RU2530549C1 - Dry selective nutrient medium for isolation of pseudomonades - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: nutrient medium comprises the pancreatic digest of casein, meat peptone, yeast extract, sodium chloride, dibasic potassium phosphate, monobasic potassium phosphate, magnesium sulphate, cetrimide, bacteriological agar, sodium carbonate, nalidixic acid, glycerol, and distilled water in a predetermined ratio of components.

EFFECT: invention enables to simplify the identification of pseudomonades and to increase the efficiency of diagnosis in carrying out clinical and sanitary-microbiological studies.

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Description

The invention relates to sanitary and clinical microbiology and can be used to isolate Pseudomonas aeruginosa from clinical material, environmental objects, food products, etc.

Leading foreign companies produce agar culture media with cetrimide based on gelatin, meat peptone and lactose, as well as potassium sulfate, magnesium chloride, cetrimide, agar, nalidixic acid and glycerin. Cetrimide Agar (Pseudomonas Selectice Agar Base) company Fluka (Scientific research 2003/2004, p. 361), LLC "GEM" (Bacteriological media. Catalog 2012,). Cetrimide Agar Base HiMedia (HiMedia Laboratories Pvt. Limited (India), http://www.himedialabs.com). Selective agar for pseudomonas, the basis of Merck "Microbiology, 2004/2005, p. 236.

The closest to the intended purpose of the proposed environment is Cetrimide Agar (Pseudomonas Selectice Agar Base) company Fluka, consisting of the following components, g / l:

Gelatin Peptone 20.0 Potassium sulfate 10.0 Magnesium chloride 1.4 Cetrimide 0.3 Agar 15.0 Glycerol 10.0 ml

Due to the lack of a domestic commercial environment with cetrimide for isolation of Pseudomonas aeruginosa from various objects, food control is carried out on agar with cetrimide, laboratory preparation GOST 54755-2011 “Food products. Methods for the identification and quantification of bacteria of the species Pseudomonas aeruginosa. " M .: Standartinform 2012, XII State Pharmacopoeia of the Russian Federation. - M .: 2007.

The component content of Agar with cetrimide of various manufacturers in g / l are presented in table 1.

Table 1 The composition of Agar with cetrimide of various manufacturers, g / l No. p / p Component composition Fluka Ltd
"GEM" Merck HiMedia GOST 54755-2011 "Food Products one. Gelatin Peptone 20.0 20,0 20,0 20,0 20,0 2. Meat peptone - 10.0 - - - 3. Potassium sulfate 10.0 10.0 10.0 10.0 10.0 four. Magnesium chloride 1.4 1.4 1.4 1.4 1.4 5. Cetrimide 0.3 0.3 0.3 0.3 0.3 6. Agar fifteen 13.6 13.0 15.0 15.0 7. Nalidixic acid 0.15 mcg Glycerol 10.0 ml pH 7.2 ± 0.2

The technical result of the invention is the creation of a domestic dry nutrient medium for the isolation of pseudomonads, dry (Cetrimide agar), which ensures the stability of the results by growth, differentiating and inhibitory properties.

The technical result is achieved by balancing the component composition of the medium, which contains pancreatic casein hydrolyzate produced by NPO PS, meat peptone, yeast extract, sodium chloride, magnesium sulfate, cetrimide, microbiological agar, sodium carbonate, a buffer mixture consisting of potassium phosphate disubstituted and disubstituted and and also additional nalidixic acid in the following ratio of components, g / l:

Casein Pancreatic Hydrolyzate 10.0 Meat peptone 10.0 Yeast extract 2.0 Sodium Chloride 5,0 Potassium Disubstituted Potassium Phosphate 1,0 Potassium phosphate monosubstituted 1,0 Magnesium sulfate 1,5 Cetrimide (hexadecyltrimethylammonium bromide) 0.3 Bacteriological agar 10.0 Sodium carbonate 0.3 Nalidixic acid 0.01 Glycerol 10.0 ml

The difference between the proposed environment from the prototype is

- replacement of magnesium chloride with magnesium sulfate, which in the presence of potassium phosphates leads to increased formation of the pigment of pyocyanin;

- replacement of potassium sulfate with mono- and disubstituted potassium phosphates, which improves differentiating properties by maintaining the buffer capacity of the medium;

- the possibility of replacing peptones from gelatin with pancreatic casein hydrolyzate, produced at NPO PS, with preservation of all biological properties of the nutrient medium, has been proven

- a yeast extract containing vitamins of group B is additionally added to the composition of the nutrient medium, which stimulates optimal growth conditions for pseudomonads;

- nalidixic acid is additionally added to the nutrient medium to enhance the inhibitory effect against concomitant microflora;

- sodium chloride was introduced into the composition of the nutrient medium to maintain the molar balance of ions.

The nutrient medium is prepared as follows:

Example 1: To prepare a nutrient medium take the following weighed components

Casein Pancreatic Hydrolyzate 10.0 ± 2.0 Meat peptone 10.0 ± 2.0 Yeast extract 2.0 ± 0.5 Sodium Chloride 5.0 ± 1.0 Potassium Disubstituted Potassium Phosphate 1.0 ± 0.1 Potassium phosphate monosubstituted 1.0 ± 0.1 Magnesium sulfate 1.5 ± 0.1 Cetrimide (hexadecyltrimethylammonium bromide) 0.3 ± 0.05 Bacteriological agar 10.0 ± 3.0 Sodium carbonate 0.3 ± 0.1 Nalidixic acid 0.01 ± 0.002 Glycerol 10.0 ± 2.0 ml

Samples are carefully mixed in 1000 ml of distilled water, heated until the agar melts and 10 ml of glycerol are added, boiled for 2 min until the agar is completely melted, filtered through a cotton-gauze filter. Autoclave for 15 minutes at a temperature of 121 ° C. The medium is cooled to a temperature of 45-50 ° C, poured into sterile Petri dishes. After the medium has solidified, the plates are dried.

