CN103667160A - High-density fermentation culture medium for haemophilus parasuis - Google Patents

Abstract

The invention relates to the technical field of microorganism culture mediums and more particularly relates to a preparation method of a high-density fermentation culture medium for haemophilus parasuis. The high-density fermentation culture medium is composed of following components according to a weight/volume ratio: 2-6g/L of glutamine, 15-35g/L of yeast extract, 5-10g/L of sodium chloride, 0.01-0.05g/L of calcium carbonate, 0.01-0.05g/L of magnesium sulfate, 1-10g/L of tryptone and 0.5-10g/L of dipotassium phosphate; the high-density fermentation culture medium further comprises following components in a volume ratio: 0.001%-0.01% of NAD (Nicotinamide Adenine Dinucleotide), 10%-20% of bovine serum and residual amount of water; before sterilization, the pH (Potential of Hydrogen) of the culture medium is 7.0-7.2. The preparation method of the high-density fermentation culture medium for the haemophilus parasuis has the beneficial effects that nutritional ingredients needed by various microorganisms are supplemented to the culture medium and the pH in the culture medium is effectively controlled by the calcium carbonate; a carbon source and a small part of nitrogen are provided by the glutamine so that the high-density fermentation culture medium is good for the rapid growth of the haemophilus parasuis.

Description

A kind of haemophilus parasuis high density fermentation culture medium

Technical field

The present invention relates to microbiological culture media technical field, more specifically a kind of preparation method of haemophilus parasuis high density fermentation culture medium.

Background technology

The secondary influenzae disease of pig claims again multiple fiber disposition serositis and sacroiliitis, also claims Ge Lazeshi sick.To be caused by the secondary influenzae of pig.This bacterium ubiquity in environment, has all over the world, or even also can find in the middle of healthy swinery.The swinery that does not have the secondary influenzae of pig to pollute for adopting specific pathogen free or medication getting up early wean technology, when this bacterium is arrived in primary infection, consequence can be quite serious.

And haemophilus parasuis: belong to Gram-negative bacillus pumilis, form is changeable, has 15 above serotype, wherein serotypes 5,4,13 the most common (accounting for more than 70%).During this bacteria growing, strictly need Reduced nicotinamide-adenine dinucleotide (NAD or the V factor), do not need the X factor (protoheme or other porphyrin substance), on blood culture medium and chocolate culture-medium, grow, bacterium colony is little and transparent, on blood culture medium without haemolysis; Well-grown around staphylococcus bacterium platform, forms satellitism.Under general condition, be difficult to separation and cultivation, especially apply the pathological material of disease that antibiotic therapy is crossed disease pig, thereby bring difficulty to the diagnosis of this disease; It is reported: the true sickness rate of the secondary influenzae of pig may be actual 10 times more than of making a definite diagnosis.

There are problems in the cultivation for haemophilus parasuis at present, such as nutritional needs is high, pH requires strict, and culture density is low etc., and conventional substratum is TSB substratum at present, but can not meet large-scale cultivation, and this is deficiency of the prior art.

Summary of the invention

Goal of the invention of the present invention is for deficiency of the prior art, a kind of haemophilus parasuis high density fermentation culture medium is provided, this substratum is by supplementing the required nutritive ingredient of various microorganisms, and effectively control pH in culture medium by calcium carbonate, and provide carbon source and small portion nitrogen by glutamine, be conducive to the Fast Growth of haemophilus parasuis.

The present invention is achieved by the following technical solutions:

A kind of haemophilus parasuis high density fermentation culture medium, by following component and proportioning, according to weight/volume meter, glutamine 2-6g/L, yeast extract paste 15-35 g/L, sodium-chlor 5-10 g/L, calcium carbonate 0.01-0.05 g/L, magnesium sulfate 0.01-0.05 g/L, Tryptones 1-10g/L, dipotassium hydrogen phosphate 0.5-10 g/L, count by volume: the bovine serum of 0.001-0.01NAD and 10-20%, surplus is water, and before sterilizing, the PH of substratum is 7.0-7.2.

