RU2792438C1 - Selective nutrient medium with mannitol, bile and polymyxin to detect bacteria of the genera proteus, morganella, providencia, dry - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: essence of the invention is the fact that pancreatic hydrolyzate of fish meal (PGRM K₂HPO₄) demineralized from calcium and magnesium cations with additional precipitation of high molecular weight proteins and peptides, purified bile (JOG) is used as a protein base in the following quantitative ratio of components, g/l: dry pancreatic hydrolyzate of fish meal (PGRM K₂HPO₄ dry) 8.8-12.8, dry purified bile 3.0-7.0, mannitol D(-) 0.5-1.5, sodium chloride 4.0-6.0, bromthymol blue 0.03-0.05, crystal violet 0.0015-0.0025. The composition of SD, g/l: polymyxin B sulfate 0.01-0.014.

EFFECT: finished medium after sterilization, the introduction of urea and a selective additive (polymyxin B) is transparent, green, which improves the visual interpretation of the research results.

1 cl, 1 tbl, 5 ex

Description

The invention relates to sanitary and clinical microbiology and can be used for selective detection of bacteria of the genera Proteus, Morganella, Providencia and their differentiation by the ability to break down urea in sanitary and bacteriological studies.

In accordance with GOST 28560-90 “Food products. The method for detecting bacteria of the genera Proteus, Morganella, Providencia is recommended. Liquid selective medium. Medium base: to 1 dm3 of meat-peptone broth add 1.0 g of mannitol, 50.0 ^cm3 of natural bovine bile or an equivalent amount of dry bile, 0.8 g of dibasic potassium phosphate, 2.0 cm3 of crystal violet solution, 2.0 cm ³ alcohol solution of bromthymol blue. Adjust the pH so that after sterilization it is (6.9±0.1) at 25°C. Sterilize with flowing steam at 100°C for 15 minutes. To prepare the medium, 10.0 cm ³ of urea solution and 100,000 units of polymyxin B sulfate or 120,000 units of polymyxin M sulfate are added aseptically to the base. The prepared medium is clear, yellow-brown in color.

The selective accumulation of bacteria of the genera Proteus, Morganella, Providencia is ensured by the presence of bile, crystal violet and polymyxin, which largely inhibit the growth of gram-positive and gram-negative concomitant microflora, and mannitol and urea impart differentiating properties of the medium in the presence of the bromthymol blue indicator.

The disadvantages of liquid selective laboratory-made media are:

- the complexity of preparation (preliminary preparation of meat-peptone broth, preparation of mother solutions of crystal violet and bromthymol blue, which significantly increases time and labor costs);

- use of untreated cattle bile as an elective factor;

- lack of domestic dry preparations - peptone and bile, which have a high degree of transparency in solutions;

- the difficulty of observing the conditions of sterilization with flowing steam: - the absence of chemical (thermal-time) indicators of steam sterilization, with the help of which, subject to the specified duration of sterilization exposure and temperature, the color changes irreversibly, and the indicator mark reaches the final color corresponding to the color of the standard.

Known commercial Selective medium for determining the genera Proteus, Morganella, Providencia

Currently, in Russia, LLC "NPTs" Biokompas-S "is producing a Selective medium for determining childbirthProteus, Morganella, Providencia,for the preparation of liquid selective medium according to GOST 28560-90, which is a set of dry medium with attached vials of solutions: bromthymol blue indicator and crystal violet.

The disadvantage of commercial environments is:

- limited availability;

- the difficulty of observing the conditions of sterilization with flowing steam: - the absence of chemical (thermal-time) indicators of steam sterilization, with the help of which, subject to the specified duration of sterilization exposure and temperature, the color changes irreversibly, and the indicator mark reaches the final color corresponding to the color of the standard.

- instability of the inhibitory ability of bovine bile.

The closest to the proposed medium is a liquid selective medium according to GOST 28560-90 of laboratory preparation, based on meat enzymatic peptone,

technical resultis to create a domestic dry selective nutrient medium with mannitol, bile and polymyxin to detect bacteria of childbirthProteus, Morganella, Providencia, transparent in a ready-to-use state, with high specific activity and selectivity, providing accessibility and obtaining objective results of bacteriological control.

