Nutrient medium for isolation of hemoculture

FIELD: medicine, medicinal microbiology.

SUBSTANCE: invention proposes a medium that comprises para-aminobenzoic acid and agar, citrate or sodium heparinate as an organic salt, tris-(hydroxymethyl)-aminomethane as a buffer, and fish nutritive broth and casein pancreatic hydrolyzate, nutrient yeast as sources of nitrogen nutrition, and glucose. By sensitivity and accumulative effect with respect to strains of microorganisms causing bacteremia and sepsis exceeds the known medium and allows detecting microorganisms in the minimal their parent content in blood. Indicated advantages of the proposed medium enhance the effectiveness in diagnosis in research of blood for hemoculture. Invention can be used in isolation of hemocultures in sepsis and bacteremia.

EFFECT: valuable properties of medium.

2 tbl, 3 ex

 

The invention relates to medicine, namely to medical Microbiology, and can be used to highlight hemoculture sepsis and bacteremia.

Bacteremia and sepsis continues to be one of the urgent problems of modern medicine due to the steady upward trend in the number of patients and stable high mortality even in the most reputable domestic and foreign clinics.

The causative agents of sepsis and bacteremia can be bacteria, fungi, protozoa and viruses. But the share of bacteria account for more than 95% of cases. The largest share among the causative agents of bacteraemia and sepsis are conditionally pathogenic microorganisms, while the proportion of gram-positive and gram-negative bacteria are approximately the same (1).

Check bacteraemia and sepsis (highlighting hemoculture) is required to confirm the diagnosis and determine the etiology of the infectious process, justification of the choice of regimen of antibiotic therapy and to assess its effectiveness.

Known breeding ground for allocation hemoculture. However, this environment is intended to highlight from the blood only of Salmonella, which limits its use (2).

Also known nutrient medium to highlight hemoculture (3, 4), contains a source of nitrogen nutrition in the form of parivara of Hottinger or broth Martin and glucose is sugar broth). The disadvantages of this environment should include the formation of a clot of fibrin in blood cultures and low cumulative effect due to the absence in the composition of the medium growth stimulants highly demanding microorganisms.

The closest solution to the same destination on the totality of symptoms and the achieved effect is a nutrient medium for selection of hemoculture and cultivation of streptococci containing sources of nitrogen nutrition in the form of enzymatic hydrolysate of blood and acid hydrolysate of casein, growth - extract fodder yeast, organic and inorganic salts (5).

For reasons that impede the achievement of specified following technical result of using a known environment, adopted for the prototype, is a narrow list of allocated microorganisms (mainly Streptococcus), the absence in the environment of anticoagulants and blood substances, neutralizing the antimicrobial effect of drugs that can accumulate in the blood during antibiotic therapy.

The absence in the environment anticoagulants leads to the formation of a clot of fibrin in blood cultures, limiting the access of nutrients to the microbial cell, which inhibits the growth of microorganisms. The absence in the environment of substances that neutralize antimicrobial the step of medicines, negatively influence Visavuori of hemoculture.

The consequence of these shortcomings is the poor sensitivity and low cumulative effect of environment and narrow the list of allocated microorganisms limits the use of the known environment in bacteriological practice.

The purpose of the invention is the increased sensitivity of the cumulative effect of the environment and expansion of the spectrum allocated microorganisms.

This technical result in the implementation of the invention is achieved by the fact that the proposed environment further comprises a para-aminobenzoic acid and agar, as an organic salt contains sodium citrate or sodium heparinate, as the buffer is Tris-(oxymethyl)-aminomethan, and as sources of nitrogen nutrition - nutrient broth fish and pancreatic hydrolysate of casein in the following ratio of components in g/l of distilled water:

Nutrient broth fish10,0-20,0
Pancreatic hydrolysate
casein10,0-20,0
Extract fodder yeast2,0-4,0
Glucose8,0-12,0
Sodium citrate or0,3-0,5
Sodium heparinate 0,02-0,04
Para-aminobenzoic acid0,005-0,015
Tris-(oxymethyl)-aminomethan0.5 to 1.5
Agar0,5-1,0

Introduction to the environment, the advanced para-aminobenzoic acid helps neutralize the antimicrobial action of drugs that may be contained in the blood during antibiotic therapy.

