CN106047781B - A kind of enriched medium and preparation method thereof, Zengjing Granule method - Google Patents
A kind of enriched medium and preparation method thereof, Zengjing Granule method Download PDFInfo
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Abstract
The invention discloses a kind of enriched medium, it include mother liquor and selectivity addition in mother liquor to inhibit Gram-negative bacteria growth selective additives, mother liquor by count in parts by weight 1.0~3.0 parts of 1.5~3.0 parts of sodium chloride, peptone, 0.2~0.8 part of yeast extract, 0.5~1.5 part of sodium dihydrogen phosphate, 0.1~0.5 part of potassium dihydrogen phosphate, combination 0.7~2.0 part of accelerating agent be added in 100 parts of water it is heated dissolving cool down again it is obtained.The enriched medium can be carried out at the same time Zengjing Granule to salmonella, hemorrhagic escherichia coli O157, Enterobacter sakazakii, staphylococcus aureus, Shigella, comma bacillus, vibrio parahemolyticus and single listeria spp that increases, and saved workload for Food Inspection and improved the efficiency of inspection.In addition, the preparation method the invention also discloses this enriched medium.
Description
Technical field
The present invention relates to field of microbial culture technology, in particular to a kind of enriched medium and preparation method thereof,
Zengjing Granule method.
Background technology
At present, the biologic contamination problem in food either developed country or developing country are pacified on food
Complete main reason.Detection currently for the pathogenic bacteria in food is typically all to be carried out according to related standard method, and phase
The establishment for closing standard method depends on traditional isolation and identification method.It is directed to each pathogenic bacteria and is required to its specific increasing
Bacterium culture medium carries out increasing bacterium, selective enrichment and etc.;Same sample needs to be carried out at the same time the detection of various pathogens, thus
The increasing bacterium for carrying out sample on a variety of specific enriched medium simultaneously is needed to handle, causes detection work increasing early period bacterium process numerous
It is trivial.
The method of traditional microculture is still the authoritative method for screening microorganism so far.National standard, which increases bacterium method, to be needed
3-4 time carried out to sample increase bacterium that the increasing bacterium process of bacterium could be completed, and it is compound increase that bacterium only needs once to increase bacterium process can be complete
Into the breeding of bacterium, it is easy to operate to be compared to national standard increasing bacterium method.Existing enriched medium single increases bacterium and can only increase
1~2 kind of pathogenic bacteria of bacterium, can not realize the purpose quickly detected, therefore, to realize that the quick of pathogenic bacteria increases bacterium detection, it is necessary to
It is improved for conventional medium and method.
The content of the invention
It is an object of the invention to provide a kind of enriched medium, this enriched medium is enough in the same of various pathogens
When quick Zengjing Granule, realize the purpose that quickly detects.
Another object of the present invention is to provide the preparation method of above-mentioned enriched medium, to obtain enriched medium, this
Enriched medium quick Zengjing Granule while being enough in various pathogens realizes the purpose quickly detected.
It is still another object of the present invention to provide a kind of Zengjing Granule method, which can be simultaneously to a variety of
Pathogenic bacteria carry out Zengjing Granule, realize the purpose quickly detected.
The present invention is solved its technical problem and is realized using following technical scheme.
A kind of enriched medium, including mother liquor, mother liquor is by 1.5~3.0 parts of sodium chloride, the peptone counted in parts by weight
1.0~3.0 parts, 0.2~0.8 part of yeast extract, 0.5~1.5 part of sodium dihydrogen phosphate, 0.1~0.5 part of potassium dihydrogen phosphate, combination
0.7~2.0 part of accelerating agent be added in 100 parts of water it is heated dissolving cool down again it is obtained, combination accelerating agent be glucose and breast
The combination of one or both of sugar, enriched medium is counted in parts by weight be additionally included in microbionation to mother liquor culture after add
Into mother liquor with inhibit Gram-negative bacteria growth 0.001~0.003 part of selective additives.
