CN105176823B - The preparation method of microorganism proficiency testing salmonella sample - Google Patents
The preparation method of microorganism proficiency testing salmonella sample Download PDFInfo
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- CN105176823B CN105176823B CN201510313160.6A CN201510313160A CN105176823B CN 105176823 B CN105176823 B CN 105176823B CN 201510313160 A CN201510313160 A CN 201510313160A CN 105176823 B CN105176823 B CN 105176823B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 76
- 241000607142 Salmonella Species 0.000 title claims abstract description 72
- 238000012360 testing method Methods 0.000 title claims abstract description 33
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Abstract
The present invention relates to microorganism proficiency testing technical fields, disclose a kind of preparation method of microorganism proficiency testing salmonella sample, are interference bacterium with serratia marcescens, citrobacter freundii, Escherichia coli using salmonella as object bacteria, including：(A), the recovery of bacterial strain, increasing bacterium；(B), the separation of bacterial strain；(C), the preparation of bacteria suspension；(D), it is freeze-dried and packs.Finally prepd sample includes：Simple level negative sample, simple level positive；Intermediate negative sample, intermediate positive；Difficult grade negative sample, difficult grade positive.The sample pairing of same grade uses.Sample stability prepared by the method for the present invention is good, and strain survival rate is high, and with rational different grade of difficulty standards.
Description
Technical field
The present invention relates to microorganism proficiency testing technical field more particularly to microorganism proficiency testing salmonella samples
Preparation method.
Background technology
Proficiency testing is one of the effective measures of laboratory external mass control, and measures from card and Laboratory Accreditation
Necessary requirement.The technology energy for participating in laboratory progress related microorganisms inspection can effectively be assessed by implementing microorganism proficiency testing
Power.
The salmonella pathogen common as China's sitotoxismus in recent years and food origin disease, is that proficiency testing project is normal
The object bacteria of selection.But currently, microbiological Test field there is no high confidence level, high-throughput microorganism proficiency testing to prepare
Technology, uncertain factor are more so that the confidence level of laboratory monitoring proficiency testing is had a greatly reduced quality.
Cao Wenbo etc. exists《Food security quality testing journal》The 1st phases of volume 6 in 2015 disclose a kind of salmonella
The preparation method of proficiency testing sample：Sample is prepared by the way of vacuum freeze drying, this method improves to a certain extent
The stability and uniformity of sample, but it is still not ideal enough, and lack grade of difficulty contrast standard.
Existing microorganism proficiency testing salmonella sample, the strain stability of sample is poor, the survival rate of strain
It is relatively low, and the validation difficulty of variety classes sample differs, and lacks contrast standard, so that the meaning of microorganism proficiency testing
It has a greatly reduced quality.
Invention content
In order to solve the above technical problem, the present invention provides a kind of preparations of microorganism proficiency testing salmonella sample
Method, sample stability prepared by this method is good, and strain survival rate is high, has rational different grade of difficulty standards.
The specific technical solution of the present invention is：The preparation method of microorganism proficiency testing salmonella sample, with Salmonella
Bacterium is object bacteria, is interference bacterium with serratia marcescens, citrobacter freundii, Escherichia coli, includes the following steps：
(A), the recovery of bacterial strain, increasing bacterium：The standard bacteria of the standard bacteria of object bacteria and each interference bacterium is added separately to nutrition
In meat soup or nutrient agar slant medium, each standard bacteria recovery, growth are made at 36 ± 1 DEG C and increases bacterium, during culture
And strain idenfication is carried out to each bacterial strain.
(B), the separation of bacterial strain：Centrifugation point is carried out to its culture medium when the salmonella of above-mentioned culture was cultivated to stationary phase
From obtaining salmonella bacterium mud；After each interference bacterium is cultivated to exponential phase, its culture medium is centrifuged,
Obtain each interference bacterium bacterium mud.
(C), the preparation of bacteria suspension：
The preparation of simple level negative sample bacteria suspension：1 part of Escherichia coli bacterium mud is added to the de- of a concentration of 8-12wt%
In fat cow's milk aqueous solution, control a concentration of (3-7) × 10 of bacteria suspension4Cfu/mL obtains simple level feminine gender sample after stirring evenly
Product bacteria suspension.
The preparation of simple level positive bacteria suspension：1 part of salmonella bacterium mud and 1 part of Escherichia coli bacterium mud are added to dense
Degree is to be controlled a concentration of (3-7) × 10 of object bacteria in bacteria suspension in the skimmed milk aqueous solution of 8-12wt%2Cfu/mL, respectively
It interferes a concentration of (3-7) × 10 of bacterium4Cfu/mL obtains simple level positive bacteria suspension after stirring evenly.
The preparation of intermediate negative sample bacteria suspension：By 1 part of Escherichia coli bacterium mud and 1 part of citrobacter freundii bacterium mud addition
Into the skimmed milk aqueous solution of a concentration of 8-12wt%, control a concentration of (3-7) × 10 of object bacteria in bacteria suspension2cfu/
ML, each a concentration of (3-7) × 10 for interfering bacterium4Cfu/mL obtains intermediate negative sample bacteria suspension after stirring evenly.
The preparation of intermediate positive bacteria suspension：By 1 part of salmonella bacterium mud and 1 part of citrobacter freundii bacterium mud addition
Into the skimmed milk aqueous solution of a concentration of 8-12wt%, control a concentration of (3-7) × 10 of object bacteria in bacteria suspension2cfu/
ML, each a concentration of (3-7) × 10 for interfering bacterium4Cfu/mL obtains intermediate positive bacteria suspension after stirring evenly.
The preparation of difficult grade negative sample bacteria suspension：By 1 part of Escherichia coli bacterium mud, 1 part of serratia marcescens bacterium mud and 1 part
Citrobacter freundii bacterium mud is added in the skimmed milk aqueous solution of a concentration of 8-12wt%, controls object bacteria in bacteria suspension
A concentration of (3-7) × 102Cfu/mL, each a concentration of (3-7) × 10 for interfering bacterium4It is cloudy to obtain difficult grade by cfu/mL after stirring evenly
Property sample bacteria suspension.