The technical result is achieved by the fact that it is proposed Selective nutrient medium with mannitol, bile and polymyxin to detect bacteria of the genera Proteus, Morganella, Providencia dry containing a protein base, mannitol, ox bile, crystal violet, bromthymol blue, polymyxin B sulfate and urea, and as a protein base contains pancreatic hydrolyzate of fish meal (PGRM K ₂ HPO ₄ ) demineralized from calcium and magnesium cations with additional precipitation of high molecular weight proteins and peptides, purified bile (CHG), in the following quantitative ratio of components, g / l:

Pancreatic hydrolyzate of fishmeal dry (PGRM K ₂ HPO ₄ dry) 8.8-12.8 Dry purified bile 3.0-7.0 Mannitol D(-) 0.5-1.5 sodium chloride 4.0-6.0 Bromothymol blue 0.03-0.05 Crystal violet 0.0015-0.0025

Composition of SD, g/l:

Polymyxin B sulfate 0.01-0.014

The addition of potassium phosphate to the liquid PGRM makes it possible to exclude its additional introduction into the composition of the medium, ensures transparency and high buffer capacity of the finished medium. The balance of the component composition of the medium ensures good growth and selective properties of the Selective Nutrient Medium with mannitol, bile and polymyxin to detect bacteria of childbirthProteus, Morganella, Providencia.

Method for obtaining selective nutrient medium with mannitol, bile and polymyxin to detect bacteria of childbirthProteus, Morganella, Providencia provides mixing of dry components, g/l:

Pancreatic hydrolyzate of fishmeal, dry
(PGRM K ₂ HPO ₄ dry) 8.8-12.8 Bile purified dry 3.0-7.0 Mannitol D(-) 0.5-1.5 sodium chloride 4.0-6.0 Bromothymol blue 0.03-0.05 Crystal violet 0.0015-0.0025

Composition of SD, g/l:

Polymyxin B sulfate 0.01-0.014

Additive - urea (urea according to GOST 6691-77 or imported of similar quality) at the rate of 5.0 g/l. Urea is not included in the composition of the dry medium.

The difference between the proposed environment and the prototype is the use as a nutritional base specially made pancreatic hydrolyzate of fish meal with dibasic potassium phosphate. The calculated amount, in terms of dry matter of dibasic potassium phosphate, is added to liquid PGRM, the reaction mixture is heated to a temperature of 100°C, held for 15–20 min, filtered and dried in a drying plant in a vibrofluidized bed.

Another difference is the presence of purified dry bile, which ensures the transparency of the finished medium and the inhibitory properties of the medium.

Pancreatic hydrolyzate of fishmeal (PGRM K ₂ HPO ₄ ) is a source of nutrients necessary for the growth of microorganisms: nitrogen, vitamins, mineral salts and amino acids. Mannitol is a fermentable and differentiating substrate as well as a source of carbon. The medium provides selectivity for gram-positive and gram-negative associated microflora due to the presence of purified bovine bile, crystal violet and polymyxin in the medium.

Example 1. To prepare the medium, weigh the following ingredients, g/l:

Pancreatic hydrolyzate of fishmeal, dry
(PGRM K ₂ HPO ₄ dry) 8.8 Bile purified dry 3.0 Mannitol D(-) 0.5 sodium chloride 4.0 Bromothymol blue 0.03 Crystal violet 0.0015 pH ready medium 6.9-7.1

SD, g/l:

Polymyxin B sulfate 0.01

Samples are mixed in 1 liter of distilled water, boiled for 2 minutes, sterilized by autoclaving at 121°C for 15 minutes. After cooling the nutrient medium to a temperature of 40-45°C under aseptic conditions, add a sterile 50% solution of urea at the rate of 10 ml/l and the contents of the vial with SD at the rate of 5 ml per 1 liter of the nutrient medium, pour 5 ml into sterile glass test tubes GOST 25336-82.