A kind of preparation method of enriched medium, including:1.5~3.0 parts of sodium chloride, peptone are taken by weight
1.0~3.0 parts, 0.2~0.8 part of yeast extract, 0.5~1.5 part of sodium dihydrogen phosphate, 0.1~0.5 part of potassium dihydrogen phosphate, combination
0.7~2.0 part of accelerating agent is added in into 100 parts of water, is dissolved by heating, sterilized rear obtained mother liquor, combination accelerating agent is glucose
With the combination of one or both of lactose;
0.001~0.003 part of selective additives for inhibiting Gram-negative bacteria growth are taken by weight, for causing
Germ is added to after being seeded to mother liquor culture in mother liquor.
Pathogenic bacteria, are first seeded in mother liquor and cultivate by a kind of Zengjing Granule method, then toward being inoculated in the mother liquor of pathogenic bacteria
It adds in inhibit the selective additives that Gram-negative bacteria is grown, the sodium chloride 1.5~3.0 that mother liquor will be counted in parts by weight
Part, 1.0~3.0 parts of peptone, 0.2~0.8 part of yeast extract, 0.5~1.5 part of sodium dihydrogen phosphate, potassium dihydrogen phosphate 0.1~
0.5 part, combination 0.7~2.0 part of accelerating agent be added in 100 parts of water it is heated dissolving cool down again it is obtained, combination accelerating agent be
The combination of one or both of glucose and lactose, selective additives are calculated by weight as 0.001~0.003 part.
Enriched medium provided by the invention and preparation method thereof, the advantageous effect of Zengjing Granule method are:Zengjing Granule
The mother liquor of base by count in parts by weight 1.0~3.0 parts of 1.5~3.0 parts of sodium chloride, peptone, 0.2~0.8 part of yeast extract,
0.5~1.5 part of sodium dihydrogen phosphate, 0.1~0.5 part of potassium dihydrogen phosphate, combination 0.7~2.0 part of water for being added to 100 parts of accelerating agent
In it is heated dissolving cool down again it is obtained, combination accelerating agent be one or both of glucose and lactose combination, the increasing bacterium training
Foster base contains the peptone and yeast extract of suitable proportion, and rich in nutrition content is comprehensive, suitable life is provided for pathogenic bacteria
Long environment disclosure satisfy that the growth of various pathogens;In addition, the enriched medium is counted be additionally included in pathogenic bacteria in parts by weight
Be seeded to be added to after mother liquor culture in mother liquor with inhibit the selective additives 0.001 of Gram-negative bacteria growth~
0.003 part, selective additives can inhibit Gram-negative bacteria growth, help to realize the balance life of various target pathogenic bacteria
It is long, prevent competition of the non-pathogenic bacteria to nutrition.Therefore, enriched medium of the invention can be realized to salmonella, large intestine angstrom
Uncommon Salmonella O157, Enterobacter sakazakii, staphylococcus aureus, Shigella, comma bacillus, vibrio parahemolyticus and single increasing Li Si
The various pathogens such as special Salmonella carry out Zengjing Granule simultaneously, and increasing bacterium is efficient, without carrying out individually increasing bacterium training to each pathogenic bacteria
It supports, has saved the workload of Food Inspection.
Specific embodiment
It, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, builds according to normal condition or manufacturer
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Enriched medium of the embodiment of the present invention and preparation method thereof, Zengjing Granule method are specifically described below.
A kind of enriched medium, including mother liquor, mother liquor is by 1.5~3.0 parts of sodium chloride, the peptone counted in parts by weight
1.0~3.0 parts, 0.2~0.8 part of yeast extract, 0.5~1.5 part of sodium dihydrogen phosphate, 0.1~0.5 part of potassium dihydrogen phosphate, combination
0.7~2.0 part of accelerating agent be added in 100 parts of water it is heated dissolving cool down again it is obtained, combination accelerating agent be glucose and breast
The combination of one or both of sugar, enriched medium is counted in parts by weight is additionally included in microbionation to 3~4h of mother liquor culture
Afterwards be added to mother liquor in inhibit Gram-negative bacteria growth 0.001~0.003 part of selective additives.Need what is illustrated
It is that, if combination accelerating agent selects glucose and lactose simultaneously, the weight ratio of glucose and lactose is 1:1.
