CN1699592A - Two phase Roe's culture medium and preparation method thereof - Google Patents

Abstract

The present invention discloses a preparation method of a new type incubation medium, belonging to the medical inspection field. The method can promote growth of mycobacteria. The incubation medium comprises a solid part and a liquid part: the solid part is the modified Roche's incubation medium that is generally used at home and abroad and the liquid part consists of basic incubation medium, growth promoter and bacteria inhibitor. The incubation medium can not only promote growth of mycobacteria, but also inhibit other bacteria. The growing mycobacteria can form violet red bacteria colony on the solid inclined plane of incubation medium, which is beneficial to elementary appraisal. The incubation medium can shorten incubation time obviously, increase incubation positive rate considerably, very suitable for fast inspection of mycobacteria for the hospitals and the sanitation and anti-epidemic institutions.

Description

Two phase Roe's culture medium and preparation method thereof

Technical field

The invention belongs to the medical test technical field, be specifically related to a kind of novel culture medium that can promote the quick growth of mycobacterium and preparation method thereof.

Background of invention

Mycobacterium is that a class is distributed widely in natural bacterium, and wherein some mycobacterium can make humans and animals cause a disease.In recent years, widespread reports are increasing by the disease that the mycobacterium infection causes both at home and abroad.Particularly the outbreak of epidemic of immunologic hypofunction patient's secondary infection and nosocomial infection significantly increases.The diagnosis of the infectious diseases that causes for mycobacterium can be made a definite diagnosis as long as turn out mycobacterium from sample to be checked.

At present, generally use modified Russell medium separation and Culture mycobacterium (see that Chinese Medical Association writes clinical technology working specification-tuberculosis fascicle, People's Medical Officer Press October in 2004 the 1st edition 30～33 pages) both at home and abroad.This culture medium culturing takes (needing 4-8 week usually) for a long time, and positive rate not high (being generally 10%-30%) far can not satisfy the needs of clinical diagnosis and treatment and diseases prevention.Therefore, press for a kind of quick, sensitive mycobacterium substratum of development.

Summary of the invention

The objective of the invention is to propose a kind of substratum that can promote the quick growth of mycobacterium and preparation method thereof.

The mycobacterium substratum that the present invention proposes is based on traditional improvement Russell medium, adds liquid nutrient medium again and cultivates composition, and this liquid nutrient medium comprises basic medium, growth promoter and three kinds of compositions of fungistat, and it is as follows that it forms proportioning:

Basic medium: copper sulfate 0.001～0.002 gram, zinc sulfate 0.001～0.002 gram, calcium chloride 0.001～0.002 gram, sal epsom 0.01～0.02 gram, oleic acid 0.01～0.02 gram, Ferric Ammonium Citrate 0.05～0.1 gram, Sodium Citrate 0.1～0.2 gram, ammonium sulfate 0.2～0.4 gram, Sodium.alpha.-ketopropionate 0.5～1 gram, tween-80 0.5～1 gram, asparagine 1～2 gram, potassium primary phosphate 1～2 gram, SODIUM PHOSPHATE, MONOBASIC 2～4 grams, 8～12 milliliters of neutral glycerines, 1000 milliliters of distilled water.

Growth promoter: pyridoxine hydrochloride 0.01 gram～0.02 gram, vitamin H 0.01 gram～0.02 gram, coenzyme A 0.1 gram～0.2 gram, Triphosaden 0.1 gram～0.2 gram, catalase 0.1 gram～0.2 gram, α-Nai Yisuan 0.1 gram～0.2 gram, peptone 1 gram～2 grams, glucose 10 grams～20 grams, the bovine serum albumin V5 factor 100 grams～200 grams.

Fungistat: by concentration be respectively the penbritin, amphotericin B, PXB of 50 μ g～100 μ g/ml, how the distilled water solution balanced mix of pyridine ketone acid and azlocillin forms.

The component ratio of modified Russell medium and aforesaid liquid substratum, growth promoter, fungistat is:

Modified Russell medium 8ml

Basic medium 4-8ml

Growth promoter 1-2ml

Fungistat 0.1-0.2ml.

Also add oxidation-reduction indicator (also claiming developer) in the above-mentioned mycobacterium substratum, add-on is: corresponding to the 1000ml modified Russell medium, adding 4-6ml concentration is 0.1% chromogenic reagent solution.Obtain the substratum that dyes, be used for being convenient to biochemical identification and drug sensitive test cultivating the bacterium colony colour developing.The developer that adds can require to change according to actual detected.Mainly contain TCC (TCC) etc.