Preferably, above-mentioned substratum is by following component and proportioning, according to weight/volume meter, glutamine 3-5g/L, yeast extract paste 20-30 g/L, sodium-chlor 6-8 g/L, calcium carbonate 0.01-0.02 g/L, magnesium sulfate 0.01-0.02 g/L, Tryptones 2-8g/L, dipotassium hydrogen phosphate 1.3-8.7 g/L, count by volume: the bovine serum of 0.005-0.008%NAD and 12-18%, surplus is water, and before sterilizing, the PH of substratum is 7.0-7.2.

Preferably, above-mentioned substratum is by following component and proportioning, according to weight/volume meter, glutamine 4g/L, yeast extract paste 25 g/L, sodium-chlor 7g/L, calcium carbonate 0.015 g/L, magnesium sulfate 0.015g/L, Tryptones 5g/L, dipotassium hydrogen phosphate 5.5g/L, meter: 0.006%NAD and 16% bovine serum by volume, surplus is water, and before sterilizing, the PH of substratum is 7.0-7.2.

The preparation method of above-mentioned a kind of haemophilus parasuis high density fermentation culture medium, comprises the steps:

(1) get yeast extract paste 15-35 g/L, sodium-chlor 5-10 g/L, calcium carbonate 0.01-0.05 g/L, magnesium sulfate 0.01-0.05 g/L, Tryptones 1-10g/L, dipotassium hydrogen phosphate 0.5-10 g/L is settled to 1000ml with distilled water; adjusting pH is 7.0-7.2, sterilizing 20-30min under 120 ℃ of high-temperature steams;

(2) get glutamine 2-6g/L mix with the bovine serum of 0.001-0.01NAD and 10-20% after with 100 ℃ at sterilizing 30min;

(3) solution after step (1) and step (2) gained sterilizing was both mixed and obtained.

The invention has the beneficial effects as follows: this substratum passes through to supplement the required nutritive ingredient of various microorganisms, and effectively controls pH in culture medium by calcium carbonate, and provides carbon source and small portion nitrogen by glutamine, is conducive to the Fast Growth of haemophilus parasuis.

Accompanying drawing explanation

Fig. 1 is embodiment 3 haemophilus parasuis growth curves.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention will be further described, so that those skilled in the art can better understand the present invention, but therefore do not limit the present invention.

Embodiment 1

A kind of haemophilus parasuis high density fermentation culture medium, by following component and proportioning, according to weight/volume meter, glutamine 2g/L, yeast extract paste 15g/L, sodium-chlor 5 g/L, calcium carbonate 0.01g/L, magnesium sulfate 0.01g/L, Tryptones 1g/L, dipotassium hydrogen phosphate 0.5g/L, meter: 0.001NAD and 10% bovine serum by volume, surplus is water, and before sterilizing, the PH of substratum is 7.0.

Embodiment 2

A kind of haemophilus parasuis high density fermentation culture medium, by following component and proportioning, according to weight/volume meter, glutamy 6g/L, yeast extract paste 35 g/L, sodium-chlor 10 g/L, calcium carbonate 0.05 g/L, magnesium sulfate 0.05 g/L, Tryptones 10g/L, dipotassium hydrogen phosphate 10 g/L, meter: 0.01NAD and 20% bovine serum by volume, surplus is water, and before sterilizing, the PH of substratum is 7.2.

Embodiment 3

Substratum is by following component and proportioning, according to weight/volume meter, glutamine 3-5g/L, yeast extract paste 20-30 g/L, sodium-chlor 6-8 g/L, calcium carbonate 0.01-0.02 g/L, magnesium sulfate 0.01-0.02 g/L, Tryptones 2-8g/L, dipotassium hydrogen phosphate 1.3-8.7 g/L, count by volume: the bovine serum of 0.005-0.008%NAD and 12-18%, surplus is water, and before sterilizing, the PH of substratum is 7.0.

