CN105092838A - Paratyphoid c and salmonella cholerae identification kit and preparation and use methods thereof - Google Patents
Paratyphoid c and salmonella cholerae identification kit and preparation and use methods thereof Download PDFInfo
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- CN105092838A CN105092838A CN201510512571.8A CN201510512571A CN105092838A CN 105092838 A CN105092838 A CN 105092838A CN 201510512571 A CN201510512571 A CN 201510512571A CN 105092838 A CN105092838 A CN 105092838A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/255—Salmonella (G)
Abstract
The invention relates to a paratyphoid c and salmonella cholerae identification kit and preparation and use methods thereof, and mainly solves the technical difficulty in quick screening and identification of paratyphoid c and salmonella cholerae in clinical infective cases and specimens such as an environment source, a food source and a livestock animal source. The paratyphoid c and salmonella cholerae identification kit comprises 1, salmonella C-group immune rabbit antiserum; 2, salmonella H-phase immune rabbit antiserum; 3, salmonella H-phase Vi immune rabbit antiserum; 4-6, dulcite, arabinose and tan sugar fermentation pipe; 7, mucic acid biochemical identification pipe; 8, potassium tartrate biochemical identification pipe; 9, sterilization mineral oil; 10, H-phase special induction soft agar; 11, induction serum H: c and H: 5 factor. The paratyphoid c and salmonella cholerae identification kit is mainly used for screening and identifying paratyphoid c and salmonella cholerae in conventional work.
Description
Technical field
The present invention relates to the qualification of salmonella paratyphi C and Salmonella choleraesuis in Salmonella, particularly paratyphoid C and Salmonella choleraesuis identification kit, method of preparation and use.
Background technology
Detection of Salmonella is important Diet_induced obesity pathogen, extensively exist in cultivated animals and environment, have nearly 2000 various serotypes, the detection of Salmonella majority infecting relevant subgenus I in it with people relies on serotype diagnosis, and wherein the antigen formula of some serotype is identical.As C group's 6,7:c:1, the serotype that 5 correspondences 5 are different: paratyphoid C, hog cholera, hog cholera hole become doffer's mutation, hog cholera must conjecture too mutation, pig typhoid fever, especially with salmonella paratyphi C 6,7:c (Vi): 1,5 and hog cholera there is very important clinical and veterinary diagnostic significance.But both have identical O and H phase antigen, be therefore difficult to rely on serological method to be differentiated." People's Republic of China's law on the prevention and control of infectious diseases " is different with public hygienics meaning to both diseases induced biohazard classifications: salmonella paratyphi C belongs to Class B pathogen, can cause typhoid fever sample case, has important clinical and public health meaning; Salmonella choleraesuis belongs to Class C infectious disease, and it is detected in animality (piggy) epidemic disease and mankind's bacteremia and deep infection case, and have no the Case report of the food borne outbreak the mankind, therefore its public hygienics meaning is relatively little.For a long time, qualification result cannot be differentiated and accurately be reported in clinical labororatory, public health and veterinary laboratory due to both similaritys on antigen, or incur loss through delay correct report time because of the induction experiment of antigen, directly affect national infection disease notification system and the accurate case of paratyphoid C is added up and the diagnosis of animality infectious disease and popular assessment.External many specialized laboratories use fluorescent PCR (RT-PCR) method to detect usually
oriCgene is to distinguish paratyphoid C and hog cholera, but this technology need rely on the instrument configuration of molecular diagnostic laboratories and reliable reference strains in contrast, and this exists application popularization obstacle concerning most of domestic laboratories.In view of this, we devise a kind of kit of simple and easy biochemical test composition to meet clinical, public health and veterinary laboratory main screening and discriminating paratyphoid C and Salmonella choleraesuis in routine work technology needs, save time, laborsaving, be easy to operation, comprehensive cost is cheap, has good Social and economic benef@.