The preparation method of the mycobacterium substratum that the present invention proposes is as follows:

1, with the each component dissolved in distilled water of basic medium, sterilization is 10-15 minute under 115-125 ℃ of high temperature, and cooling places 4 ℃ of refrigerators to preserve;

2, with the each component dissolved in distilled water of growth promoter, sterilising filtration, packing then places 4 ℃ of refrigerators to preserve;

3, the each component with fungistat is mixed with the solution that concentration is 50-100 μ g/ml, balanced mix then with distilled water respectively;

4, at last basic medium, growth promoter, fungistat are added modified Russell medium, the ingredient proportion of each component is:

Modified Russell medium 8ml

Basic medium 4-8ml

Growth promoter 1-2ml

Fungistat 0.1-0.2ml.

In addition, in above-mentioned mixed culture medium, add developer again, evenly mixed, promptly get the substratum that dyes.Developer is a TCC, and add-on is: corresponding to the 1000ml modified Russell medium, adding 4-6ml concentration is 1% chromogenic reagent solution.

The using method of two phase Roe's culture medium provided by the invention is as follows:

A. sample to be checked carries out centrifuge washing after anti-soil is handled;

B. get precipitation 0.1ml, drip and plant in the liquid of two phase Roe's culture medium;

C. tilting cultivation is based on 35-37 ℃ of incubation;

D. every day observations once, see have bacterial growth to be to cultivate positive;

E. be cultured to 30 days and do not see that yet bacterial growth is to cultivate feminine gender;

F. cultivate the positive, contain the particulate state goods and materials that bacterium forms in the visible liquid nutrient medium, occur mauve bacterium colony on the culture medium slant.

The substratum that can promote that mycobacterium grows fast provided by the invention based on modified Russell medium, adds mycobacterium liquid culture medium and developer, makes it to become not only to contain solids component but also contain liquid component, so be called two phase Roe's culture medium.Because this substratum contains the nutritive ingredient of dual substratum simultaneously, therefore can promote the quick growth of mycobacterium, improve and cultivate positive rate; Again because contain developer, can make mycobacterium form mauve bacterium colony simultaneously, very easily observe on the Russell medium surface.By observing colony characteristics, help the quick preliminary evaluation of mycobacterium.Also can carry out smear staining, biochemical identification and drug sensitive test, can make antibacterial therapy targetedly to the patient as early as possible like this from Russell medium surface picking colony.

Embodiment

Russell medium among the present invention is exactly at present domestic and international general modified Russell medium, adds the developer TTC solution of (also can not adding) 4-6ml 0.1% in the 1000ml modified Russell medium; Liquid nutrient medium is made up of basic medium, growth promoter and fungistat.

The basic medium moiety is as follows: copper sulfate 0.001 gram, 0.0015 gram or 0.002 gram, zinc sulfate 0.001 gram, 0.0015 gram or 0.002 gram, calcium chloride 0.001 gram, 0.0016 gram or 0.002 gram, sal epsom 0.01 gram, 0.018 gram or 0.02 gram, oleic acid 0.01 gram, 0.016 gram or 0.02 gram, Ferric Ammonium Citrate 0.05 gram, 0.07 gram or 0.1 gram, Sodium Citrate 0.1 gram, 0.15 gram or 0.2 gram, ammonium sulfate 0.2 gram, 0.3 gram or 0.4 gram, Sodium.alpha.-ketopropionate 0.5 gram, 0.7 gram or 1 gram, tween-80 0.5 gram, 0.7 gram or 1 gram, asparagine 1 gram, 1.3 gram or 2 grams, potassium primary phosphate 1 gram, 1.5 gram or 2 grams, SODIUM PHOSPHATE, MONOBASIC 2 grams, 3 grams or 4 grams, 8 milliliters of neutral glycerines, 10 milliliters or 12 milliliters, 1000 milliliters of distilled water.Compound method is earlier above-mentioned each composition to be dissolved with distilled water, 115 ℃ then～121 ℃ autoclavings 10～15 minutes, and cooling, standby through the rearmounted 4 ℃ of refrigerators preservations of sterility test.

The growth promoter moiety is as follows: pyridoxine hydrochloride 0.01 gram, 0.015 gram or 0.02 gram, vitamin H 0.01 gram, 0.015 gram or 0.02 gram, coenzyme A 0.1 gram, 0.15 gram or 0.2 gram, Triphosaden 0.1 gram, 0.15 gram or 0.2 gram, catalase 0.1 gram, 0.15 gram or 0.2 gram, α-Nai Yisuan 0.1 gram, 0.15 gram or 0.2 gram, peptone 1 gram, 1.5 grams or 2 grams, glucose 10 grams, 15 grams or 20 grams, the bovine serum albumin V5 factor 100 grams, 150 grams or 200 grams.Compound method is with distilled water mentioned component to be dissolved earlier, and sterile filtration then, packing are preserved through the rearmounted 4 ℃ of refrigerators of sterility test.