Introduction in the proposed environment, the advanced agar with high sorption capacity, promotes neutralization products of metabolism and prevents microbial cells from the toxic effect.

Introduction to the environment of sodium citrate or sodium of heparinate with anticoagulant properties, instead of sodium acetate, do not have such, prevents coagulation (formation of a fibrin clot), which facilitates the access of nutrients to the microbial cell, thereby promoting active reproduction of microorganisms in the environment.

Given that the patients with positive hemoculture concentration of pathogens in the blood usually does not exceed 102/ml, providing conditions for the penetration of nutrients to the environment by microbial cell is necessary for the active accumulation of microorganisms in the environment.

Using the environment as a buffer of Tris-(oxymethyl)-am is Nomentana, with greater buffer capacity compared with phosphate contributes to the fact that the acid formed by fermentation of microorganisms of glucose, fully neutralized, resulting in no shift of pH in the acidic side, preventing the growth of microorganisms.

The high nutritional value of the nitrogen components of the proposed environment - nutrient broth of fish and pancreatic hydrolysate of casein provides full nutritional requirements of microorganisms of different taxonomic groups, causing bacteremia.

On the whole, the collection of characteristics contributes to the expansion of the spectrum allocated microorganisms increases the sensitivity of the environment and provides a high cumulative effect that allows you to select hemoculture with a minimum initial amount in their blood.

The medium was prepared as follows.

Example 1. In 1 l of distilled water successively dissolve 10 g of nutrient broth fish, 10 g of pancreatic hydrolysate of casein, 2 g yeast extrat, 8 g of glucose, 0.3 g of sodium citrate or 0.02 g of sodium heparinate, 0.005 g of para-aminobenzoic acid, 0.5 g of Tris-(oxymethyl)-aminomethane, 0.5 g of agar. The mixture is heated until complete unfolding of the agar, boil for 1-2 minutes, poured into vials to 100.0 ml and sterile the comfort at 120° C for 30 min environmental quality performed by seeding vials of test strains of microorganisms most frequently isolated in sepsis and bacteremia: staphylococci, streptococci, salmonel, Escherichia, meningococci, Klebsiella, Brucella, Yersinia, Pseudomonas aeruginosa, Listeria and others from breeding 10-6and 10-7(10-10 respectively of cells) (6).

After incubation of cultures in an incubator at 37°C for 18-24 h are seeing an increase both visually and by seeding away from the vials loop on nutrient, blood and serum agars. The cumulative effect is determined by the formulawhere

p1the number of colonies on plates seeded from the environment after 18-20 h of incubation;

p0the number of viable cells in the seed dose;

to the degree of cultivation.

Example 2. In 1 l of distilled water successively dissolve 15 g of nutrient broth fish, 15 g of pancreatic hydrolysate of casein, 3 g of fodder yeast extract, 10 g glucose, 0.4 g of sodium citrate or 0.04 sodium of heparinate, 0.01 grams of para-aminobenzoic acid, 1.0 g of Tris-(oxymethyl)-aminomethane, 0.75 agar. Further, according to the example 1.

Example 3. In 1 l of distilled water successively dissolve 20 g of nutrient broth fish, 20 g of pancreatic hydrolysate of casein, 4 g of fodder yeast extract, 12 is glucose, 0.5 g of sodium citrate or 0.04 g of sodium heparinate, 0.015 g of para-aminobenzoic acid, 1.5 g of Tris-(oxymethyl)-aminomethane, 1.0 g of agar. Further, according to the example 1.

The results of bacteriological monitoring proposed and known environments is presented in table 1, 2.