Summary of the invention
The object of the invention is open one mainly for paratyphoid C and Salmonella choleraesuis differential diagnosis kit, method of preparation and use.Mainly solve rapid screening in the samples such as clinical infection case and environment, food source, poultry animal sources and screen paratyphoid C and Salmonella choleraesuis technical barrier.The somatotype effect that the present invention utilizes serodiagnosis to reach fast in conjunction with the combination primary dcreening operation of different biochemical reaction, simple differentiation 5 kinds has identical C group's detection of Salmonella antigen.
Technical scheme of the present invention is: salmonella paratyphi C and Salmonella choleraesuis identification kit, comprising:
1, detection of Salmonella C group immunize rabbit antiserum: constituent: O:6, O:7;
2, detection of Salmonella H phase immunize rabbit antiserum, constituent: H:c, H:5;
3, detection of Salmonella H phase Vi immunize rabbit antiserum, constituent: H:Vi;
4-6, dulcitol, arabinose, ALPHA-D-glycopyranosyl-ALPHA-D-glycopyranoside fermentation tube, composition: peptone 1.0g, sodium chloride 0.5g, 1.6%(v/v) bromcresol purple alcoholic solution 0.1ml, dulcitol, arabinose or ALPHA-D-glycopyranosyl-ALPHA-D-glycopyranoside 0.5g, distilled water 100.0ml;
7, mucic acid biochemistry differentiates pipe, composition: composition: peptone 1.0g, 0.2%(v/v) bromothymol blue solution 1.2ml, mucic acid 1.0g, distilled water 100.0ml;
8, potassium tartrate biochemistry differentiates pipe, composition: peptone 10.0g, sodium potassium tartrate tetrahydrate 10.0g, sodium chloride 5.0g, phenol red 0.024g, agar 15.0g, distilled water 1000.0ml;
9, sterilizing mineral oil, composition: mineral oil 10ml;
10, the special induced soft agar of H phase, composition: pancreas soya peptone peptone 10.0g, casein 5.0g, potassium nitrate 1.0g, agar powder 2.0g, distilled water 1000.0ml;
11, blood serum induced H:c and the H:5 factor: H:c and the H:5 factor serum packing 0.5ml/ bottle after cross agglutinin of learning from else's experience absorbs.
The invention has the beneficial effects as follows: standardization formulation kit can meet clinical, public health, environment food, farming animals, veterinary laboratories to carry out screening and the technology needs screened for salmonella paratyphi C and hog cholera in routine work, save time, laborsaving, be easy to operation, comprehensive cost is cheap, has good Social and economic benef@.
Embodiment
Salmonella paratyphi C and Salmonella choleraesuis identification kit, comprising:
1, detection of Salmonella C group immunize rabbit antiserum: constituent: O:6, O:7.Method for making: O:6 immunizing antigen bacterial strain (Newport detection of Salmonella) and O:7 immunizing antigen bacterial strain (Salmonella thompson) are inoculated ordinary nutrient agar nutrient culture media, lawn is collected after cultivating 20h through 36 ± 1 DEG C, the bacterium liquid of suitable concentration is made with physiological saline, put in 100 DEG C of water-baths and boil 2.5h, atrichia somatic antigen is made after destroying flagellar antigen, according to 1d,(50 hundred million bacterium amount) abdominal cavity hypodermic injection, 3d(,100 hundred million bacterium amount) ear vein injection, 1,0d(,200 hundred million bacterium amount) ear vein injection, 1,7d(,400 hundred million bacterium amount) ear vein injection, 2,4d(,800 hundred million bacterium amount) ear vein injection bacterial concentration and the time interval program immunity rabbit, 25d gathers rabbit blood to rabbit row heart sterile blood sampling or arteria carotis sterile blood sampling art, put 4 DEG C of separation of serum after clot solidification shrinkage.Rough o antiserum needs to absorb other nonspecific antigen.Serum absorbs removing cross agglutinin: get crude serum and do 10 times of dilutions with physiological saline and do slide agglutination test with corresponding bacterial strain (by the requirement of Products in China norm standard), as there is the cross reaction of other cross agglutinins, the absorption bacterium (absorption) that must add cross reaction relevant in serum kind removes these cross agglutinins.Method: get rough antiserum, first use the normal saline dilution of 5 times, then appropriate washed absorption bacterium (absorbing bacterium antigen method for making the same: the absorption bacterium of O:6 is the absorption bacterium Newport detection of Salmonella of salmonella kentucky, O:7) is added, put 36 ± 1 DEG C of insulation 3h(and shake up 1 time every 15min), 4 DEG C of refrigerator overnight are put in taking-up, centrifuging next day serum, by serum-dilution 10 times, and does slide agglutination test calibrating with cross agglutination bacterial strain.When once absorbing not to the utmost, can re-absorption effect, until there is not cross agglutination.General needs absorb repeatedly just can be made.Kit configuration serum packing 2ml/ bottle, working dilution is 1:10.