The preparation of difficult grade positive bacteria suspension：By 1 part of salmonella bacterium mud, 1 part of Escherichia coli bacterium mud, 1 part of cement
Serratieae bacterium mud and 1 part of citrobacter freundii bacterium mud are added in the skimmed milk aqueous solution of a concentration of 8-12wt%, control
A concentration of (3-7) × 10 of object bacteria in bacteria suspension processed2Cfu/mL, each a concentration of (3-7) × 10 for interfering bacterium4Cfu/mL, stirring
Difficult grade positive bacteria suspension is obtained after uniformly.
Above-mentioned number is parts by weight；
(D), it is freeze-dried and packs：Each bacteria suspension 1mL of above-mentioned preparation is weighed respectively, and is respectively added to respective
In ampulla, the ampulla is opened flat lid and is put into freeze drier, freezes 7-9h in advance at a temperature of -22 DEG C to -18 DEG C, then
It is transferred in the freeze drier that temperature is -50 DEG C to -40 DEG C, vacuumizes and vacuum degree is made to reach 100 μm of Hg or more, then will
Ampulla is uniformly positioned on the indoor partition board of freezing, lyophilization 45-55h；Wait for that substance is creamy white loose spongy in ampulla
When, parsing-desiccation 1.5-2.5h is continued to ampulla；After ampulla is sealed, gland, labeling, microorganism energy is made
Power verifies salmonella sample, the sample is stored in 4 DEG C of environment.
It is object bacteria that the present invention, which selects salmonella, and selection serratia marcescens, citrobacter freundii, Escherichia coli are
Bacterium is interfered, and according to interference bacterium and similarity degree when Salmeterol fluticasone propionate, is prepared into three grade of difficulty sample sets, each
Sample sets include positive and negative sample.By sample prepared by the method for the present invention, by controlling the items in preparation process
Parameter so that the stability and survival rate of strain are higher in sample.
Preferably, being added with trehalose in the step (A) in the culture medium of each bacterial strain, the trehalose is in culture medium
In content be 2-4g/mL.The purpose of addition trehalose is that mushroom is absorbed using trehalose as carbon source in the medium
Afterwards, trehalose can carry out inside the cell membrane of mushroom, and when carrying out subsequent freezing dry process, the presence of trehalose can
Improve the freezing tolerance of mushroom so that intracellular cell liquid is not easy to form ice crystal, avoids the cell liquid due to cell interior
Become ice crystal, increase the osmotic pressure of cell and leads to the situation of mushroom death.
Preferably, before thering is each culture medium of bacterial strain to detach culture in the step (B), by the culture
Base is positioned over 1-2h in 45-50 DEG C of environment.Above-mentioned steps are carried out to culture medium before being detached, are conducive to bacterial strain to culture
The absorption of trehalose in base, is conducive to trehalose and enters inside strain cell.
Preferably, when the step (C) prepares each bacteria suspension, it is also poly- added with shell in the skimmed milk aqueous solution
It is one or more in sugar and polyvinyl alcohol.The presence of chitosan or polyvinyl alcohol is made when being freeze-dried to bacteria suspension
For the supplement to cryoprotector skimmed milk, chitosan or polyvinyl alcohol become gel in freezing dry process, have crowd
More three-dimensional open-frameworks can carry out a degree of embedding and absorption to bacterial strain, form a buffer protection layer, play
One buffer action, weakens influence of the extremely low temperature to bacterial strain to a certain extent.In addition chitosan and polyvinyl alcohol can
It is harmless to mushroom by mushroom natural decomposition.
Preferably, the concentration of the chitosan or polyvinyl alcohol in the skimmed milk aqueous solution is 0.2-
0.5wt%.
Preferably, the relative molecular weight of the polyvinyl alcohol is 16000-20000.In the polyethylene of this molecular weight ranges
Alcohol viscosity is relatively low, is suitable as protective agent.
Preferably, carrying out sealing and when gland in the drying of freeze drier to the ampulla in the step (D)
Operation in case, and be vacuum state in ampulla after sealing.
Preferably, when carrying out recovery culture to each standard bacteria in the step (A), with one ring bacterium of oese picking
Strain, and be inoculated into the culture medium of 10mL.
Preferably, when preparing each bacteria suspension in the step (B), the skimmed milk aqueous solution it is a concentration of
10wt%, a concentration of the 5 × 10 of object bacteria in bacteria suspension2Cfu/mL, it is each to interfere a concentration of the 5 × 10 of bacterium4cfu/mL。
Preferably, in the step (D), ampulla advance solidification point be -20 DEG C, freeze 8h in advance；Then
The temperature for the freeze drier being transferred to is -45 DEG C；The lyophilization time to ampulla is 50h；When to the parsing-desiccation of ampulla
Between be 2h.
It is compared with the prior art, the beneficial effects of the invention are as follows：Sample stability prepared by the method for the present invention is good, and strain is deposited
Motility rate is high, and with rational different grade of difficulty standards.
Description of the drawings
Fig. 1 is the influence of storage temperature and holding time to simple level positive stability in embodiment 2.
Specific implementation mode
With reference to embodiment, the invention will be further described.
Embodiment 1
The preparation method of microorganism proficiency testing salmonella sample, using salmonella as object bacteria, with Serratia
Bacterium, citrobacter freundii, Escherichia coli are interference bacterium, are included the following steps：
(A), the recovery of bacterial strain, increasing bacterium：With each bacterial strain of one ring of oese picking, by the standard bacteria of object bacteria and each interference bacterium
Standard bacteria be added separately in the nutrient broth or nutrient agar slant medium of 10mL, each mark is made at 36 ± 1 DEG C
Quasi- bacterium recovery, growth and increasing bacterium, strain idenfication is carried out during culture and to each bacterial strain.
(B), the separation of bacterial strain：Centrifugation point is carried out to its culture medium when the salmonella of above-mentioned culture was cultivated to stationary phase
From obtaining salmonella bacterium mud；After each interference bacterium is cultivated to exponential phase, its culture medium is centrifuged,
Obtain each interference bacterium bacterium mud.
