there is a sharp decrease in sensitivity, a change in the diameter and morphology of the colonies of enterobacteria.

Thus, in comparison with the prototype environment, the proposed nutrient medium has several advantages:

- for its manufacture, a dry nutrient base was first developed, containing pancreatic hydrolyzate of fish meal with tergitol 7;

- easy to prepare, does not require autoclaving, sterilized by boiling for 3-4 minutes and can be used for 10-12 days when stored at a temperature of 2-8 ° C, while maintaining differentiating properties with respect to lactose-negative, but restoring TTX of bacteria that are stained red-brown;

- does not require fractional introduction of components and separate sterilization of sodium hepta-decyl sulfate (tergitol 7) by autoclaving for 15 min at a temperature of 121 ° C and a solution of 2,3,5-triphenyltetrazolium chloride (TTX) by membrane filtration;

- does not require special equipment and supplies for sterilization of components by membrane filtration;

- on the proposed enterobacteria medium, after 20 h of incubation of crops at a temperature of (37 ± 1) ° С, colonies of larger diameter are formed, which provides more accurate differentiation and, as a result, reliable interpretation of the results.

Table 1. The composition of the nutrient media of various manufacturers, g / l No. p / p Component composition of culture media Lactose TTX agar with sodium heptadecyl sulfate (medium with Tergitol-7) GOST R 52426-2005; ISO 9308-1,2000 Lactose agar with Tergito-scrap-7 and TTX Merck Tergitol-7 agar HiMedi » Lactose TTS agar with Tergito-scrap-7 GEM LLC Tergitol ^@ -7 agar Fluka one Peptone 10.0 10.0 5,0 10.0 5,0 2 Peptic hydrolyzate of animal tissue - - - - - 3 Casein Pancreatic Hydrolyzate - - - - - four Meat extract 5,0 5,0 - 5,0 - 5 Yeast extract 6.0 6.0 3.0 6.0 3.0 6 Lactose 20,0 20,0 10.0 20,0 10.0 7 Bromothymol blue 0.05 0.05 0,025 0.05 8 Agar 15.0-20.0 12.7 15.0 12.0 15.0 9 Tergitol-7 5 ml of 0.2% solution per 100 ml 0.1 0.1 0.1 0.1 10 TTX 5 ml of a 0.05% solution per 100 ml 0,025 0,025

Table 2. Comparative characteristics of the biological parameters of Lactose TTX agar with tergitol 7 p / p The name of the test strains Proposed Environment Example Comparison environments Control the growth of test strains on timing agar one 2 3 four 5 Lactose TTC agar with Tergitol 7, Merck Tergitol-7 Agar, Fluka one 2 3 four 5 6 7 8 9 10 one E.coli 055 3912/41 74 81 72 65 34 77 80 85 2.5-3.0 2.5-3.0 2.5-3.0 2.3-2.5 1.0-1.5 2.2-2.5 1.4-1.8 2.0-3.0 Round, yellow-orange with a yellow area around Round, yellow-orange with a yellow area around Round, yellow-orange with a yellow area around Round, light yellow with a faint yellow area around Round, yellow-orange with a yellow area around Round, yellow-orange with a yellow area around Round, yellow-orange with a yellow area around Round, colorless, smooth 2 E.coliATCC 25922 82 79 85 36 28 68 81 96 2.5-3.2 2.5-3.2 2.5-3.2 2.0-2.7 0.8-1.4 2.5-3.0 1.4-1.6 2.2-2.8 Round, yellow-orange with a yellow area around Round, yellow-orange with a yellow area around Round, yellow-orange with a yellow area around Round, light yellow with a faint yellow area around Round, yellow-orange with a yellow area around Round, yellow-orange with a yellow area around Round, yellow-orange with a yellow area around Round, colorless, smooth 3 S.enteritidis 11276 60 65 63 60 60 60 64 71 2.3-2.7 2.3-2.7 2.3-2.7 2.3-2.7 1.2-1.4 3.0-3.5 1.4-2.0 1.2-1.5 reddish brown with a rough edge and a blue area reddish brown with a rough edge and a blue area reddish brown with a rough edge and a blue area Pale brown with a rough edge and a blue area reddish brown with a rough edge and a blue area reddish brown with a rough edge and a blue area reddish brown with a rough edge and a blue area Round, colorless, smooth