For biological control of the proposed nutrient medium, cultures of museum strains are used: P. aeruginosa 453, P. aeruginosa 27/99, P. aeruginosa 10145, P. aeruginosa 27853 from a dilution of 10 ~ 6 S. typhimurium 79, E. coli 055.K59 3912 / 41, P. mirabilis 3177 from a 10 ^-3 dilution, S. aureus 209-P from a 10 ^-6 dilution. All crops are incubated aerobically at a temperature of 37 ± 1 ° C for 44-48 hours. When taking into account the results, the presence and nature of the growth of colonies are taken into account. The results of biological control are presented in table 2.

Selective nutrient medium for the isolation of pseudomonads, dry (Cetrimide agar), prepared in accordance with the proposed formulation, provides the growth of P. aeruginosa in the form of yellow-green colonies. The growth of concomitant gram-positive and gram-negative bacteria is significantly inhibited. The developed medium meets the requirements for cetrimide agar in selectivity and differentiating properties against P. aeruginosa.

The results of biological control are presented in table 2.

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

Casein Pancreatic Hydrolyzate 8.0 Meat peptone 8.0 Yeast extract 1,5 Sodium Chloride 4.0 Potassium Disubstituted Potassium Phosphate 0.9 Potassium phosphate monosubstituted 0.9 Magnesium sulfate 1.4 Cetrimide (hexadecyltrimethylammonium bromide) 0.25 Bacteriological agar 7.0 Sodium carbonate 0.2 Nalidixic acid 0.008 Glycerol 8.0 ml

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

Casein Pancreatic Hydrolyzate 10.0 Meat peptone 10.0 Yeast extract 2.0 Sodium Chloride 5,0 Potassium Disubstituted Potassium Phosphate 1,0 Potassium phosphate monosubstituted 1,0 Magnesium sulfate 1,5 Cetrimide (hexadecyltrimethylammonium bromide) 0.3 Bacteriological agar 10.0 Sodium carbonate 0.3 Nalidixic acid 0.01 Glycerol 10.0 ml

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.

Casein Pancreatic Hydrolyzate 12.0 Meat peptone 12.0 Yeast extract 2,5 Sodium Chloride 6.0 Potassium Disubstituted Potassium Phosphate 1,1 Potassium phosphate monosubstituted 1,1 Magnesium sulfate 1,6 Cetrimide (Hexadecyltrimethylammonium bromide) 0.35 Bacteriological agar 13.0 Sodium carbonate 0.4 Nalidixic acid 0.012 Glycerol 12.0 ml

The results of biological control are presented in table 2.

The table shows that the proposed nutrient medium prepared in accordance with examples 1, 2, 3, has good growth properties against P. aeruginosa, inhibits the growth of Salmonella, Proteus and staphylococci.

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.

Thus, the proposed nutrient medium ensures the stability of the results in terms of growth, differentiating and inhibitory properties. Using the proposed environment simplifies the identification of the pathogen from objects with a high degree of contamination of P. aeruginosa with the possibility of direct seeding on cetrimide agar, which increases the efficiency of diagnosis in clinical and sanitary-microbiological studies.

table 2 The results of the biological control of Cetrimide agar No. p / p Test strains Biological indicators (diameter (mm), colony morphology) Breeding Selective Agar for Pseudomonas (Cetrimide Agar) Cetrimide Agar (Pseudomonas Selectice Agar Base) Fluka one. P. aeruginosa 453 10 ^-6 3.0-4.0 colonies yellow-green 3.0-4.0 colonies yellow-green 2. P. aeruginosa 27/99 10 ^-6 3.0-4.0 colonies yellow-green 3.0-4.0 colonies yellow-green 3. P. aeruginosa 10145 10 ^-6 4.0
yellow-green colonies 4.0
yellow-green colonies four. P. aeruginosa 27853 10 ^-6 4.5-5.0 yellow-green colonies 4.5-5.0 yellow-green colonies 5. S. typhimurium 79 10 ^-3 no growth no growth 6. E. coli 055.K59 3912/41 10 ^-3 no growth no growth 7. P. mirabilis 3177 10 ^-3 no growth no growth 8. S. aureus 209-P 10 ^-1 no growth no growth

Claims (1)

Selective nutrient medium for the isolation of pseudomonads, dry, containing a nutrient base, potassium and magnesium salts, cetrimide and glycerin, characterized in that it contains pancreatic hydrolyzate of casein and additionally baking yeast extract, potassium phosphate disubstituted, potassium phosphate, monosodium sulfate, sodium chloride, sodium carbonate, nalidixic acid in the following ratio of components, g / l:
Casein Pancreatic Hydrolyzate 10.0 ± 2.0 Meat peptone 10.0 ± 2.0 Yeast extract 2.0 ± 0.5 Sodium Chloride 5.0 ± 1.0 Potassium Disubstituted Potassium Phosphate 1.0 ± 0.1 Potassium phosphate monosubstituted 1.0 ± 0.1 Magnesium sulfate 1.5 ± 0.1 Cetrimide (Hexadecyltrimethylammonium bromide) 0.3 ± 0.05 Bacteriological agar 10.0 ± 3.0 Sodium carbonate 0.3 ± 0.1 Nalidixic acid 0.01 ± 0.002 Glycerol
Distilled water 10.0 ± 2.0 ml
up to 1 l