Fungistat preparation:, get balanced mix and form respectively with penbritin, amphotericin B, PXB, how pyridine ketone acid and azlocillin are mixed with the distilled water solution of 50 μ g～100 μ g/ml concentration.

Get the modified Russell medium 8ml that is added with developer TTC solution then, get the liquid nutrient medium of a kind of assembly in the above-mentioned various assembly, wherein 4-8ml is cultivated on the basis, growth promoter 1-2ml, fungistat 0.1-0.2ml mixes, and promptly gets required two phase Roe's culture medium.These substratum all are that good effect is arranged through experiment.

Claims (5)

1, a kind of two phase Roe's culture medium based on traditional improvement Russell medium, adds liquid and stops the substratum composition, and this liquid nutrient medium comprises basic medium, growth promoter and fungistat, and its component proportioning is as follows:

Basic medium: copper sulfate 0.001～0.002 gram, zinc sulfate 0.001～0.002 gram, calcium chloride 0.001～0.002 gram, sal epsom 0.01～0.02 gram, oleic acid 0.01～0.02 gram, Ferric Ammonium Citrate 0.05～0.1 gram, Sodium Citrate 0.1～0.2 gram, ammonium sulfate 0.2～0.4 gram, Sodium.alpha.-ketopropionate 0.5～1 gram, tween-80 0.5～1 gram, asparagine 1～2 gram, potassium primary phosphate 1～2 gram, SODIUM PHOSPHATE, MONOBASIC 2～4 grams, 8～12 milliliters of neutral glycerines, 1000 milliliters of distilled water;

Growth promoter: pyridoxine hydrochloride 0.01 gram～0.02 gram, vitamin H 0.01 gram～0.02 gram, coenzyme A 0.1 gram～0.2 gram, Triphosaden 0.1 gram～0.2 gram, catalase 0.1 gram～0.2 gram, α-Nai Yisuan 0.1 gram～0.2 gram, peptone 1 gram～2 grams, glucose 10 grams～20 grams, the bovine serum albumin V5 factor 100 grams～200 grams;

Fungistat: by concentration be respectively the penbritin, amphotericin B, PXB of 50 μ g～100 μ g/ml concentration, how the distilled water solution balanced mix of pyridine ketone acid and azlocillin forms;

The component ratio of modified Russell medium and aforesaid liquid substratum, growth promoter, fungistat is:

Modified Russell medium 8ml

Liquid nutrient medium 4-8ml

Growth promoter 1-2ml

Fungistat 0.1-0.2ml.

2, two phase Roe's culture medium according to claim 1 is characterized in that also adding the developer TCC is arranged, and its add-on is: corresponding to the 1000ml modified Russell medium, add 4-6ml concentration and be 0.1% chromogenic reagent solution.

3, a kind of preparation method of two phase Roe's culture medium according to claim 1 is as follows:

(1) with the each component dissolved in distilled water of basic medium, sterilization is 10-15 minute under 115-125 ℃ of high temperature, and cooling places 4 ℃ of refrigerators to preserve;

(2) with the each component dissolved in distilled water of growth promoter, sterilising filtration, packing then places 4 ℃ of refrigerators to preserve;

(3) each component with fungistat is mixed with the solution that concentration is 50-100 μ g/ml, balanced mix then with distilled water respectively;

(4) at last basic medium, growth promoter, fungistat are added modified Russell medium, the ingredient proportion of each component is:

Modified Russell medium 8ml

Basic medium 4-8ml

Growth promoter 1-2ml

Fungistat 0.1-0.2ml.

4, the preparation method of two phase Roe's culture medium according to claim 3 is characterized in that also adding the developer TCC, and add-on is: corresponding to the 1000ml modified Russell medium, adding 4-6ml concentration is 0.1% chromogenic reagent solution.

5, a kind of using method of two phase Roe's culture medium as claimed in claim 1 is characterized in that concrete steps are as follows:

A. sample to be checked carries out centrifuge washing after anti-soil is handled;

B. get precipitation 0.1ml, drip and plant in the liquid of two phase Roe's culture medium;

C. tilting cultivation is based on 35-37 ℃ of incubation;

D. every day observations once, see have bacterial growth to be to cultivate positive;

E. be cultured to 30 days and do not see that yet bacterial growth is to cultivate feminine gender;

F. cultivate the positive, contain the particulate state goods and materials that bacterium forms in the visible liquid nutrient medium, occur mauve bacterium colony on the culture medium slant.