2, detection of Salmonella H phase immunize rabbit antiserum, constituent: H:c, H:5.Method for making: by H:c immunizing antigen bacterial strain (Salmonella choleraesuis rough type) and H:5 immunizing antigen bacterial strain (Salmonella thompson) recovery, single-phase or diphasic antigen inoculates mass percentage 0.8% soft agar medium after providing slide agglutination to test the agglutination titer reaching certain, thalline is collected after 36 ± 1 DEG C of cultivation 20h, the bacterium liquid of suitable concentration is made with physiological saline, adding mass percentage concentration is that immunity antigen is made in 0.5% formalin deactivation, according to 1d,(50 hundred million) abdominal cavity hypodermic injection, 3d(,100 hundred million) ear vein injection, 1,0d(,200 hundred million) ear vein injection, 1,7d(,400 hundred million) ear vein injection, 2,4d(,800 hundred million) ear vein injection bacterial concentration and time interval immunizing rabbit, 25d gathers rabbit blood to rabbit row heart sterile blood sampling or arteria carotis sterile blood sampling art, put 4 DEG C of separation of serum after clot solidification shrinkage.Rough anti-H factor serum needs to absorb other nonspecific antigen.Serum absorbs removing cross agglutinin: get crude serum and do 10 times of dilutions with physiological saline and do slide agglutination test with corresponding bacterial strain (by the requirement of Products in China norm standard), as there is the cross reaction of other cross agglutinins, the absorption bacterium (absorption) that must add cross reaction relevant in serum kind removes these cross agglutinins.Method: get rough antiserum, first use the normal saline dilution of 5 times, then appropriate washed absorption bacterium (absorbing bacterium antigen method for making the same: the absorption bacterium of H:c is Salmonella choleraesuis rough type, the absorption bacterium of H:5 is the single-phase mutation of Salmonella typhimurtum) is added, put 36 ± 1 DEG C of insulation 3h(and shake up 1 time every 15min), 4 DEG C of refrigerator overnight are put in taking-up, centrifuging next day serum, by serum-dilution 10 times, and does slide agglutination test calibrating with cross agglutination bacterial strain.When once absorbing not to the utmost, can re-absorption effect, until there is not cross agglutination.General needs absorb repeatedly just can be made.Kit configuration serum packing 2ml/ bottle, working dilution is 1:10.
3, detection of Salmonella H phase Vi immunize rabbit antiserum, constituent: H:Vi.Method for making: H:Vi immunizing antigen bacterial strain (Salmonella typhi 6S strain) is recovered, single-phase or diphasic antigen inoculates mass percentage 0.8% soft agar medium after providing slide agglutination to test the agglutination titer reaching certain, thalline is collected after 36 ± 1 DEG C of cultivation 20h, the bacterium liquid of suitable concentration is made with physiological saline, adding mass percentage concentration is that immunity antigen is made in 0.5% formalin deactivation, according to 1d,(50 hundred million) abdominal cavity hypodermic injection, 3d(,100 hundred million) ear vein injection, 1,0d(,200 hundred million) ear vein injection, 1,7d(,400 hundred million) ear vein injection, 2,4d(,800 hundred million) ear vein injection bacterial concentration and time interval immunizing rabbit, 25d gathers rabbit blood to rabbit row heart sterile blood sampling or arteria carotis sterile blood sampling art, put 4 DEG C of separation of serum after clot solidification shrinkage.The Vi of immunity does not need to absorb other nonspecific antigen.Kit configuration serum packing 2ml/ bottle, working dilution is 1:10.