Wherein, peptone and yeast extract provide bacterial growth and are metabolized the carbon source and nitrogen source needed for producing enzyme, cause a disease to be each
The growth of bacterium provides nutrition comprehensively, balanced;It is preferred that peptone is tryptone and the group for showing one or both of peptone the moon
It closes.It should be noted that if peptone selects tryptone and shows peptone the moon simultaneously, tryptone is with the weight ratio for showing peptone the moon
1:1。
Sodium chloride provides certain osmotic pressure;Disodium hydrogen phosphate and potassium dihydrogen phosphate provide buffering for cultivating system, adjust
Medium pH in bacteria growth process.It is suitable that sodium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate provide for the growth of each pathogenic bacteria
Growing environment, be adapted to various pathogenic bacteria and grow simultaneously.In addition, combination accelerating agent preferentially uses carbon source, energy as bacterium
Promote bacterial growth and metabolism producing enzyme.
It is preferred that selective additives are the combinations of one or both of Nalidixate Sodium and acriflavine.Naphthyridones
Sour sodium and acriflavine can inhibit Gram-negative bacteria growth to a certain extent, enable to need the target pathogenic bacteria for increasing bacterium
There are sufficient growing nutrient and space, and the mutual growth of each pathogenic bacteria is made to avoid vying each other in equilibrium state.It needs
It is bright, if selective additives are Nalidixate Sodium and acriflavine, by the weight of Nalidixate Sodium and acriflavine
Than for 1:1 ratio combines to form selective additives.
It is preferred that the pH of mother liquor is 7.0~7.4.The mother liquor being in neutrality provides suitable growth for the growth of each pathogenic bacteria
Environment is adapted to various pathogenic bacteria and grows simultaneously.
The preparation method of above-mentioned enriched medium, including:
1.5~3.0 parts of sodium chloride, 1.0~3.0 parts of peptone, 0.2~0.8 part of yeast extract, phosphoric acid are taken by weight
0.5~1.5 part of sodium dihydrogen, 0.1~0.5 part of potassium dihydrogen phosphate, 0.7~2.0 part of combination accelerating agent are added in into 100 parts of water, are added
Thermal agitation dissolving, sterilized rear obtained mother liquor, obtain mother liquor.It is one or both of glucose and lactose to combine accelerating agent
Combination.
Wherein, heating stirring dissolving after, mother liquor need to through being cooled to room temperature, then adjust mother liquor pH to 7.0~
7.4, to ensure the stability of pH value;Then, by mother liquor at 120 DEG C~122 DEG C 20~25min of high pressure sterilization, be cooled to room
Temperature obtains sterile mother liquor.
Then, 0.001~0.003 part of selective additives for inhibiting Gram-negative bacteria growth are taken by weight, are used
It is added to after the mother liquor culture is seeded in pathogenic bacteria in the mother liquor.Selective additives are Nalidixate Sodium and acridine
The combination of one or both of flavine.
Preferably, first selective additives are added to the water, fully dissolve, be filtered degerming, obtained selectivity and add
Add agent solution.In use, selective additives solution is directly directly appended to mother liquor.
A kind of Zengjing Granule method, including:
First pathogenic bacteria are seeded in above-mentioned mother liquor and are cultivated, specifically, directly sample to be tested is seeded in mother liquor, are carried out
Culture;
It adds in inhibit the above-mentioned selective additives that Gram-negative bacteria is grown into the mother liquor for be inoculated with pathogenic bacteria again.