To prepare a 50% urea solution, 50.0 g of urea is placed in a 100 ml flask, dissolved in a small amount of distilled water, then the volume of the solution is adjusted to the mark. The urea solution is sterilized by exposure at room temperature for 2-3 days. The urea solution is stored for a month at a temperature of 2-8°C.

To control the resulting environment, test strains of microorganisms from the State Collection of Pathogenic Microorganisms and Cell Cultures "GKPM-Obolensk" are used.

A standard culture suspension of each test strain is prepared, corresponding to 10 units according to the standard turbidity sample OSO 42-28-85 P, using a sterile 0.9% sodium chloride solution. The resulting culture suspensions were diluted tenfold (4.5 ml of 0.9% sodium chloride solution with 0.5 ml of microbial suspension) to a dilution of 10^-6 (ForEscherichia coliATCC 25922 AndStaphylococcus aureus Wood-46 АТСС 10832 - before dilution 10^-4)

To determine the growth properties of seeding produced 0.5 ml of microbial suspension of each of the test strains from a dilution in 5.0 ml of medium.

The inoculations were incubated at a temperature of (37 ± 1)°C for 20-24 hours. The prepared nutrient medium is transparent green in color, ensures the growth and accumulation of the following test strains: Proteus vulgaris HX 19 222, Proteus mirabilis 3177, Morganella morganii ATCC 25830 DSM 30164 .

The nutrient medium significantly inhibits the growth of test strains Escherichia coli ATCC 25922 , Staphylococcus aureus Wood-46 ATCC 10832.

The results of biological control of laboratory samples of the Selective medium for the determination of the genera Proteus, Morganella, Providencia on a set of test strains are presented in Table 2.

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

Pancreatic hydrolyzate of fishmeal, dry
(PGRM K ₂ HPO ₄ dry) 10.8 Bile purified dry 5.0 Mannitol D(-) 1.0 sodium chloride 5.0 Bromothymol blue 0.04 Crystal violet 0.002 pH ready medium 6.9-7.1

SD, g/l:

Polymyxin B sulfate 0.012

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

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

Pancreatic hydrolyzate of fishmeal, dry
(PGRM K ₂ HPO ₄ dry) 12.8 Bile purified dry 7.0 Mannitol D(-) 1.5 sodium chloride 6.0 Bromothymol blue 0.05 Crystal violet 0.0025 pH ready medium 6.9-7.1

SD, g/l:

Polymyxin B sulfate 0.014

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

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

Pancreatic hydrolyzate of fishmeal, dry
(PGRM K ₂ HPO ₄ dry) 6.8 Bile purified dry 2.0 Mannitol D(-) 0.25 sodium chloride 3.0 Bromothymol blue 0.02 Crystal violet 0.001 pH ready medium 6.9-7.1

SD, g/l:

Polymyxin B sulfate 0.008

Violation of the quantitative ratio of the ingredients of the medium in example 4 leads to a deterioration in growth and inhibitory properties.

The results of biological control are presented in table 2.

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

Pancreatic hydrolyzate of fishmeal, dry
(PGRM K ₂ HPO ₄ dry) 14.8 Bile purified dry 7.0 Mannitol D(-) 1.75 sodium chloride 7.0 Bromothymol blue 0.06 Crystal violet 0.003 pH ready medium 6.9-7.1

SD, g/l:

Polymyxin B sulfate 0.016

The results of biological control are presented in table 2.

Violation of the quantitative ratio of the ingredients of the medium in a larger direction in example 5 leads to a significant increase in inhibitory properties, which negatively affects the specific activity of the medium.

The table shows that the proposed nutrient medium in accordance with examples 1, 2, 3 has good growth properties, is effective in the accumulation of representatives of the genus Proteus and inhibits the growth of a number of gram-positive and gram-negative microorganisms.

Visual accounting of the results of detection of bacteria of the genus Proteus and their differentiation by the ability to break down urea at the end of the incubation period of crops showed identical results in the test and control (laboratory preparation) media.

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

So, in the case of preparing the medium in accordance with examples 4.5, a decrease in specific activity and a change in the inhibitory ability of the medium are observed:

- at concentrations of components below the minimum limits, growth of Staphylococcus aureus Wood-46 ATCC 10832 was observed.