(C), the preparation of bacteria suspension：
The preparation of simple level negative sample bacteria suspension：1 part of Escherichia coli bacterium mud is added to the degreasing of a concentration of 10wt%
In cow's milk aqueous solution, a concentration of the 5 × 10 of bacteria suspension are controlled4It is outstanding to obtain simple level negative sample bacterium by cfu/mL after stirring evenly
Liquid.
The preparation of simple level positive bacteria suspension：1 part of salmonella bacterium mud and 1 part of Escherichia coli bacterium mud are added to dense
Degree is to control a concentration of 5 × 10 of object bacteria in bacteria suspension in the skimmed milk aqueous solution of 10wt%2Cfu/mL respectively interferes bacterium
A concentration of 5 × 104Cfu/mL obtains simple level positive bacteria suspension after stirring evenly.
The preparation of intermediate negative sample bacteria suspension：By 1 part of Escherichia coli bacterium mud and 1 part of citrobacter freundii bacterium mud addition
Into the skimmed milk aqueous solution of a concentration of 10wt%, a concentration of 5 × 10 of object bacteria in bacteria suspension are controlled2Cfu/mL, it is each dry
Disturb a concentration of the 5 × 10 of bacterium4Cfu/mL obtains intermediate negative sample bacteria suspension after stirring evenly.
The preparation of intermediate positive bacteria suspension：By 1 part of salmonella bacterium mud and 1 part of citrobacter freundii bacterium mud addition
Into the skimmed milk aqueous solution of a concentration of 10wt%, a concentration of 5 × 10 of object bacteria in bacteria suspension are controlled2Cfu/mL, it is each dry
Disturb a concentration of the 5 × 10 of bacterium4Cfu/mL obtains intermediate positive bacteria suspension after stirring evenly.
The preparation of difficult grade negative sample bacteria suspension：By 1 part of Escherichia coli bacterium mud, 1 part of serratia marcescens bacterium mud and 1 part
Citrobacter freundii bacterium mud is added in the skimmed milk aqueous solution of a concentration of 10wt%, and object bacteria is dense in control bacteria suspension
Degree is 5 × 102Cfu/mL, it is each to interfere a concentration of the 5 × 10 of bacterium4It is outstanding to obtain difficult grade negative sample bacterium by cfu/mL after stirring evenly
Liquid.
The preparation of difficult grade positive bacteria suspension：By 1 part of salmonella bacterium mud, 1 part of Escherichia coli bacterium mud, 1 part of cement
Serratieae bacterium mud and 1 part of citrobacter freundii bacterium mud are added in the skimmed milk aqueous solution of a concentration of 10wt%, control
A concentration of the 5 × 10 of object bacteria in bacteria suspension2Cfu/mL, it is each to interfere a concentration of the 5 × 10 of bacterium4Cfu/mL is obtained after stirring evenly
Difficult grade positive bacteria suspension.
Above-mentioned number is parts by weight；
(D), it is freeze-dried and packs：Each bacteria suspension 1mL of above-mentioned preparation is weighed respectively, and is respectively added to respective
In ampulla, the ampulla is opened flat lid and is put into freeze drier, freezes 8h in advance at a temperature of -20 DEG C, is then transferred to temperature
In the freeze drier that degree is -45 DEG C, vacuumizes and vacuum degree is made to reach 100 μm of Hg or more, be then uniformly positioned over ampulla
It freezes on indoor partition board, lyophilization 50h；When substance in ampulla is creamy white loose spongy, ampulla is continued
Parsing-desiccation 2h；After ampulla is sealed in the drying box of freeze drier, gland, labeling, microorganism energy is made
Power verifies salmonella sample, and it is vacuum state that ampulla is interior after sealing, the sample is stored in 4 DEG C of environment.
Embodiment 2
The preparation method of microorganism proficiency testing salmonella sample, using salmonella as object bacteria, with Serratia
Bacterium, citrobacter freundii, Escherichia coli are interference bacterium, are included the following steps：
(A), the recovery of bacterial strain, increasing bacterium：With each bacterial strain of one ring of oese picking, by the standard bacteria of object bacteria and each interference bacterium
Standard bacteria be added separately in the nutrient broth or nutrient agar slant medium of 10mL, each mark is made at 36 ± 1 DEG C
Quasi- bacterium recovery, growth and increasing bacterium, strain idenfication is carried out during culture and to each bacterial strain.Trehalose is added in the culture medium,
The content of the trehalose in the medium is 3g/mL.
(B), the separation of bacterial strain：When the salmonella of above-mentioned culture was cultivated to stationary phase, culture medium is positioned over 48 DEG C
Environment in 1.5h, then culture medium is centrifuged, obtains salmonella bacterium mud；When each interference bacterium is cultivated to right
After number growth period, culture medium is positioned over 1.5h in 48 DEG C of environment, culture medium is centrifuged, obtains each interference bacterium bacterium
Mud.
(C), the preparation of bacteria suspension：
The preparation of simple level negative sample bacteria suspension：1 part of Escherichia coli bacterium mud is added to the degreasing of a concentration of 10wt%
In cow's milk aqueous solution, a concentration of the 5 × 10 of bacteria suspension are controlled4It is outstanding to obtain simple level negative sample bacterium by cfu/mL after stirring evenly
Liquid.The chitosan and polyvinyl alcohol that concentration is 0.3wt% are also added in the skimmed milk aqueous solution.The polyvinyl alcohol
Relative molecular weight be 16000-20000.
The preparation of simple level positive bacteria suspension：1 part of salmonella bacterium mud and 1 part of Escherichia coli bacterium mud are added to dense
Degree is to control a concentration of 5 × 10 of object bacteria in bacteria suspension in the skimmed milk aqueous solution of 10wt%2Cfu/mL respectively interferes bacterium
A concentration of 5 × 104Cfu/mL obtains simple level positive bacteria suspension after stirring evenly.In the skimmed milk aqueous solution
Also it is added with the chitosan and polyvinyl alcohol that concentration is 0.3wt%.
