CN102827795B - Culture medium for enriching salmonella, shigella and staphylococcus aureus in composite way and preparation method thereof - Google Patents

Culture medium for enriching salmonella, shigella and staphylococcus aureus in composite way and preparation method thereof Download PDF

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CN102827795B
CN102827795B CN201210313462.XA CN201210313462A CN102827795B CN 102827795 B CN102827795 B CN 102827795B CN 201210313462 A CN201210313462 A CN 201210313462A CN 102827795 B CN102827795 B CN 102827795B
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parts
distilled water
substratum
salmonellas
shigellae
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CN102827795A (en
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余以刚
肖性龙
王永志
吴晖
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South China University of Technology SCUT
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Abstract

The invention discloses a culture medium for enriching salmonella, shigella and staphylococcus aureus in a composite way and a preparation method thereof. The culture medium comprises 13-16 parts of tryptone, 4-7 parts of soy peptone, 2-3 parts of monosodium orthophosphate, 2-3 parts of glucose, 1,000 parts of distilled water, 0.07-0.13 part of bile salt, 25-40 parts of sodium chloride, 0.4-1 part of lithium chloride, 1.5-3.5 parts of mannite and 0.0002-0.0004 part of potassium tellurite, and the pH of the culture medium is 7.1-7.3. The culture medium can be used for restraining the growth of other pathogenic microorganisms while enriching three target pathogenic bacteria simultaneously, can be directly applied to separate culturing of target bacteria and biological assay experiments, and can be directly applied to a detection technology for a plurality of pathogenic bacteria based on one detection platform such as multiple PCRs (Polymerase Chain Reactions) and the like for making a diagnosis report.

Description

Substratum and the preparation method of bacterium is increased for Salmonellas, Shigellae and streptococcus aureus compound
Technical field
The invention belongs to the front enrichment medium of pathogenic bacterium, particularly relate to and a kind ofly substratum of bacterium and preparation method thereof is increased to Salmonellas, Shigellae and streptococcus aureus compound simultaneously.
Technical background
In global range, the lethal number of the gastrointestinal tract disease brought by Salmonellas and shigella infection every year, up to 1,000,000, all brings larger burden to various countries.One of streptococcus aureus can not only contaminated food products, or the major source that infects of clinical patient.The investigation display of China, Salmonellas, Shigellae and streptococcus aureus all occupy the row of the highest food-borne pathogens of infection rate, and wherein Salmonellas proportion in whole cases of infection is maximum.
Depend on the detection method widespread use now of gene, become one of main method detecting food-borne pathogens in the world.Rapidly, quantitative fluorescent PCR is also ripe, and the application of multiple fluorescence quantitative PCR also specifies direction for detecting various bacteria simultaneously in round pcr development.This method efficiency is high, and accuracy is high, and flux is high, but increases bacterium process before generally all needing one.Current front enrichment medium is existing a lot, and Salmonellas, Shigellae and streptococcus aureus all have enrichment medium before respective selectivity, growth that can be special.The general enrichment medium such as nutrient broth can increase various bacteria simultaneously, but lack inhibitor and go select target bacterium, do not conform to the starting material and undressed sample that are suitable for high background microorganism, therefore develop and a kind ofly can carry out increasing to Salmonellas, Shigellae and streptococcus aureus necessity that bacterium simultaneously can suppress the substratum of other non-targeted bacterium to become current simultaneously.
Summary of the invention
The present invention aims to provide can support Salmonellas simultaneously, grow while Shigellae and streptococcus aureus three kinds of food origin diseases, and suppress the substratum of other non-targeted bacterium, realize to increase bacterium step before in common detection methods and selective enrichment step unites two into one, for detecting multiple pathogenic bacterium in a detection platform.