It is preferred that selective additives are added to after pathogenic bacteria are seeded to mother liquor culture 3-4h in mother liquor.Add in selection
Property additive after, Gram-negative bacteria growth is suppressed so that the growths of each pathogenic bacteria is mutually coordinated, balance, can obtain
To rapidly breeding, and then realize single quickly to the enriching effect of various pathogens.Each pathogenic bacteria are carried out without again independent
Ground Zengjing Granule, and then help to improve the efficiency for examining work.
To sum up, enriched medium of the invention, the growth characteristics of each pathogenic bacteria in based food, species variation simultaneously combine food
Pathogenic bacteria easily damaged the features such as need to repairing, pointedly optimize and arrange in pairs or groups to the component of enriched medium in product.Make
Obtaining enriched medium of the invention can be effectively to salmonella, escherichia coli O157, Enterobacter sakazakii, golden yellow grape
Coccus, Shigella, comma bacillus, vibrio parahemolyticus and single listeria spp that increases carry out Zengjing Granule.When having saved inspection
Between and workload, to realize that the quick detection to food provides the foundation.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
Present embodiments provide the preparation method of enriched medium and as the enriched medium obtained by the preparation method.
The preparation method of enriched medium includes:
Sodium chloride 1.5g, peptone 1.0g, yeast extract 0.2g, sodium dihydrogen phosphate 0.5g, phosphoric acid are taken by the formula of mother liquor
Potassium dihydrogen 0.1g, accelerating agent 0.7g are added in distilled water 100ml, and heating stirring is to being completely dissolved.Accelerating agent is glucose.
PH to 7.2 or so is adjusted after being cooled to room temperature, in 121 DEG C of high pressure sterilization 20min, is cooled to room temperature, obtains mother liquor
100ml。
The selective additives 0.002g that can inhibit Gram-negative bacteria growth is taken, is added to (distilled water in 2ml distilled water
It is 1000 with selective additives volume mass ratio:1, in other embodiments, which is not construed as limiting, and main function is
Dissolve selective additives in advance, it is ensured that when in use selective additives can rapid dispersion into mother liquor, as long as per 100ml
Mother liquor is added with 0.001g~0.003g selective additives), it fully dissolves, obtains selective additives solution, select
Property additive is Nalidixate Sodium..
It is filtered degerming to selective additives solution, filter membrane 0.22um is obtained sterile containing selective addition
The selective additives solution 2ml of agent.The selection additive solution can all add again after pathogenic bacteria are seeded to mother liquor culture
Enter into mother liquor.
Embodiment 2~7
The corresponding increasing bacterium that embodiment 2~7 provides the preparation method of enriched medium and obtained by the preparation method is trained
Base is supported, the preparation method for the enriched medium that embodiment 2~7 provides is substantially the same manner as Example 1, the difference is that mother liquor and selection
The ingredient of property additive, the mother liquor core selective additives of embodiment 2~7 into being grouped into and dosage is shown in Table 1.
The mother liquor of 1. 2-7 of the embodiment of the present invention of table and the ingredient and dosage of selective additives
Note:The dosage unit of each ingredient is g in table, and "-" representative does not add in.
Embodiment 8
The present embodiment illustrates Zengjing Granule method, the increasing bacterium of culture medium used obtained by above-described embodiment 1
Culture medium is illustrated with activation bacterium solution containing pathogenic bacteria.
The Zengjing Granule method of the present embodiment, including:
After the activation bacterium solutions of each pathogenic bacteria is diluted ten times, be added to together in the mother liquor of the offer of embodiment 1, in 35 DEG C,
It is cultivated under the conditions of 150rpm.And the pathogenic bacteria in ten times of activation bacterium solution of dilution are counted, as in the culture solution before increasing bacterium
Total plate count.Certainly, in specific practical work process, when carrying out increasing bacterium, directly sample to be tested (solid) 1mg can also be connect
Kind carries out increasing bacterium into mother liquor.