The preparation of intermediate negative sample bacteria suspension：By 1 part of Escherichia coli bacterium mud and 1 part of citrobacter freundii bacterium mud addition
Into the skimmed milk aqueous solution of a concentration of 10wt%, a concentration of 5 × 10 of object bacteria in bacteria suspension are controlled2Cfu/mL, it is each dry
Disturb a concentration of the 5 × 10 of bacterium4Cfu/mL obtains intermediate negative sample bacteria suspension after stirring evenly.The skimmed milk aqueous solution
In also be added with concentration be 0.3wt% chitosan and polyvinyl alcohol.
The preparation of intermediate positive bacteria suspension：By 1 part of salmonella bacterium mud and 1 part of citrobacter freundii bacterium mud addition
Into the skimmed milk aqueous solution of a concentration of 10wt%, a concentration of 5 × 10 of object bacteria in bacteria suspension are controlled2Cfu/mL, it is each dry
Disturb a concentration of the 5 × 10 of bacterium4Cfu/mL obtains intermediate positive bacteria suspension after stirring evenly.The skimmed milk aqueous solution
In be also 0.3wt% chitosans and polyvinyl alcohol added with concentration.
The preparation of difficult grade negative sample bacteria suspension：By 1 part of Escherichia coli bacterium mud, 1 part of serratia marcescens bacterium mud and 1 part
Citrobacter freundii bacterium mud is added in the skimmed milk aqueous solution of a concentration of 10wt%, and object bacteria is dense in control bacteria suspension
Degree is 5 × 102Cfu/mL, it is each to interfere a concentration of the 5 × 10 of bacterium4It is outstanding to obtain difficult grade negative sample bacterium by cfu/mL after stirring evenly
Liquid.The chitosan and polyvinyl alcohol that concentration is 0.3wt% are also added in the skimmed milk aqueous solution.
The preparation of difficult grade positive bacteria suspension：By 1 part of salmonella bacterium mud, 1 part of Escherichia coli bacterium mud, 1 part of cement
Serratieae bacterium mud and 1 part of citrobacter freundii bacterium mud are added in the skimmed milk aqueous solution of a concentration of 10wt%, control
A concentration of the 5 × 10 of object bacteria in bacteria suspension2Cfu/mL, it is each to interfere a concentration of the 5 × 10 of bacterium4Cfu/mL is obtained after stirring evenly
Difficult grade positive bacteria suspension.The chitosan and gather that concentration is 0.3wt% are also added in the skimmed milk aqueous solution
Vinyl alcohol.
Above-mentioned number is parts by weight；
(D), it is freeze-dried and packs：Each bacteria suspension 1mL of above-mentioned preparation is weighed respectively, and is respectively added to respective
In ampulla, the ampulla is opened flat lid and is put into freeze drier, freezes 8h in advance at a temperature of -20 DEG C, is then transferred to temperature
In the freeze drier that degree is -45 DEG C, vacuumizes and vacuum degree is made to reach 100 μm of Hg or more, be then uniformly positioned over ampulla
It freezes on indoor partition board, lyophilization 50h；When substance in ampulla is creamy white loose spongy, ampulla is continued
Parsing-desiccation 2h；After ampulla is sealed in the drying box of freeze drier, gland, labeling, microorganism energy is made
Power verifies salmonella sample, and it is vacuum state that ampulla is interior after sealing, the sample is stored in 4 DEG C of environment.
Embodiment 3
The preparation method of microorganism proficiency testing salmonella sample, using salmonella as object bacteria, with Serratia
Bacterium, citrobacter freundii, Escherichia coli are interference bacterium, are included the following steps：
(A), the recovery of bacterial strain, increasing bacterium：With each bacterial strain of one ring of oese picking, by the standard bacteria of object bacteria and each interference bacterium
Standard bacteria be added separately in the nutrient broth or nutrient agar slant medium of 10mL, each mark is made at 36 ± 1 DEG C
Quasi- bacterium recovery, growth and increasing bacterium, strain idenfication is carried out during culture and to each bacterial strain.Trehalose is added in the culture medium,
The content of the trehalose in the medium is 4g/mL.
(B), the separation of bacterial strain：When the salmonella of above-mentioned culture was cultivated to stationary phase, culture medium is positioned over 45 DEG C
Environment in 2h, then culture medium is centrifuged, obtains salmonella bacterium mud；When each interference bacterium is cultivated to logarithm
After growth period, culture medium is positioned over 2h in 45 DEG C of environment, culture medium is centrifuged, obtains each interference bacterium bacterium mud.
(C), the preparation of bacteria suspension：
The preparation of simple level negative sample bacteria suspension：1 part of Escherichia coli bacterium mud is added to the degreasing of a concentration of 12wt%
In cow's milk aqueous solution, a concentration of the 7 × 10 of bacteria suspension are controlled4It is outstanding to obtain simple level negative sample bacterium by cfu/mL after stirring evenly
Liquid.A concentration of 0.5wt% chitosans are also added in the skimmed milk aqueous solution.
The preparation of simple level positive bacteria suspension：1 part of salmonella bacterium mud and 1 part of Escherichia coli bacterium mud are added to dense
Degree is to control a concentration of 7 × 10 of object bacteria in bacteria suspension in the skimmed milk aqueous solution of 12wt%2Cfu/mL respectively interferes bacterium
A concentration of 7 × 104Cfu/mL obtains simple level positive bacteria suspension after stirring evenly.In the skimmed milk aqueous solution
Also it is added with a concentration of 0.5wt% chitosans.
The preparation of intermediate negative sample bacteria suspension：By 1 part of Escherichia coli bacterium mud and 1 part of citrobacter freundii bacterium mud addition
Into the skimmed milk aqueous solution of a concentration of 12wt%, a concentration of 7 × 10 of object bacteria in bacteria suspension are controlled2Cfu/mL, it is each dry
Disturb a concentration of the 7 × 10 of bacterium4Cfu/mL obtains intermediate negative sample bacteria suspension after stirring evenly.The skimmed milk aqueous solution
In also be added with a concentration of 0.5wt% chitosans.