Continuation of table 2 one 2 3 four 5 6 7 8 9 10 four S.typhimurium 69 75 65 39 26 72 84 96 79 2.5-3.5 2.8-3.5 2.5-3.2 3.0-4.0 1.5-2.0 2.5-3.0 1.8-2.2 2.0-3.0 red red red Light red red red brown with a rough edge and a blue area brown with a rough edge and a blue area brown with a rough edge and a blue area brown with a rough edge and a blue area brown with a rough edge and a blue area brown with a rough edge and a blue area brown with a rough edge and a blue area Round, colorless, smooth 5 K. pneumonia 418 53 58 59 43 26 49 61 53 3.0-4.0 3.0-4.0 3.0-3.5 2.5-4.0 3.0-3.5 2.5-3.7 1.8-2.0 3.0-3.5 Round, slimy, orange., Faint yellow Round, slimy, orange., Faint yellow Round, slimy, orange., Faint yellow Round, slimy, yellow, faint yellow Round, slimy, yellow, faint yellow Shiny, slimy, orange, yellow, S-f Shiny, slimy, orange., Round, slimy, colorless 6 K.oxytoca 2102 64 68
2.3-2.7
Round, yellow with a yellow area around 59
2.3-2.7
Round, yellow with a yellow area around 24
2.3-2.7
Round, light yellow with a faint yellow area around fourteen 61 48 72 2.3-2.7 2.3-2.7 2.0-2.3 2.5-3.2 2.5-3.0 Round, yellow with a yellow area around Round, yellow with a yellow area around Round, yellow-orange with a yellow area around Round, yellow-orange with a yellow area around Round, slimy, colorless

Continuation of table 2 one 2 3 four 5 6 7 8 9 10 7 K pneumonia 3534/51 46 52 43 26 16 45 42 63 3.0-4.0 3.0-4.0 3.0-4.0 3.0-4.5 1.5-2.5 3.0-3.5 2.0-2.5 2.5-3.5 Round, red-brown with a blue area around Round, red-brown with a blue area around Round, red-brown with a blue area around Round, red-brown with a blue area around Round, red-brown with a blue area around Round, red-brown with a blue area around Round, red-brown with a blue area around Round, slimy, colorless 8 S.sonnei "S-form" 58 55 52 25 fifteen 45 43 64 2.0-2.5 2.0-2.5 2.0-2.5 2.0-3.0 1.0-1.5 3,5-3,7 1.6-2.0 Round, red-brown with a blue area around Round, red-brown with a blue area around Round, red-brown with a blue area around Round, light brown with a faint blue area around Round, red-brown with a blue area around Round, red-brown with a blue area around the SR-shape Round, red-brown with a blue area around the SR-shape 1.5-2.0 Round, colorless, smooth 9 C.freundii 101/57 51 57 53 53 53 48 51 56 1.6-1.8 1.6-1.8 1.6-1.8 1.2-1.5 0.8-1.2 1.8-2.0 1.2-1.5 Round, yellow-orange with a yellow area around Round, yellow-orange with a yellow area around Round, yellow-orange with a yellow area around Round, yellow - with a faint yellow area around Round, yellow-orange with a yellow area around Round, yellow-orange with a yellow area around Round, yellow-orange with a yellow area around 1.5-2.0 Round, slimy, colorless

Continuation of table 2 one 2 3 four 5 6 7 8 9 10 10 P.aervginosa 27/99 48 46 fifty 38 eighteen 22 thirty 59 1.2-2.0 1.2-2.0 1.2-2.0 0.8-1.6 1.2-2.0 1.6-2.5 0.6-1.0 2.0-3.0 reddish brown with a rough edge and a blue area reddish brown with a rough edge and a blue area reddish brown with a rough edge and a blue area light brown with a rough edge and a faint blue area reddish brown with a rough edge and a blue area red-brown color with a rough edge and a blue zone R-shape red-brown color with a rough edge and a blue zone R-shape Round, translucent, gray-green eleven P.mirabilis 3177 51 58 53 21 fifteen fifty No growth Continuous swarming 1.2-1.8 1.2-1.8 1.2-1.8 0.8-1.2 0.5-1.0 red-brown color with an uneven edge and with a blue zone Focal swarm reddish brown with a rough edge and a blue area reddish brown with a rough edge and a blue area reddish brown with a rough edge and a blue area Light brown with a rough edge and blue area reddish brown with a rough edge and a blue area

Claims (1)

Dry differential diagnostic nutrient medium for detecting and counting E. coli and coliform bacteria, containing a nutrient base, yeast extract, lactose, 2,3,5-triphenyltetrazolium chloride, bromothymol blue, microbiological agar, characterized in that it is a nutrient base contains pancreatic hydrolyzate of fish meal, dried with tergitol 7 at the rate of 0.1 g of tergitol 7 per 6 g of dry pancreatic hydrolyzate of fish meal, and additionally sodium dodecyl sulfate, sodium carbonate, with the following amount Hinnom ratio, g / l:
Pancreatic hydrolyzate of fish meal with tergitol 7 (PGRM with tergitol 7) 5.5-6.7 Yeast extract 4.0-6.0 D (+) - lactose, 1-water 8.0-12.0 Bromothymol blue 0.07-0.09 Sodium dodecyl sulfate 0.08-0.12 2,3,5-triphenyltetrazolium chloride 0,023-0,026 Sodium carbonate 0.14-0.16 Microbiological agar 8.0-14.0 pH 7.4 ± 0.2