CN101597582B - Culture medium of meningitis Neisseria - Google Patents

Abstract

The invention provides a culture medium of meningitis Neisseria, which is characterized in that the culture medium consists of the following components: 3.0-8.0g/L of sodium chloride, 0.05-0.2g/L of potassium chloride, 1.0-6.0g/L of disodium hydrogen phosphate, 5-20g/L of L-glutamic acid, 0.01-0.1g/L of L-arginine, 0.001-0.01g/L of L-tryptophan, 0.005-0.030g/L of L-aminothiopropionic acid, 0.01-0.1g/L of calcium chloride, 0.001-0.008g/L of ferrous sulphate, 0.1-1/0 g/L of magnesium sulfate, 5.0-20.0 g/L of soya peptone and 5.0-20.0g/L of glucose. The invention is good for the growth and the metabolism of bacterium, can obtain higher output, has a wide material source, is free of structural constituents of the other animals, can effectively remove exogenous interference, simplifies technology on manufacture, is easy to operate, can effectively amplify the meningitis neisseria, and satisfies the mass production of vaccines.

Description

A kind of culture medium of meningitis Neisseria

Technical field

The present invention relates to a kind of substratum, the improved culture medium of the fairly large cultivation of especially a kind of suitable Neisseria meningitidis belongs to biological technical field.

Technical background

Meningococcal meningitis is called for short epidemic meningitis, is by the caused acute respiratory transmissible disease of Neisserial Neisseria meningitidis.Because morbidity is anxious, PD is fast, the mortality ratio height, to Susceptible population---children's fulminant type case fatality rate can reach 40%～60%, thereby is subjected to generally attention both domestic and external always.

Though to Neisseria meningitidis research very early, and obtain certain achievement, the relevant report of its cultivation aspect seldom, and each producer maintains secrecy especially to its substratum.Kan Fangqi etc. (CN 101089190A) have invented a kind of nutrition blood culture medium, general sample mainly is provided and had used the patient specimen separation and Culture usefulness of microbiotic and sulfa drug, are not suitable for large scale culturing, have limitation.At present both at home and abroad the chocolate two anti-agar of more application are as isolation medium, and the separation test effect of this substratum is fine, but need use fresh defiber sheep blood when making, having material source is difficult for, the cost height, shortcomings such as complex manufacturing process are not suitable for increasing on a large scale bacterium and use.Present in addition meningitis substratum mostly is isolation medium, and its main purpose is separation and detects that so detect in order to separate, existing substratum will use antibiotic.

Because Neisseria meningitidis is very harsh to environment requirement, its growth is not only relevant with the bacterial classification self character, as belong to obligate aerobe, and often, thalline causes self cracking death in the culturing process because of producing autolytic enzyme, and must give suitable medium and just can make its good growth metabolism.Therefore, be necessary that fully development and exploitation are fit to industrial scale production, and material source is wide, price is low, can avoid thalline to produce the substratum of the adaptation Neisseria meningitidis growth metabolism of autolytic enzyme.

Summary of the invention

The present invention provides a kind of and can guarantee that the Neisseria meningitidis growth is vigorous just for overcoming the prior art above shortcomings, carries out eubolism, can provide again to increase bacterium on a large scale to be used to produce the culture medium of meningitis Neisseria of vaccine.

The present invention finishes by following technical proposal: a kind of culture medium of meningitis Neisseria is characterized in that being made up of following component:

Sodium-chlor 3.0～8.0g/L Repone K 0.05～0.2g/L

Sodium phosphate dibasic 1.0～6.0g/L L-L-glutamic acid 5～20g/L

L-arginine 0.01～0.1g/L L-tryptophane 0.001～0.01g/L

L-halfcystine 0.005～0.030g/L calcium chloride 0.01～0.1g/L

Ferrous sulfate 0.001～0.008g/L sal epsom 0.1～1.0g/L

Soy peptone 5.0～20.0g/L glucose 5.0～20.0g/L.

Substratum provided by the invention prepares by following method:

A, with sodium-chlor, Repone K, Sodium phosphate dibasic, L-L-glutamic acid, L-arginine, L-halfcystine, calcium chloride, ferrous sulfate, soy peptone mixture with dissolved in distilled water after, constant volume, regulate pH to 7.2-7.4, at 113～117 ℃, 0.06 under～0.08MPa the condition, sterilization 15～25min;

B, with L-tryptophane, sal epsom, glucose mixture with the 15mL dissolved in distilled water after, after the 0.2 μ m disposable filter filtration sterilization, add in the mixture of A step of high pressure degerming, liquid nutrient medium.

Substratum provided by the invention has following characteristics: contain the required nutritional requirement of Neisseria meningitidis growth metabolism, the Neisseria meningitidis of purifying is used for increasing on a large scale, wherein L-L-glutamic acid is one of primary amino acid of nitrogen metabolism in the organism, Neisseria meningitidis does not have the sulphate reducing ability, so will allocate the L-halfcystine that belongs to reduced form sulfide into, the L-arginine is the moiety of urea cycle in the bacterium, the effect with the nitrogen equilibrium kept; Calcium ion in the calcium chloride can promote growth; Magnesium ion in the sal epsom can shorten lag phase; Iron ion in the ferrous sulfate can influence the virulence of bacterial strain and help the expansion of bacterium colony; Glucose helps the breeding growth of Neisseria meningitidis.