The preparation of intermediate positive bacteria suspension：By 1 part of salmonella bacterium mud and 1 part of citrobacter freundii bacterium mud addition
Into the skimmed milk aqueous solution of a concentration of 12wt%, a concentration of 7 × 10 of object bacteria in bacteria suspension are controlled2Cfu/mL, it is each dry
Disturb a concentration of the 7 × 10 of bacterium4Cfu/mL obtains intermediate positive bacteria suspension after stirring evenly.The skimmed milk aqueous solution
In also be added with a concentration of 0.5wt% chitosans.
The preparation of difficult grade negative sample bacteria suspension：By 1 part of Escherichia coli bacterium mud, 1 part of serratia marcescens bacterium mud and 1 part
Citrobacter freundii bacterium mud is added in the skimmed milk aqueous solution of a concentration of 12wt%, and object bacteria is dense in control bacteria suspension
Degree is 7 × 102Cfu/mL, it is each to interfere a concentration of the 7 × 10 of bacterium4It is outstanding to obtain difficult grade negative sample bacterium by cfu/mL after stirring evenly
Liquid.A concentration of 0.5wt% chitosans are also added in the skimmed milk aqueous solution.
The preparation of difficult grade positive bacteria suspension：By 1 part of salmonella bacterium mud, 1 part of Escherichia coli bacterium mud, 1 part of cement
Serratieae bacterium mud and 1 part of citrobacter freundii bacterium mud are added in the skimmed milk aqueous solution of a concentration of 12wt%, control
A concentration of the 7 × 10 of object bacteria in bacteria suspension2Cfu/mL, it is each to interfere a concentration of the 7 × 10 of bacterium4Cfu/mL is obtained after stirring evenly
Difficult grade positive bacteria suspension.A concentration of 0.5wt% chitosans are also added in the skimmed milk aqueous solution.
Above-mentioned number is parts by weight；
(D), it is freeze-dried and packs：Each bacteria suspension 1mL of above-mentioned preparation is weighed respectively, and is respectively added to respective
In ampulla, the ampulla is opened flat lid and is put into freeze drier, freezes 7h in advance at a temperature of -22 DEG C, is then transferred to temperature
In the freeze drier that degree is -50 DEG C, vacuumizes and vacuum degree is made to reach 100 μm of Hg or more, be then uniformly positioned over ampulla
It freezes on indoor partition board, lyophilization 45h；When substance in ampulla is creamy white loose spongy, ampulla is continued
Parsing-desiccation 1.5h；After ampulla is sealed in the drying box of freeze drier, gland, labeling, be made microorganism
Proficiency testing salmonella sample, it is vacuum state that ampulla is interior after sealing, the sample is stored in 4 DEG C of environment.
Embodiment 4
The preparation method of microorganism proficiency testing salmonella sample, using salmonella as object bacteria, with Serratia
Bacterium, citrobacter freundii, Escherichia coli are interference bacterium, are included the following steps：
(A), the recovery of bacterial strain, increasing bacterium：With each bacterial strain of one ring of oese picking, by the standard bacteria of object bacteria and each interference bacterium
Standard bacteria be added separately in the nutrient broth or nutrient agar slant medium of 10mL, each mark is made at 36 ± 1 DEG C
Quasi- bacterium recovery, growth and increasing bacterium, strain idenfication is carried out during culture and to each bacterial strain.Trehalose is added in the culture medium,
The content of the trehalose in the medium is 2g/mL.
(B), the separation of bacterial strain：When the salmonella of above-mentioned culture was cultivated to stationary phase, culture medium is positioned over 50 DEG C
Environment in 1h, then culture medium is centrifuged, obtains salmonella bacterium mud；When each interference bacterium is cultivated to logarithm
After growth period, culture medium is positioned over 1h in 50 DEG C of environment, culture medium is centrifuged, obtains each interference bacterium bacterium mud.
(C), the preparation of bacteria suspension：
The preparation of simple level negative sample bacteria suspension：1 part of Escherichia coli bacterium mud is added to the degreasing ox of a concentration of 8wt%
In whey solution, a concentration of the 3 × 10 of bacteria suspension are controlled4Cfu/mL obtains simple level negative sample bacteria suspension after stirring evenly.
The polyvinyl alcohol of a concentration of 0.2wt% is also added in the skimmed milk aqueous solution.The relative molecular weight of the polyvinyl alcohol
For 16000-20000.
The preparation of simple level positive bacteria suspension：1 part of salmonella bacterium mud and 1 part of Escherichia coli bacterium mud are added to dense
Degree is to control a concentration of 3 × 10 of object bacteria in bacteria suspension in the skimmed milk aqueous solution of 8wt%2Cfu/mL, each interference bacterium
A concentration of 3 × 104Cfu/mL obtains simple level positive bacteria suspension after stirring evenly.In the skimmed milk aqueous solution also
Polyvinyl alcohol added with a concentration of 0.2wt%.
The preparation of intermediate negative sample bacteria suspension：By 1 part of Escherichia coli bacterium mud and 1 part of citrobacter freundii bacterium mud addition
Into the skimmed milk aqueous solution of a concentration of 8wt%, a concentration of 3- × 10 of object bacteria in bacteria suspension are controlled2Cfu/mL, it is each dry
Disturb a concentration of the 3 × 10 of bacterium4Cfu/mL obtains intermediate negative sample bacteria suspension after stirring evenly.The skimmed milk aqueous solution
In also be added with a concentration of 0.2wt% polyvinyl alcohol.
The preparation of intermediate positive bacteria suspension：By 1 part of salmonella bacterium mud and 1 part of citrobacter freundii bacterium mud addition
Into the skimmed milk aqueous solution of a concentration of 8wt%, a concentration of 3 × 10 of object bacteria in bacteria suspension are controlled2Cfu/mL, it is each to interfere
A concentration of the 3 × 10 of bacterium4Cfu/mL obtains intermediate positive bacteria suspension after stirring evenly.In the skimmed milk aqueous solution
Also it is added with the polyvinyl alcohol of a concentration of 0.2wt%.