Embodiment 4

A kind of haemophilus parasuis high density fermentation culture medium, by following component and proportioning, according to weight/volume meter, glutamine 3g/L, yeast extract paste 20 g/L, sodium-chlor 6 g/L, calcium carbonate 0.02g/L, magnesium sulfate 0.02 g/L, Tryptones 3g/L, dipotassium hydrogen phosphate 2.35 g/L, meter: 0.006NAD and 13% bovine serum by volume, surplus is water, and before sterilizing, the PH of substratum is 7.0.

Embodiment 5

A kind of haemophilus parasuis high density fermentation culture medium, by following component and proportioning, according to weight/volume meter, glutamine 4g/L, yeast extract paste 30 g/L, sodium-chlor 8 g/L, calcium carbonate 0.01g/L, magnesium sulfate 0.02 g/L, Tryptones 8g/L, dipotassium hydrogen phosphate 8.7 g/L, meter: 0.008NAD and 18% bovine serum by volume, surplus is water, and before sterilizing, the PH of substratum is 7.2.

The experiment of haemophilus parasuis 3L fermentor tank, and contrast with TSB substratum.

By scheme and watch in can find out that substratum of the present invention grows up soon in culturing process mid-early stage, amount of survival is high, is applicable to very much the extensive cultivation of haemophilus parasuis.

Claims (4)

1. a haemophilus parasuis high density fermentation culture medium, by following component and proportioning, according to weight/volume meter, glutamine 2-6g/L, yeast extract paste 15-35 g/L, sodium-chlor 5-10 g/L, calcium carbonate 0.01-0.05 g/L, magnesium sulfate 0.01-0.05 g/L, Tryptones 1-10g/L, dipotassium hydrogen phosphate 0.5-10 g/L, count by volume: the bovine serum of 0.001-0.01NAD and 10-20%, surplus is water, and before sterilizing, the PH of substratum is 7.0-7.2.

2. a kind of haemophilus parasuis high density fermentation culture medium according to claim 1, it is characterized in that: described substratum is by following component and proportioning, according to weight/volume meter, glutamine 3-5g/L, yeast extract paste 20-30 g/L, sodium-chlor 6-8 g/L, calcium carbonate 0.01-0.02 g/L, magnesium sulfate 0.01-0.02 g/L, Tryptones 2-8g/L, dipotassium hydrogen phosphate 1.3-8.7 g/L, count by volume: the bovine serum of 0.005-0.008%NAD and 12-18%, surplus is water, and before sterilizing, the PH of substratum is 7.0-7.2.

3. a kind of haemophilus parasuis high density fermentation culture medium according to claim 1, it is characterized in that: described substratum is by following component and proportioning, according to weight/volume meter, glutamine 4g/L, yeast extract paste 25 g/L, sodium-chlor 7g/L, calcium carbonate 0.015 g/L, magnesium sulfate 0.015g/L, Tryptones 5g/L, dipotassium hydrogen phosphate 5.5g/L, meter: 0.006%NAD and 16% bovine serum by volume, surplus is water, and before sterilizing, the PH of substratum is 7.0-7.2.

4. the preparation method of a kind of haemophilus parasuis high density fermentation culture medium according to claim 1, comprises the steps:

(1) get yeast extract paste 15-35 g/L, sodium-chlor 5-10 g/L, calcium carbonate 0.01-0.05 g/L, magnesium sulfate 0.01-0.05 g/L, Tryptones 1-10g/L, dipotassium hydrogen phosphate 0.5-10 g/L is settled to 1000ml with distilled water; adjusting pH is 7.0-7.2, sterilizing 20-30min under 120 ℃ of high-temperature steams;

(2) get glutamine 2-6g/L mix with the bovine serum of 0.001-0.01NAD and 10-20% after with 100 ℃ at sterilizing 30min;

(3) solution after step (1) and step (2) gained sterilizing was both mixed and obtained.

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