Culture is treated to 3h, the selective additives that embodiment 1 is provided all are added in into the mother liquor after inoculated and cultured, after
Continuous culture 12h.After culture, the pathogenic bacteria classification and quantity of culture solution are detected.The method of detection can refer to as follows:
Culture solution is subjected to gradient dilution in ten times of ratios, by dilution with the corresponding chromogenic culture medium of various pathogenic bacteria in 35 DEG C of cultures
12 it is small when, counted using plate count method, the total plate count increased in entire culture solution after bacterium calculated, as a result such as table 2
It is shown.
The enriched medium that 2. embodiment 1 of table provides is to the result of pathogenic bacteria Zengjing Granule
It can be with the total plate count of the pathogenic bacteria after being cultivated using enriched medium provided by the invention can by table 2
Meet testing requirements, and the classification for being capable of the pathogenic bacteria of Zengjing Granule is more, includes salmonella, hemorrhagic escherichia coli
Bacterium O157, Enterobacter sakazakii, staphylococcus aureus, Shigella, comma bacillus, vibrio parahemolyticus and single increasing Listeria
Bacterium.
In addition, in actual detection process, if, it is necessary to which further Molecular Detection, can refer to as follows after Zengjing Granule
Method carries out:When carrying out Zengjing Granule to sample to be tested, sample to be tested is divided into two parts of phase homogenous quantities, and (such as every part treated test sample
This 1~5mg), it is seeded in identical mother liquor A and mother liquor B that (ingredient and dosage and the present invention of mother liquor A and mother liquor B are arbitrary respectively
The mother liquor that embodiment provides is identical).After cultivating 3~4h, selective additives are added into mother liquor A, the indiscriminate property of mother liquor B adds
Add agent.After culture, the pathogenic bacteria genomic DNA in the pathogenic bacteria genomic DNA and mother liquor B in mother liquor A is extracted respectively, it will
Two parts of genomic DNA mixing are used further in subsequent multiplex PCR equimolecular detection, and the result so detected is more accurate and can
It leans on.
Embodiment 9
Enriched medium is carried out using the enriched medium of embodiment 2~7, Zengjing Granule method and steps is the same as embodiment 8.
The enriched medium enriched medium of embodiment 2~7 the results are shown in Table 3.
The enriched medium that 3. embodiment 2~7 of table provides is to the result of pathogenic bacteria Zengjing Granule
It can be with the bacterium of the pathogenic bacteria after being cultivated using the enriched medium of the offer of the embodiment of the present invention 2~7 by table 3
Fall sum to agree to that testing requirements can be met.It it is indicated above that can be simultaneously to Salmonella using enriched medium provided by the invention
Bacterium, hemorrhagic escherichia coli O157, Enterobacter sakazakii, staphylococcus aureus, Shigella, comma bacillus, secondary haemolysis
Property vibrios and single listeria spp that increases carry out Zengjing Granule.
It to sum up, can be simultaneously to salmonella, escherichia coli O157, the rugged intestines bar of slope using culture medium provided by the invention
The pathogenic bacteria such as bacterium, staphylococcus aureus, Shigella, comma bacillus, vibrio parahemolyticus and single increasing listeria spp carry out
Compound increasing bacterium avoids the complicated procedures for each increasing bacterium from improving work efficiency;Object bacteria can directly be done by increasing the culture medium after bacterium
Be separately cultured and bioassay experiments, can also be directly used in PCR detection.
The foregoing is only a preferred embodiment of the present invention, is not intended to limit the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of enriched medium, which is characterized in that it includes mother liquor, and the mother liquor is by the sodium chloride 1.5 counted in parts by weight
0.7 part part, 1.0 parts of peptone, 0.2 part of yeast extract, 0.5 part of sodium dihydrogen phosphate, 0.1 part of potassium dihydrogen phosphate, combination of accelerating agent
Be added to heated dissolving in 100 parts of water cool down again it is obtained, the combination accelerating agent be one kind in glucose and lactose or
Two kinds of combination, the enriched medium, which is counted to be additionally included in after microbionation to the mother liquor culture in parts by weight, is added to institute
State in mother liquor with inhibit Gram-negative bacteria growth 0.001 ~ 0.003 part of selective additives;The selective additives
It is the combination of one or both of Nalidixate Sodium and acriflavine.