The object of the invention is achieved through the following technical solutions:
The substratum of bacterium is increased for Salmonellas, Shigellae and streptococcus aureus compound, in mass fraction, its composition of raw materials consists of: Tryptones 13-16 part, soy peptone 4-7 part, SODIUM PHOSPHATE, MONOBASIC 2-3 part, glucose 2-3 part, distilled water 1000 parts, bovine bile 0.07-0.13 part, sodium-chlor 25-40 part, lithium chloride 0.4-1 part, N.F,USP MANNITOL 1.5-3.5 part and potassium tellurite 0.0002-0.0004 part, pH value is 7.1-7.3.
For realizing the object of the invention further, in mass fraction, described substratum formula optimization be Tryptones 15 parts, soy peptone 5 parts, SODIUM PHOSPHATE, MONOBASIC 2.5 parts, glucose 2.5 parts, distilled water 1000 parts, 35.0 parts, sodium-chlor, lithium chloride 0.5 part, bovine bile 0.1 part, 2 parts, N.F,USP MANNITOL, potassium tellurite 0.0003 part, pH value 7.2.
Increase the preparation method of the substratum of bacterium for Salmonellas, Shigellae and streptococcus aureus compound, comprise the steps:
(1) according to composition of raw materials, Tryptones, peptone, glucose, potassium primary phosphate, N.F,USP MANNITOL, lithium chloride, sodium-chlor, bovine bile are joined in distilled water, be heated to dissolve, after being cooled to room temperature, carrying out acidity with the NaOH of 1mol/L and correct pH value to 7.1-7.3;
(2) mix, 121-125 DEG C of sterilizing 15-18min;
(3) by composition of raw materials, take potassium tellurite and join in sterilized distilled water, obtain potassium tellurite solution; The total amount controlling distilled water is 1000 mass parts;
(4) the potassium tellurite solution of preparation in step 3 aseptically, is added; Packing is for subsequent use at being stored in 4-6 DEG C.
The distilled water of described step (1) is preferably 985-990 part.
The sterilized distilled water of described step (3) is preferably 10-15 part.
The present invention by selecting suitable component, and carries out rational mixture, obtains a kind of substratum making Salmonellas, Listeria monocytogenes and streptococcus aureus compound increase bacterium through steps such as heating, dissolving, mixing, high-temperature sterilizations.Substratum of the present invention contains inhibitor lithium chloride, bovine bile and potassium tellurite.Wherein lithium chloride Salmonellas and streptococcus aureus significantly do not have restraining effect, have certain restraining effect to Shigellae, but can suppress the food-borne pathogens such as Pseudomonas aeruginosa, small intestine Yersinia.Bovine bile can suppress most gram-positive microorganism, but during low concentration, streptococcus aureus also can grow.Salmonellas, Shigellae and streptococcus aureus all have Tellurite resistance, and being added with the substratum forcing down hydrochlorate can as the Selective agar medium of Shigellae and streptococcus aureus.Potassium tellurite makes it become one of composition of enrichment medium altogether for this reason to O157, common enterobacteria, vibrio cholerae and the restraining effect of diphtheria corynebacterium.Shigellae and Salmonellas have certain tolerance effect to sodium-chlor, and streptococcus aureus is salt tolerant inherently, therefore select the sodium-chlor adding higher concentration.Potassium primary phosphate is for regulating pH.Tryptones, peptone, glucose, be nutrition basic in substratum and support composition.Select N.F,USP MANNITOL as promotor.Compare Salmonellas and streptococcus aureus, the promotion situation of N.F,USP MANNITOL to Shigellae is more obvious, can weaken the restraining effect of lithium chloride to Shigellae to a certain extent.After specimen inoculation, 37 DEG C of cultivations, increase bacterium after 24 hours, be scoring on three kinds of object bacteria selective medium separately, be separated and differentiate.
Relative to prior art, the present invention has the following advantages:
Basal culture medium can merge front increasing bacterium and selective enrichment two steps of pathogenic bacterium, shortens and increases the bacterium time to 18 hour; Make that three kinds of common pathogenic bacterium increase bacterium simultaneously and other pathogenic microorganism growth is subject to certain suppression;
Culture of the present invention directly can do separation and Culture and the bioassay experiments of object bacteria, also can be directly used in multiplex PCR and detect, make diagnosis report.