The preparation of difficult grade negative sample bacteria suspension：By 1 part of Escherichia coli bacterium mud, 1 part of serratia marcescens bacterium mud and 1 part
Citrobacter freundii bacterium mud is added in the skimmed milk aqueous solution of a concentration of 8wt%, and object bacteria is dense in control bacteria suspension
Degree is 3 × 102Cfu/mL, it is each to interfere a concentration of the 3 × 10 of bacterium4It is outstanding to obtain difficult grade negative sample bacterium by cfu/mL after stirring evenly
Liquid.The polyvinyl alcohol of a concentration of 0.2wt% is also added in the skimmed milk aqueous solution.
The preparation of difficult grade positive bacteria suspension：By 1 part of salmonella bacterium mud, 1 part of Escherichia coli bacterium mud, 1 part of cement
Serratieae bacterium mud and 1 part of citrobacter freundii bacterium mud are added in the skimmed milk aqueous solution of a concentration of 8wt%, control bacterium
A concentration of the 3 × 10 of object bacteria in suspension2Cfu/mL, it is each to interfere a concentration of the 3 × 10 of bacterium4Cfu/mL is stranded after stirring evenly
Difficult grade positive bacteria suspension.The polyvinyl alcohol of a concentration of 0.2wt% is also added in the skimmed milk aqueous solution.
Above-mentioned number is parts by weight；
(D), it is freeze-dried and packs：Each bacteria suspension 1mL of above-mentioned preparation is weighed respectively, and is respectively added to respective
In ampulla, the ampulla is opened flat lid and is put into freeze drier, freezes 9h in advance at a temperature of -18 DEG C, is then transferred to temperature
In the freeze drier that degree is -40 DEG C, vacuumizes and vacuum degree is made to reach 100 μm of Hg or more, be then uniformly positioned over ampulla
It freezes on indoor partition board, lyophilization 55h；When substance in ampulla is creamy white loose spongy, ampulla is continued
Parsing-desiccation 2.5h；After ampulla is sealed in the drying box of freeze drier, gland, labeling, be made microorganism
Proficiency testing salmonella sample, it is vacuum state that ampulla is interior after sealing, the sample is stored in 4 DEG C of environment.
Embodiment 5
The preparation method of microorganism proficiency testing salmonella sample, using salmonella as object bacteria, with Serratia
Bacterium, citrobacter freundii, Escherichia coli are interference bacterium, are included the following steps：
(A), the recovery of bacterial strain, increasing bacterium：With each bacterial strain of one ring of oese picking, by the standard bacteria of object bacteria and each interference bacterium
Standard bacteria be added separately in the nutrient broth or nutrient agar slant medium of 10mL, each mark is made at 36 ± 1 DEG C
Quasi- bacterium recovery, growth and increasing bacterium, strain idenfication is carried out during culture and to each bacterial strain.Trehalose is added in the culture medium,
The content of the trehalose in the medium is 3g/mL.
(B), the separation of bacterial strain：When the salmonella of above-mentioned culture was cultivated to stationary phase, culture medium is positioned over 46 DEG C
Environment in 1.5h, then culture medium is centrifuged, obtains salmonella bacterium mud；When each interference bacterium is cultivated to right
After number growth period, culture medium is positioned over 1.5h in 46 DEG C of environment, culture medium is centrifuged, obtains each interference bacterium bacterium
Mud.
(C), the preparation of bacteria suspension：
The preparation of simple level negative sample bacteria suspension：1 part of Escherichia coli bacterium mud is added to the degreasing ox of a concentration of 9wt%
In whey solution, a concentration of the 4 × 10 of bacteria suspension are controlled4Cfu/mL obtains simple level negative sample bacteria suspension after stirring evenly.
The chitosan and polyvinyl alcohol that concentration is 0.4wt% are also added in the skimmed milk aqueous solution.The polyvinyl alcohol
Relative molecular weight is 16000-20000.
The preparation of simple level positive bacteria suspension：1 part of salmonella bacterium mud and 1 part of Escherichia coli bacterium mud are added to dense
Degree is to control a concentration of 4 × 10 of object bacteria in bacteria suspension in the skimmed milk aqueous solution of 9wt%2Cfu/mL, each interference bacterium
A concentration of 4 × 104Cfu/mL obtains simple level positive bacteria suspension after stirring evenly.In the skimmed milk aqueous solution also
It is the chitosan and polyvinyl alcohol of 0.4wt% added with concentration.
The preparation of intermediate negative sample bacteria suspension：By 1 part of Escherichia coli bacterium mud and 1 part of citrobacter freundii bacterium mud addition
Into the skimmed milk aqueous solution of a concentration of 9wt%, a concentration of 4 × 10 of object bacteria in bacteria suspension are controlled2Cfu/mL, it is each to interfere
A concentration of the 4 × 10 of bacterium4Cfu/mL obtains intermediate negative sample bacteria suspension after stirring evenly.In the skimmed milk aqueous solution
Also it is added with the chitosan and polyvinyl alcohol that concentration is 0.4%.
The preparation of intermediate positive bacteria suspension：By 1 part of salmonella bacterium mud and 1 part of citrobacter freundii bacterium mud addition
Into the skimmed milk aqueous solution of a concentration of 9wt%, a concentration of 4 × 10 of object bacteria in bacteria suspension are controlled2Cfu/mL, it is each to interfere
A concentration of the 4 × 10 of bacterium4Cfu/mL obtains intermediate positive bacteria suspension after stirring evenly.In the skimmed milk aqueous solution
Also it is added with the chitosan and polyvinyl alcohol that concentration is 0.4wt%.
The preparation of difficult grade negative sample bacteria suspension：By 1 part of Escherichia coli bacterium mud, 1 part of serratia marcescens bacterium mud and 1 part
Citrobacter freundii bacterium mud is added in the skimmed milk aqueous solution of a concentration of 9wt%, and object bacteria is dense in control bacteria suspension
Degree is 4 × 102Cfu/mL, it is each to interfere a concentration of the 4 × 10 of bacterium4It is outstanding to obtain difficult grade negative sample bacterium by cfu/mL after stirring evenly
Liquid.The chitosan and polyvinyl alcohol that concentration is 0.4wt% are also added in the skimmed milk aqueous solution.