4, dulcitol fermentation tube, composition: peptone 1.0g, sodium chloride 0.5g, 1.6%(v/v) bromcresol purple alcoholic solution 0.1ml, dulcitol 0.5g, distilled water 100.0ml.Method for making: heat soluble in water by peptone, sodium chloride, adjusts pH to 7.6, filters, continue to add dulcitol after adding bromcresol purple solution, waits packing test tube, often pipe 2.0ml after dissolving completely, for subsequent use after 115 DEG C of 15min sterilizings.
5, arabinose fermentation tube, arabinose substitutes dulcitol, and all the other are with 4.
6, ALPHA-D-glycopyranosyl-ALPHA-D-glycopyranoside fermentation tube, ALPHA-D-glycopyranosyl-ALPHA-D-glycopyranoside substitutes dulcitol, and all the other are with 4.
7, mucic acid biochemistry differentiates pipe, composition: composition: peptone 1.0g, 0.2%(v/v) bromothymol blue solution 1.2ml, mucic acid 1.0g, distilled water 100.0ml.Method for making: except mucic acid, mentioned component is mixed, in 121 DEG C of 15min sterilizings.Take out and add mucic acid while hot, slowly add NaOH and adjust pH to 7.4, packing test tube, often pipe 3.0ml, finally need sterility test to verify.
8, potassium tartrate biochemistry differentiates pipe, composition: peptone 10.0g, sodium potassium tartrate tetrahydrate 10.0g, sodium chloride 5.0g, phenol red 0.024g, agar 15.0g, distilled water 1000.0ml.Method for making: mixed by mentioned component (except phenol red), heating is dissolved, adjustment pH is 7.8, and add packing test tube after phenol red filtration, often pipe is about 10.0ml, in 115 DEG C of 20min sterilizings, uprightly solidifies rear for subsequent use.
9, sterilizing mineral oil, composition: the rearmounted 100 DEG C of baking 2h of mineral oil 10ml packing.Using method: puncture potassium tartrate developmental tube to deep layer with oese or transfer needle picking pure culture, slowly adds sterilizing paraffin oil along test tube wall and is about rearmounted 36 ± 1 DEG C of cultivation 24-48h observationss of 1ml.Positive is in yellow, and negative patient color is constant.
10, the special induced soft agar of H phase, composition: pancreas soya peptone peptone 10.0g, casein 5.0g, potassium nitrate 1.0g, agar powder 2.0g, distilled water 1000.0ml.Method for making: mixed by mentioned component, heating is dissolved, adjustment pH is 7.4, and packing 5ml/ props up, for subsequent use in 110 DEG C of 15min sterilizings.
11, blood serum induced H:c and the H:5 factor: H:c and the H:5 factor serum packing 0.5ml/ bottle after cross agglutinin of learning from else's experience absorbs.
using method
Dulcitol, arabinose, ALPHA-D-glycopyranosyl-ALPHA-D-glycopyranoside fermentation tube using method: get pure culture and be inoculated in candy ferments pipe, put 36 DEG C ± 1 DEG C and cultivate 24h-48h, nutrient culture media is yellow person by purple stain look is positive reaction, and nondiscolouring person is negative reaction, judged result after feminine gender can extend at most 7 days.
Mucic acid biochemistry differentiates pipe using method: get rearmounted 36 DEG C ± 1 DEG C of fresh cultured thing inoculation and cultivate 24h observations, nutrient culture media is yellow by blue-green variable color is positive reaction, and nondiscolouring person is negative reaction.