The present invention compared with prior art has following advantage and effect: the invention provides a kind of extensive enrichment medium that can be used for production of vaccine, material that is adopted and technology all are final purpose with the production of vaccine.Adopt above-mentioned culture medium culturing Neisseria meningitidis, the growth metabolism that helps bacterium, with other substratum mutually specific energy obtain high yield, and material source is extensive, do not contain other animal tissues's compositions, as serum etc., can effectively get rid of the interference of external source, the bacterium that can obtain higher degree on producing is used for production of vaccine, make work simplification, easy handling uses liquid nutrient medium provided by the invention to carry out large scale culturing, the Neisseria meningitidis that can effectively increase, the bacterium that obtains purifying is for production of vaccine.

Embodiment

Embodiment 1

Get the raw materials ready by following prescription:

Sodium-chlor 5.8g Repone K 0.09g

Sodium phosphate dibasic 2.6g L-L-glutamic acid 15.0g

L-arginine 0.03g L-tryptophane 0.0025g

L-halfcystine 0.012g calcium chloride 0.021g

Ferrous sulfate 0.002g sal epsom 0.6g

Soy peptone 15.0g glucose 10.0g

Make through following method:

A, with sodium-chlor, Repone K, Sodium phosphate dibasic, L-L-glutamic acid, L-arginine, L-halfcystine, calcium chloride, ferrous sulfate, soy peptone mixture with dissolved in distilled water after, be settled to 1L, regulate pH to 7.4, at 113 ℃, 0.08MPa sterilization 15min down;

B, with L-tryptophane, sal epsom, glucose mixture with the 15mL dissolved in distilled water after, after the 0.2 μ m disposable filter filtration sterilization, add in the mixture of A step of high pressure degerming, liquid nutrient medium.

Embodiment 2

Get the raw materials ready by following prescription:

Sodium-chlor 3.0g Repone K 0.05g

Sodium phosphate dibasic 6.0g L-L-glutamic acid 5.0g

L-arginine 0.01g L-tryptophane 0.001g

L-halfcystine 0.005g calcium chloride 0.01g

Ferrous sulfate 0.008g sal epsom 1.0g

Soy peptone 5.0g glucose 5.0g

Make through following method:

A, with sodium-chlor, Repone K, Sodium phosphate dibasic, L-L-glutamic acid, L-arginine, L-halfcystine, calcium chloride, ferrous sulfate, soy peptone mixture with dissolved in distilled water after, be settled to 1L, regulate pH to 7.2, at 117 ℃, 0.06MPa sterilization 15min down;

B, with L-tryptophane, sal epsom, glucose mixture with the 15mL dissolved in distilled water after, after the 0.2 μ m disposable filter filtration sterilization, add in the mixture of A step of high pressure degerming, liquid nutrient medium.

Embodiment 3

Get the raw materials ready by following prescription:

Sodium-chlor 8.0g Repone K 0.2g

Sodium phosphate dibasic 2.0g L-L-glutamic acid 20.0g

L-arginine 0.1g L-tryptophane 0.01g

L-halfcystine 0.03g calcium chloride 0.1g

Ferrous sulfate 0.001g sal epsom 0.1g

Soy peptone 20.0g glucose 20.0g

Make through following method:

A, with sodium-chlor, Repone K, Sodium phosphate dibasic, L-L-glutamic acid, L-arginine, L-halfcystine, calcium chloride, ferrous sulfate, soy peptone mixture with dissolved in distilled water after, be settled to 1L, regulate pH to 7.4, at 115 ℃, 0.07MPa sterilization 20min down;

B, with L-tryptophane, sal epsom, glucose mixture with the 15mL dissolved in distilled water after, after the 0.2 μ m disposable filter filtration sterilization, add in the mixture of A step of high pressure degerming, liquid nutrient medium.

The Neisseria meningitidis amplification culture

Claims (2)

1. a culture medium of meningitis Neisseria is characterized in that being made up of following component: sodium-chlor, Repone K, Sodium phosphate dibasic, L-L-glutamic acid, L-arginine, L-tryptophane, L-halfcystine, L-halfcystine L-arginine, calcium chloride, ferrous sulfate, sal epsom, soy peptone, glucose, distilled water; Sodium-chlor 3.0～8.0g/L wherein, Repone K 0.05～0.2g/L, Sodium phosphate dibasic 1.0～6.0g/L, L-L-glutamic acid 5～20g/L, L-arginine 0.01～0.1g/L, L-tryptophane 0.001～0.01g/L, L-halfcystine 0.005～0.030g/L, calcium chloride 0.01～0.1g/L, ferrous sulfate 0.001～0.008g/L, sal epsom 0.1～1.0g/L, soy peptone 5.0～20.0g/L, glucose 5.0～20.0g/L.

2. culture medium of meningitis Neisseria according to claim 1 is characterized in that it prepares by following method:

A, with sodium-chlor, Repone K, Sodium phosphate dibasic, L-L-glutamic acid, L-arginine, L-halfcystine, calcium chloride, ferrous sulfate, soy peptone mixture with dissolved in distilled water after, constant volume, regulate pH to 7.2-7.4, at 113～117 ℃, 0.06 under～0.08MPa the condition, sterilization 15～25min;

B, with L-tryptophane, sal epsom, glucose mixture with the 15mL dissolved in distilled water after, after the 0.2 μ m disposable filter filtration sterilization, add in the mixture of A step of high pressure degerming, liquid nutrient medium.