Embodiment
For better understanding the present invention, below in conjunction with embodiment the present invention done and describe in detail further, but the scope of protection of present invention being not limited to the scope that embodiment represents.
Embodiment 1
Take Tryptones 15g, soy peptone 5g, SODIUM PHOSPHATE, MONOBASIC 2.5g, glucose 2.5g, sodium-chlor 35g, lithium chloride 0.5g, bovine bile 0.1g, N.F,USP MANNITOL 2g, distilled water adds to 990ml, heating for dissolving, acidity rectification is carried out, adjusted to ph to 7.2 with the NaOH of 1mol/L after cool to room temperature; Mixing, 121 DEG C of sterilizing 15min; Take 0.3mg potassium tellurite to join in the sterilized distilled water of 10mL, be made into potassium tellurite solution.Aseptically, potassium tellurite solution 10mL is added; Packing is for subsequent use at being stored in 4 DEG C, obtains the substratum increasing bacterium for Salmonellas, Shigellae and streptococcus aureus compound.
Be all 10 by bacteria concentration 9the Salmonellas of cfu/mL, Shigellae and streptococcus aureus and miscellaneous bacteria (specifically in ten kinds of table 1) nutrient solution dilute 10 respectively 5doubly, then get the substratum increasing bacterium for Salmonellas, Shigellae and streptococcus aureus compound that 0.01mL access 10mL prepares respectively, make bacteria concentration in substratum be 10cfu/mL, cultivate 18h for 37 DEG C.Under 600nm, its OD value is measured with spectrophotometer.Take nutrient broth as blank.The results are shown in Table 1.
Table 1 object bacteria and the independent growing state in the medium of miscellaneous bacteria
0 1 2 3
Salmonellas 1.567 1.097 1.028 1.113
Shigella bogdii 1.492 0.732 0.719 0.71
Streptococcus aureus 1.505 1.135 1.15 1.176
Intestinal bacteria 1.582 0.205 0.258 0.271
Bacillus cereus 1.493 × × ×
Small intestine Yersinia 1.402 × × ×
Bacillus proteus 1.591 0.305 0.330 0.302
Shigella flexneri 1.527 × × ×
Vibrio parahemolyticus 1.534 0.401 0.384 0.379
Pseudomonas aeruginosa 1.231 × × ×
In table, × represent that thalline does not grow, No. 0 is blank, and No. 1-3 is three revision tests, and between guarantee three of trying one's best repeats, deviation is below 0.2%.The object repeated is the error result in order to avoid causing due to operation reason or test materials reason.As shown in Table 1, Salmonellas, Shigellae and streptococcus aureus three kinds of object bacteria and miscellaneous bacteria are linked into separately in compound enrichment liquid, increased the nutrient broth of bacterium compound enrichment liquids to the enriching effect of object bacteria and non-selectivity through 24 hours suitable, and miscellaneous bacteria in compound enrichment liquid do not grow or grow suppressed.
Embodiment 2
Take Tryptones 15g, soy peptone 5g, SODIUM PHOSPHATE, MONOBASIC 2.5g, glucose 2.5g, sodium-chlor 30g, lithium chloride 1g, bovine bile 0.07g, N.F,USP MANNITOL 2.5g, distilled water adds to 990ml, heating for dissolving, acidity rectification is carried out, adjusted to ph to 7.2 with the NaOH of 1mol/L after cool to room temperature; Mixing, 121 DEG C of sterilizing 15min; Take 0.25mg potassium tellurite to join in the sterilized distilled water of 10mL, be made into potassium tellurite solution.Aseptically, potassium tellurite solution 10mL is added; Packing is for subsequent use at being stored in 4 DEG C, obtains the substratum increasing bacterium for Salmonellas, Shigellae and streptococcus aureus compound.