The preparation of difficult grade positive bacteria suspension：By 1 part of salmonella bacterium mud, 1 part of Escherichia coli bacterium mud, 1 part of cement
Serratieae bacterium mud and 1 part of citrobacter freundii bacterium mud are added in the skimmed milk aqueous solution of a concentration of 9wt%, control bacterium
A concentration of the 4 × 10 of object bacteria in suspension2Cfu/mL, it is each to interfere a concentration of the 4 × 10 of bacterium4Cfu/mL is stranded after stirring evenly
Difficult grade positive bacteria suspension.Added with concentration it is also the chitosan of 0.4wt% and poly- second in the skimmed milk aqueous solution
Enol.
Above-mentioned number is parts by weight；
(D), it is freeze-dried and packs：Each bacteria suspension 1mL of above-mentioned preparation is weighed respectively, and is respectively added to respective
In ampulla, the ampulla is opened flat lid and is put into freeze drier, freezes 8h in advance at a temperature of -20 DEG C, is then transferred to temperature
In the freeze drier that degree is -45 DEG C, vacuumizes and vacuum degree is made to reach 100 μm of Hg or more, be then uniformly positioned over ampulla
It freezes on indoor partition board, lyophilization 50h；When substance in ampulla is creamy white loose spongy, ampulla is continued
Parsing-desiccation 2h；After ampulla is sealed in the drying box of freeze drier, gland, labeling, microorganism energy is made
Power verifies salmonella sample, and it is vacuum state that ampulla is interior after sealing, the sample is stored in 4 DEG C of environment.
The estimation of stability of sample
1 storage temperature influences sample stability
Microbiological specimens after freeze-drying need to be placed under refrigerated condition to ensure its stability.When in view of proficiency testing hair sample
Reason span is big, provides the process estimated 1-3d times of sample, 36 DEG C of period or more hot weather and packaging environment may be made
At the failure of sample, therefore this experiment has carried out stability test of the sample under condition of different temperatures, chooses letter in embodiment 2
Single-stage positive is as test object, and the results are shown in Figure 1.
2 holding times influenced sample stability
It chooses simple level positive in embodiment 2 and preserves sample under 4 DEG C of preservation condition as test object
180d, and 7 periods are chosen in 180d, 2-4 parts of samples are randomly selected every time and recovered, counted and is identified, as a result such as Fig. 1
It is shown.The above is only presently preferred embodiments of the present invention, is not imposed any restrictions to the present invention, every according to skill of the present invention
Art essence still falls within technical solution of the present invention to any simple modification, change and equivalent transformation made by above example
Protection domain.
Claims (6)
1. the preparation method of microorganism proficiency testing salmonella sample, using salmonella as object bacteria, with serratia marcescens,
Citrobacter freundii, Escherichia coli are interference bacterium, it is characterised in that are included the following steps：
(A), the recovery of bacterial strain, increasing bacterium：The standard bacteria of the standard bacteria of object bacteria and each interference bacterium is added separately to nutrient broth
Or in nutrient agar slant medium, each standard bacteria recovery, growth are made at 36 ± 1 DEG C and increases bacterium, it is during culture and right
Each bacterial strain carries out strain idenfication；It is added with trehalose in the culture medium of wherein each bacterial strain, the trehalose containing in the medium
Amount is 2-4g/mL；
(B), the separation of bacterial strain：Its culture medium is centrifuged when the salmonella of above-mentioned culture was cultivated to stationary phase,
Obtain salmonella bacterium mud；After each interference bacterium is cultivated to exponential phase, its culture medium is centrifuged, is obtained
The culture medium is positioned over 45-50 DEG C by each interference bacterium bacterium mud wherein before having each culture medium of bacterial strain to detach culture
Environment in 1-2h；
(C), the preparation of bacteria suspension：
The preparation of simple level negative sample bacteria suspension：1 part of Escherichia coli bacterium mud is added to the degreasing ox of a concentration of 8-12wt%
In whey solution, control a concentration of (3-7) × 10 of bacteria suspension4Cfu/mL obtains simple level negative sample bacterium after stirring evenly
Suspension；
The preparation of simple level positive bacteria suspension：1 part of salmonella bacterium mud and 1 part of Escherichia coli bacterium mud are added to a concentration of
In the skimmed milk aqueous solution of 8-12wt%, control a concentration of (3-7) × 10 of object bacteria in bacteria suspension2Cfu/mL interferes bacterium
A concentration of (3-7) × 104Cfu/mL obtains simple level positive bacteria suspension after stirring evenly；
The preparation of intermediate negative sample bacteria suspension：1 part of Escherichia coli bacterium mud and 1 part of citrobacter freundii bacterium mud are added to dense
Degree is each a concentration of (3-7) × 10 for interfering bacterium in the skimmed milk aqueous solution of 8-12wt%4Cfu/mL, after stirring evenly
To intermediate negative sample bacteria suspension；
The preparation of intermediate positive bacteria suspension：By 1 part of salmonella bacterium mud, 1 part of Escherichia coli bacterium mud and 1 part of Freund citric acid
Bacillus bacterium mud is added in the skimmed milk aqueous solution of a concentration of 8-12wt%, controls a concentration of (3- of object bacteria in bacteria suspension
7)×102Cfu/mL, each a concentration of (3-7) × 10 for interfering bacterium4It is outstanding to obtain intermediate positive bacterium by cfu/mL after stirring evenly
Liquid；
The preparation of difficult grade negative sample bacteria suspension：By 1 part of Escherichia coli bacterium mud, 1 part of serratia marcescens bacterium mud and 1 part of Freund
Citric acid bacillus bacterium mud is added in the skimmed milk aqueous solution of a concentration of 8-12wt%, and each a concentration of (3-7) for interfering bacterium ×
104Cfu/mL obtains difficult grade negative sample bacteria suspension after stirring evenly；
The preparation of difficult grade positive bacteria suspension：By 1 part of salmonella bacterium mud, 1 part of Escherichia coli bacterium mud, 1 part of cement sand thunder
Salmonella bacterium mud and 1 part of citrobacter freundii bacterium mud are added in the skimmed milk aqueous solution of a concentration of 8-12wt%, control bacterium
A concentration of (3-7) × 10 of object bacteria in suspension2Cfu/mL, each a concentration of (3-7) × 10 for interfering bacterium4Cfu/mL is stirred evenly
After obtain difficult grade positive bacteria suspension；
The one kind or two being also added with when preparing each bacteria suspension, in the skimmed milk aqueous solution in chitosan and polyvinyl alcohol
Kind；The concentration of the chitosan or polyvinyl alcohol in the skimmed milk aqueous solution is 0.2-0.5wt%；Above-mentioned number is equal
For parts by weight；
(D), it is freeze-dried and packs：Each bacteria suspension 1mL of above-mentioned preparation is weighed respectively, and is respectively added to respective ampulla
In, the ampulla is opened flat lid and is put into freeze drier, freezes 7-9h in advance at a temperature of -22 DEG C to -18 DEG C, then shifts
In the freeze drier for being -50 DEG C to -40 DEG C to temperature, vacuumizes and vacuum degree is made to reach 100 μm of Hg or more, then by ampulla
It is uniformly positioned on the indoor partition board of freezing, lyophilization 45-55h；It is right when substance in ampulla is creamy white loose spongy
Ampulla continues parsing-desiccation 1.5-2.5h；After ampulla is sealed, gland, labeling, be made microorganism ability test
Salmonella sample is demonstrate,proved, the sample is stored in 4 DEG C of environment.