Potassium tartrate biochemistry differentiates pipe using method: puncture potassium tartrate developmental tube to deep layer with oese or transfer needle picking pure culture, slowly add rearmounted 36 ± 1 DEG C of sterilizing paraffin oil 1ml along test tube wall and cultivate 24-48h observations, positive is in yellow, and negative patient color is constant.
Detection of Salmonella person's (confirming that sulfuretted hydrogen is positive or negative) is confirmed as in bacterial strain to be checked qualification that is simple through biochemistry, mass spectrum etc. or system, its fresh cultured thing carries out slide agglutination with O:6, O:7 and H:c, H:5 respectively, if see, O and H phase all has obvious, coarse grained agglutinating reaction, they are 6 years old, 7:c:1, when 5 antigen formulas are set up, should get immediately 5 kinds biochemical differentiate pipes carry out inoculating or puncture rearmounted 36 ± 1 DEG C cultivate 24-48h after take out observations, if H phase only has 1 phase aggegation better, another 1 phase negative patient, need get after 1 H phase induces soft agar heating for dissolving is poured in the aseptic plane of diameter 5cm, to be cooled to about the 50 DEG C serum adding the aggegation of 1 H phase as blood serum induced, place 20min in 4 DEG C after slight mixing to take out, with or 5 methods, bacterium to be checked is inoculated in induction agar plate surface at 3, put 16-24h in 36 ± 1 DEG C to cultivate, if through the culture of single-phase inducing in spreading growth, getting edge culture and carrying out slide agglutination with expection serum, if confirm 6, 7:c:1, 5 antigen formulas, should get immediately 5 kinds biochemical differentiate pipes carry out inoculating or puncture rearmounted 36 ± 1 DEG C cultivate 24-48h after take out observations, if single-phase aggegation still cannot confirm 6,7:c:1 to the 3rd generation, the inapplicable biochemical identification mode of 5 antigen formula person.Chemical result meets table 1, and person can report corresponding detection of Salmonella serotype.
result judges:
When serotype meets 6,7:c:1,5, and Vi slide agglutination person, sulfuretted hydrogen is positive, potassium tartrate negative patient can be judged as salmonella paratyphi C;
When serotype meets 6,7:c:1,5, Vi slide not aggegation person, and sulfuretted hydrogen is negative, potassium tartrate positive can be judged as Salmonella choleraesuis;
When serotype meets 6,7:c:1,5, Vi slide not aggegation person, if sulfuretted hydrogen and potassium tartrate are the positive, otherwise then dulcitol, mucic acid, arabinose, ALPHA-D-glycopyranosyl-ALPHA-D-glycopyranoside simultaneously negative patient be judged as that if hog cholera hole becomes the judgement of doffer's mutation detection of Salmonella candy ferments total positives to be that hog cholera must be conjectured too mutation detection of Salmonella, candy ferments and has the moon to have positive person to be judged as pig Salmonella typhi.Biochemical identification result is in table 1.
The biochemistry of table 1C group 5 kinds of same antigen formula detection of Salmonella is differentiated
| 6,7:c:1,5 | Sulfuretted hydrogen | Dulcitol | Mucic acid | Arabinose | ALPHA-D-glycopyranosyl-ALPHA-D-glycopyranoside | Potassium tartrate |
| Paratyphoid C (Vi) | + | + | - | + | +or(+) | - |
| Hog cholera | - | - | - | - | - | + |
| Hog cholera hole becomes doffer's mutation | + | - | - | - | - | + |
| Hog cholera must conjecture too mutation | + | + | + | + | + | + |
| Pig typhoid fever | (+) | (+) | - | (+) | + | - |
Note: (+) most of result is positive.