Be all 10 by bacteria concentration 9the Salmonellas of cfu/mL, Shigellae and streptococcus aureus and miscellaneous bacteria (specifically in ten kinds of table 2) nutrient solution dilute 10 respectively 5doubly, then get the substratum increasing bacterium for Salmonellas, Shigellae and streptococcus aureus compound that 0.01mL access 10mL prepares respectively, make bacteria concentration in substratum be 10cfu/mL, cultivate 18h for 37 DEG C.Under 600nm, its OD value is measured with spectrophotometer.Take nutrient broth as blank.The results are shown in Table 2.
Table 2 object bacteria and the independent growing state in the medium of miscellaneous bacteria
0 1 2 3
Salmonellas 1.567 0.951 0.96 0.947
Shigella bogdii 1.492 0.423 0.404 0.41
Streptococcus aureus 1.505 1.175 1.183 1.18
Intestinal bacteria 1.582 0.212 0.235 0.24
Bacillus cereus 1.493 × × ×
Small intestine Yersinia 1.402 × × ×
Bacillus proteus 1.591 0.254 0.23 0.227
Shigella flexneri 1.527 × × ×
Vibrio parahemolyticus 1.534 0.31 0.293 0.29
Pseudomonas aeruginosa 1.231 × × ×
In table, × represent that thalline does not grow, No. 0 is blank, and No. 1-3 is three repetitions.As shown in Table 2, Salmonellas, Shigellae and streptococcus aureus three kinds of object bacteria and miscellaneous bacteria are linked into separately in compound enrichment liquid, increased the nutrient broth of bacterium compound enrichment liquids to the enriching effect of object bacteria and non-selectivity through 24 hours suitable, and miscellaneous bacteria in compound enrichment liquid do not grow or grow suppressed.
Embodiment 3
Take Tryptones 15g, soy peptone 5g, SODIUM PHOSPHATE, MONOBASIC 2.5g, glucose 2.5g, sodium-chlor 25g, lithium chloride 0.8g, bovine bile 0.1g, N.F,USP MANNITOL 3g, distilled water adds to 990ml, heating for dissolving, acidity rectification is carried out, adjusted to ph to 7.2 with the NaOH of 1mol/L after cool to room temperature; Mixing, 121 DEG C of sterilizing 15min; Take 0.35mg potassium tellurite to join in the sterilized distilled water of 10mL, be made into potassium tellurite solution.Aseptically, potassium tellurite solution 10mL is added; Packing is for subsequent use at being stored in 4 DEG C, obtains the substratum increasing bacterium for Salmonellas, Shigellae and streptococcus aureus compound.
Be all 10 by bacteria concentration 9the Salmonellas of cfu/mL, Shigellae and streptococcus aureus and miscellaneous bacteria (specifically in ten kinds of table 3) nutrient solution dilute 10 respectively 5doubly, then get the substratum increasing bacterium for Salmonellas, Shigellae and streptococcus aureus compound that 0.01mL access 10mL prepares respectively, make bacteria concentration in substratum be 10cfu/mL, cultivate 18h for 37 DEG C.Under 600nm, its OD value is measured with spectrophotometer.Take nutrient broth as blank.The results are shown in Table 3.
Table 3 object bacteria and the independent growing state in the medium of miscellaneous bacteria
0 1 2 3
Salmonellas 1.567 1.046 1.056 1.042
Shigella bogdii 1.492 0.805 0.791 0.781
Streptococcus aureus 1.505 1.192 1.208 1.235
Intestinal bacteria 1.582 0.226 0.284 0.298
Bacillus cereus 1.493 × × ×
Small intestine Yersinia 1.402 × × ×
Bacillus proteus 1.591 0.279 0.253 0.250
Shigella flexneri 1.527 × × ×
Vibrio parahemolyticus 1.534 0.326 0.308 0.3045
Pseudomonas aeruginosa 1.231 × × ×
In table, × represent that thalline does not grow, No. 0 is blank, and No. 1-3 is three repetitions.As shown in Table 3, Salmonellas, Shigellae and streptococcus aureus three kinds of object bacteria and miscellaneous bacteria are linked into separately in compound enrichment liquid, increased the nutrient broth of bacterium compound enrichment liquids to the enriching effect of object bacteria and non-selectivity through 24 hours suitable, and miscellaneous bacteria in compound enrichment liquid do not grow or grow suppressed.