2. enriched medium according to claim 1, which is characterized in that the pH of the mother liquor is 7.0 ~ 7.4.
3. the enriched medium according to any one of claim 1 ~ 2, which is characterized in that the peptone is tryptone
With the combination for showing one or both of peptone the moon.
4. a kind of preparation method of enriched medium, which is characterized in that it includes:
1.5 parts of sodium chloride, 1.0 parts of peptone, 0.2 part of yeast extract, 0.5 part of sodium dihydrogen phosphate, di(2-ethylhexyl)phosphate are taken by weight
0.1 part of hydrogen potassium, 0.7 part of combination accelerating agent are added in into 100 parts of water, are dissolved by heating, sterilized rear obtained mother liquor, and the combination promotees
It is the combination of one or both of glucose and lactose into agent;
0.001 ~ 0.003 part of selective additives for inhibiting Gram-negative bacteria growth are taken by weight, are inoculated in pathogenic bacteria
After to the mother liquor culture, the selective additives are added in the mother liquor;The selective additives are naphthyridones
The combination of one or both of sour sodium and acriflavine.
5. the preparation method of enriched medium according to claim 4, which is characterized in that by the sodium chloride, the egg
White peptone, the yeast extract, the sodium dihydrogen phosphate, the potassium dihydrogen phosphate, the combination accelerating agent are added in into the water
Afterwards, heated stirring and dissolving, then adjust pH to 7.0 ~ 7.4 after cooling treatment is cooled to room temperature through first time.
6. the preparation method of enriched medium according to claim 5, which is characterized in that after adjusting pH to 7.0 ~ 7.4,
Sterilize 20 ~ 25min at 120 DEG C ~ 122 DEG C, then is cooled to room temperature through second of cooling treatment.
A kind of 7. Zengjing Granule method, which is characterized in that it includes:First pathogenic bacteria are seeded in mother liquor and are cultivated, then past inoculation
Have and the selective additives for inhibiting Gram-negative bacteria growth are added in the mother liquor of the pathogenic bacteria, the mother liquor is by pressing
1.0 parts of 1.5 parts of sodium chloride, the peptone of parts by weight meter, 0.2 part of yeast extract, 0.5 part of sodium dihydrogen phosphate, potassium dihydrogen phosphate
0.1 part, combination 0.7 part of accelerating agent be added in 100 parts of water it is heated dissolving cool down again it is obtained, it is described combination accelerating agent be Portugal
The combination of grape sugar and one or both of lactose, the selective additives are calculated by weight as 0.001 ~ 0.003 part;Institute
State the combination that selective additives are one or both of Nalidixate Sodium and acriflavine.
8. Zengjing Granule method according to claim 7, which is characterized in that the selective additives are in the pathogenic bacteria
It is added to after being seeded to the mother liquor culture 3-4h in the mother liquor.
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CN106434844A (en) * | 2016-11-29 | 2017-02-22 | 上海市农业科学院 | Co-enrichment medium SSEL |
CN107142300B (en) * | 2017-06-27 | 2020-05-22 | 中国疾病预防控制中心传染病预防控制所 | Listeria monocytogenes enrichment culture medium and preparation method thereof |
CN107916287B (en) * | 2017-12-13 | 2021-09-07 | 内蒙古蒙牛乳业(集团)股份有限公司 | Method for rapidly detecting enterobacteriaceae in milk powder |
CN115074271B (en) * | 2022-06-10 | 2023-09-29 | 深圳海关食品检验检疫技术中心 | Enrichment medium for salmonella in tea polyphenol food, preparation method of enrichment medium and enrichment method |
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