- slight discoloration and diffuse clouding during growth of Proteus vulgaris HX 19 222, Proteus mirabilis 3177, Morganella morganii ATCC 25830 DSM 30164

- when the concentration of components is above the maximum limits

- the growth of Proteus vulgaris HX 19 222, Proteus mirabilis 3177, Morganella morganii ATCC 25830 DSM 30164 is suppressed (slight discoloration and diffuse turbidity, when visually viewing crops after cultivation):

the inhibitory effect of the medium is pronounced.

Thus, a domestic competitive selective nutrient medium with mannitol, bile and polymyxin has been developed to detect bacteria of the genera Proteus, Morganella, Providencia dry;

a technology has been developed for obtaining a dry nutritional base from liquid pancreatic fish meal hydrolyzate (PGRM K ₂ HPO ₄ ) with disubstituted potassium phosphate, demineralized from calcium and magnesium cations with additional precipitation of high-molecular proteins and peptides provides transparency and high buffer capacity of the finished medium.

simplified procedure and reduced labor costs for the preparation of the medium;

ready-made medium after sterilization, introduction of urea and selective additive (polymyxin B) - transparent, green, which improves the visual interpretation of the research results;

the mode of sterilization of the medium has been changed, which makes it possible to control the process using chemical indicators.

Table 2. Biological indicators of Selective nutrient medium with mannitol, bile and polymyxin for the detection of bacteria of the genera Proteus, Morganella, Providencia.

No. p / p Name of the test strain Selective nutrient medium with mannitol, bile and polymyxin to detect bacteria of the genera Proteus, Morganella, Providencia. Control
Lab preparation environment Example 1 Example 2 Example 3 Example 4 Example 5 1 Proteus vulgaris HX 19 222
breeding 10 ^-6 diffuse haze
environment color change from green to blue diffuse haze
environment color change from green to blue diffuse haze
environment color change from green to blue slight diffuse haze
environment color change from green to blue slight diffuse haze
environment color change from green to blue diffuse haze
environment color change from green to blue 2 Proteus mirabilis 3177
breeding 10 ^-6 diffuse haze
environment color change from green to blue diffuse haze
environment color change from green to blue diffuse haze
environment color change from green to blue slight diffuse haze
environment color change from green to blue slight diffuse haze
environment color change from green to blue diffuse haze
environment color change from green to blue 3 Providencia alcalifaciens1068-50 NCTC
breeding 10 ^-6 diffuse haze
Without changing the color of the medium diffuse haze
Without changing the color of the medium diffuse haze
Without changing the color of the medium slight diffuse haze
Without changing the color of the medium slight diffuse haze
Without changing the color of the medium diffuse haze
Without changing the color of the medium 4 Morganella morganii ATCC 25830 DSM 30164
breeding 10 ^-6 diffuse haze
environment color change from green to blue diffuse haze
environment color change from green to blue diffuse haze
environment color change from green to blue slight diffuse haze
environment color change from green to blue slight diffuse haze
environment color change from green to blue diffuse haze
environment color change from green to blue 5 Staphylococcus aureus Wood-46 ATCC 10832
breeding 10 ^-4 No growth No growth No growth slight diffuse haze
Without changing the color of the medium No growth No growth 6 Escherichia coli ATCC 25922
breeding 10 ^-4 No growth No growth No growth No growth No growth No growth

Claims (4)

Selective nutrient medium for the detection of bacteria of the genera Proteus, Morganella, Providencia, dry, containing the following initial components, g/l: Pancreatic digest of fishmeal demineralized from calcium cations and magnesium with additional precipitation high molecular weight proteins and peptides dry (PGRM K ₂ HPO ₄ dry) 8.8-12.8 Dry purified bile 3.0-7.0 Mannitol D(-) 0.5-1.5 sodium chloride 4.0-6.0 Bromothymol blue 0.03-0.05 Crystal violet 0.0015-0.0025, selective additive (SD), g/l: Polymyxin B sulfate 0.01-0.014