Embodiment 4
Substratum is prepared, in Salmonellas for embodiment 1: Shigellae: the ratio of streptococcus aureus is in 1: 1: 1 this substratum of access, cultivates 18 hours for 37 DEG C, by bacterium liquid dilution 10 6doubly, be then applied to salmonella color culture medium flat board respectively, on the dull and stereotyped and B-P of Shigellae color developing culture medium is dull and stereotyped, carry out separation and Culture.Salmonellas is purple bacterium colony on salmonella color culture medium, and the yellowish micro-translucent color of Shigellae, streptococcus aureus does not grow.Shigellae is colourless bacterium colony on Shigellae color developing culture medium, and salmonella is black colonies, and streptococcus aureus does not grow.On B-P flat board, S. aureus L-forms is black colonies, and Shigellae, Salmonellas do not grow.Growth result is in table 4.
Table 4 compound enrichment medium enriching effect
From this table, increased bacterium through 18 hours, thalline can increase to 10 7to 10 8, the bacteria concentration requirement of late detection can be met.

Claims (4)

1. the substratum of bacterium is increased for Salmonellas, Shigellae and streptococcus aureus compound, it is characterized in that, in mass fraction, its composition of raw materials consists of: Tryptones 13 ?16 parts, soy peptone 4 ?7 parts, SODIUM PHOSPHATE, MONOBASIC 2 ?3 parts, glucose 2 ?3 parts, distilled water 1000 parts, bovine bile 0.07 ?0.13 part, sodium-chlor 25 ?40 parts, lithium chloride 0.4 ?1 part, N.F,USP MANNITOL 1.5 ?3.5 parts and potassium tellurite 0.0002 ?0.0004 part, pH value be 7.1 ?7.3;
During preparation, comprise the steps:
(1) according to composition of raw materials, Tryptones, peptone, glucose, potassium primary phosphate, N.F,USP MANNITOL, lithium chloride, sodium-chlor, bovine bile are joined in distilled water, be heated to dissolve, after being cooled to room temperature, with the NaOH of 1mol/L carry out acidity correct pH value to 7.1 ?7.3;
(2) mix, 121 ?125 DEG C of sterilizings 15 ?18min;
(3) by composition of raw materials, take potassium tellurite and join in sterilized distilled water, obtain potassium tellurite solution; The total amount controlling distilled water is 1000 mass parts;
(4) the potassium tellurite solution of preparation in step 3 aseptically, is added; Packing be stored in 4 ?for subsequent use at 6 DEG C.
2. substratum according to claim 1, it is characterized in that: in mass fraction, the formula of described substratum is Tryptones 15 parts, soy peptone 5 parts, SODIUM PHOSPHATE, MONOBASIC 2.5 parts, glucose 2.5 parts, distilled water 1000 parts, 35.0 parts, sodium-chlor, lithium chloride 0.5 part, bovine bile 0.1 part, 2 parts, N.F,USP MANNITOL, potassium tellurite 0.0003 part, pH value 7.2.
3. the substratum increasing bacterium for Salmonellas, Shigellae and streptococcus aureus compound according to claim 1, is characterized in that: the distilled water of described step (1) be 985 ?990 parts.
4. the substratum increasing bacterium for Salmonellas, Shigellae and streptococcus aureus compound according to claim 1, is characterized in that: the sterilized distilled water of described step (3) be 10 ?15 parts.
CN201210313462.XA 2012-08-29 2012-08-29 Culture medium for enriching salmonella, shigella and staphylococcus aureus in composite way and preparation method thereof Expired - Fee Related CN102827795B (en)

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