From table 1, 2, it is seen that the proposed environment has high sensitivity and significant cumulative effect against strains of microorganisms most frequently causing bacteremia and sepsis, and is superior in these indicators known environment, which allows to identify the microorganisms with minimal initial content in their blood.

Sources of information

1. Modern principles of antibiotic therapy of sepsis. Vasudev. Antibiotics and chemotherapy, Vol.45, 7, 2000, p.3-5.

2. Thissystem, Addriana, Iagoon. Nutrient medium for selection of hemoculture in the diagnosis of typhoid fever and paratyphoid. Patent No. 2175671. Registered. November 10, 2001

3. Handbook of microbiological and virological methods. Under. edit Mourer. M., 1982, s, 255.

4. Rcharles, Lberoamerica, Naselsky. Microbiology. M., 1986, s-241, 249, 261.

5. Handbook of microbiological nutrient media and Microdissection. Under. edit Div. Makhachkala, 1999, p.41 (prototype).

6. VDDIO, Laeulalia, Vpolicy. Sovershenstvovanie the methods for the microbiological examination of blood of patients with purulent-septic infections. The wedge. lab. the automotive technician., 1998, No. 7, p.17-19.

Table 1.

Comparative characteristics of growth of test strains of microorganisms on the proposed and known environments
Name of test strainsThe proposed environmentIn known environment
Sowing doseSowing dose
100 cells10 cells100 cells10 cells
Of these bacteria to antibiotics Dick 1++++-
Of these bacteria to antibiotics 1512++++-
S.epidermidis ATCC 14990+++++++-
S.aureus 46++++++++
S.aureus 209 "R".++++++++
P.aerogenes 27/99++++++++++
S.marcescens 1++++++++++
E.coli 055++++++++++
P.irabilis 71(2) ++++++++++
P.vulgaris H19++++++++++
K.pneumoniae 51++++++++++
S.typhi H 901++++++++++
S.pneumoniae LD++++-
B.abortus 19BA++++-
L.monocytogenes 56++++++++
J.enterocolitica 335++++++++++
J.pseudotuberculosis 111++++++++++
N.meningitidis 637++---
Legend: +++ - intensive growth
++ - average growth
+ - poor growth
- no growth

Table 2.

Comparative characteristics of the cumulative effect of the proposed and known environments
Name strains The cumulative effect
The proposed environmentIn known environment
Of these bacteria to antibiotics Dick 1740±15360±12
Of these bacteria to antibiotics 1512750±20250±9
S.epidermidis ATCC 14990845±18470±15
S.aureus 46780±16540±12
S.aureus 209 "P"874±20560±18
P.aerogenes 27/991284±24900±22
S.marcescens 1933±22850±16
E.coli 0553346±501900±22
P.mirabilis 71(2)3076±451500±21
P.vulgaris H·192448±351400±26
K.pneumoniae 511000±25860±12
S.typhi H 9013140±421800±26
S.pneumoniae LD766±10420±16
B.abortus 19BA950±21130±14
L.monocytogenes 561330±32900±24
J.enterocolitica 335866±26420±11
J.pseudotuberculosis 111 960±30560±13
N.meningitidis 637540±12-

Nutrient medium for selection of hemoculture containing sources of nitrogen nutrition, fodder yeast extract, glucose, buffers, organic salt, distilled water, characterized in that it further comprises para-aminobenzoic acid and agar, as an organic salt contains sodium citrate or sodium heparinate, as the buffer is Tris-(oxymethyl)-aminomethan, as sources of nitrogen nutrition - nutrient broth fish and pancreatic hydrolysate of casein in the following ratio of components, g/l distilled water:

Nutrient broth fish10,0-20,0
Pancreatic hydrolysate of casein10,0-20,0
Extract fodder yeast2,0-4,0
Glucose8,0-12,0
Sodium citrate0,3-0,5
or
Sodium heparinate0,03-0,05
Para-aminobenzoic acid0,005-0,015
Tris-(oxymethyl)-aminomethan0.5 to 1.5
Agar0,5-1,0



 

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