2. the preparation method of microorganism proficiency testing salmonella sample as described in claim 1, which is characterized in that described poly-
The relative molecular weight of vinyl alcohol is 16000-20000.
3. the preparation method of microorganism proficiency testing salmonella sample as described in claim 1, which is characterized in that described
It is operated in the drying box of freeze drier when carrying out sealing and gland to the ampulla in step (D), and after sealing in ampulla
For vacuum state.
4. the preparation method of microorganism proficiency testing salmonella sample as described in claim 1, which is characterized in that described
When carrying out recovery culture to each standard bacteria in step (A), with one ring bacterial strain of oese picking, and it is inoculated into the culture of 10mL
In base.
5. the preparation method of microorganism proficiency testing salmonella sample as described in claim 1, which is characterized in that described
When preparing each bacteria suspension in step (C), a concentration of 10wt% of the skimmed milk aqueous solution, the concentration of object bacteria in bacteria suspension
It is 5 × 102Cfu/mL, it is each to interfere a concentration of the 5 × 10 of bacterium4cfu/mL。
6. the preparation method of microorganism proficiency testing salmonella sample as described in claim 1, which is characterized in that described
In step (D), ampulla advance solidification point be -20 DEG C, freeze 8h in advance；The temperature for the freeze drier being then transferred to
It is -45 DEG C；The lyophilization time to ampulla is 50h；The parsing-desiccation time to ampulla is 2h.
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| CN107142303B (en) * | 2017-05-24 | 2020-09-22 | 中国检验检疫科学研究院 | Test sample for capacity of resisting cholate gram negative bacteria in medicine and preparation method thereof |
| CN107190048A (en) * | 2017-05-24 | 2017-09-22 | 中检科（北京）实验室能力评价有限公司 | Staphylococcus aureus proficiency testing sample and preparation method thereof in medicine |
| CN107192822A (en) * | 2017-05-24 | 2017-09-22 | 中国检验检疫科学研究院 | Detection of Salmonella proficiency testing sample and preparation method thereof in medicine |
| CN107190046A (en) * | 2017-05-24 | 2017-09-22 | 中国检验检疫科学研究院 | EHEC proficiency testing sample and preparation method thereof in medicine |
| CN107142301A (en) * | 2017-05-24 | 2017-09-08 | 中检科（北京）实验室能力评价有限公司 | Aerobic bacteria sum numerical ability verification sample and preparation method thereof in medicine |
| CN107236783B (en) * | 2017-06-28 | 2020-04-28 | 中国检验检疫科学研究院 | Salmonella standard sample in milk powder and preparation method thereof |
| CN107090489B (en) * | 2017-06-28 | 2020-08-14 | 中国检验检疫科学研究院 | Standard sample for total number of bacterial colonies in milk powder and preparation method thereof |
| CN107227336A (en) * | 2017-06-28 | 2017-10-03 | 中国检验检疫科学研究院 | Coliform standard sample and preparation method thereof in food |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1706964A (en) * | 2005-05-26 | 2005-12-14 | 卢行安 | Bacterial colony number sample for verifying microbiological capacity of food and its prepn process |
| CN101550438A (en) * | 2009-05-12 | 2009-10-07 | 天津天狮生物发展有限公司 | Method for testing and evaluating capacity of corrosion prevention system of cosmetic |
-
2015
- 2015-06-10 CN CN201510313160.6A patent/CN105176823B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1706964A (en) * | 2005-05-26 | 2005-12-14 | 卢行安 | Bacterial colony number sample for verifying microbiological capacity of food and its prepn process |
| CN101550438A (en) * | 2009-05-12 | 2009-10-07 | 天津天狮生物发展有限公司 | Method for testing and evaluating capacity of corrosion prevention system of cosmetic |
Non-Patent Citations (5)
| Title |
|---|
| "Proficiency Testing Performance in US Laboratories Results Reported to the Centers for Medicare & Medicaid Services,1994 Through 2006";Devery Howerton et al.;《Arch Pathol Lab Med》;20100531;第134卷;第751-758页 * |
| "沙门氏菌能力验证样品制备";曹文博等;《食品安全质量检测学报》;20150131;第6卷(第1期);第134页摘要、第135页引言、第135-136页材料与设备部分和第137页表2、表3 * |
| "能力验证样品中沙门氏菌的分离与鉴定";游勇来等;《检验检疫学刊》;20131231;第23卷(第1期);第45-47页 * |
| "能力验证试验中沙门菌分离与鉴定";俞慕华等;《中国卫生工程学》;20121031;第11卷(第5期);第424-426页 * |
| "菌种冷冻干燥保藏的影响因素";常金梅等;《微生物学通报》;20080620;第35卷(第6期);第959-962页 * |
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