Claims (3)
1. salmonella paratyphi C and Salmonella choleraesuis identification kit, is characterized in that: comprise
1), detection of Salmonella C group immunize rabbit antiserum: containing the dilute serum of detection of Salmonella O:6, O:7 factor antibody;
2), detection of Salmonella H phase immunize rabbit antiserum: the dilute serum containing detection of Salmonella H phase c and 5 factor antibody respectively;
3), detection of Salmonella H phase immunize rabbit antiserum: containing the dilute serum of detection of Salmonella Vi factor antibody;
4), dulcitol fermentation tube: composition: peptone 1.0g, sodium chloride 0.5g, 1.6% volumn concentration bromcresol purple alcoholic solution 0.1ml, dulcitol 0.5g, distilled water 100.0ml;
5), arabinose fermentation tube: composition: peptone 1.0g, sodium chloride 0.5g, 1.6% volumn concentration bromcresol purple alcoholic solution 0.1ml, arabinose 0.5g, distilled water 100.0ml;
6), ALPHA-D-glycopyranosyl-ALPHA-D-glycopyranoside fermentation tube: composition: peptone 1.0g, sodium chloride 0.5g, 1.6% volumn concentration bromcresol purple alcoholic solution 0.1ml, ALPHA-D-glycopyranosyl-ALPHA-D-glycopyranoside 0.5g, distilled water 100.0ml;
7), mucic acid biochemistry differentiates pipe: composition: composition: peptone 1.0g, 0.2% volumn concentration bromothymol blue solution 1.2ml, mucic acid 1.0g, distilled water 100.0ml;
8), potassium tartrate biochemistry differentiates pipe: composition: peptone 10.0g, sodium potassium tartrate tetrahydrate 10.0g, sodium chloride 5.0g, phenol red 0.024g, agar 15.0g, distilled water 1000.0ml;
9), sterilizing mineral oil, composition: mineral oil 10ml;
10), the special induced soft agar of H phase: composition: pancreas soya peptone peptone 10.0g, casein 5.0g, potassium nitrate 1.0g, agar powder 2.0g, distilled water 1000.0ml;
11), H phase c and 5 factors blood serum induced: containing blood serum induced H:c, H:5 factor.
2. the salmonella paratyphi C according to claims 1 and Salmonella choleraesuis identification kit, is characterized in that:
1), the method for making of dulcitol, arabinose, ALPHA-D-glycopyranosyl-ALPHA-D-glycopyranoside fermentation tube: peptone, sodium chloride are heated soluble in water, adjust pH to 7.6, filter after adding bromcresol purple solution, continue to add dulcitol, arabinose or ALPHA-D-glycopyranosyl-ALPHA-D-glycopyranoside, rear packing test tube is dissolved Deng completely, often pipe 2.0ml, for subsequent use after 115 DEG C of 15min sterilizings;
2), mucic acid biochemistry differentiates regulation law: mixed by described composition except mucic acid, in 121 DEG C of 15min sterilizings, taking-up adds mucic acid while hot, slowly adds NaOH and adjusts pH to 7.4, packing test tube, often pipe 3.0ml, for subsequent use finally by sterility test negative patient;
3), potassium tartrate biochemistry differentiates regulation law: mixed by described composition except phenol red, heating is dissolved, and adjustment pH is 7.8, and add packing test tube after phenol red filtration, often pipe is about 10.0ml, in 115 DEG C of 20min sterilizings, uprightly cools;
4), H phase special induced soft agar method for making: mixed by described composition, heating is dissolved, and adjustment pH is 7.4, and packing 5ml/ props up, for subsequent use in 110 DEG C of 15min sterilizings;
5), the blood serum induced method for making of the H phase b factor: the H:b factor after cross agglutinin of learning from else's experience absorbs, the soft agar for the preparation of single-phase inducing is dull and stereotyped.
3. the using method of salmonella paratyphi C according to claim 1 and Salmonella choleraesuis identification kit, is characterized in that: comprise the following steps
1), dulcitol, arabinose, ALPHA-D-glycopyranosyl-ALPHA-D-glycopyranoside fermentation tube using method: get pure culture and be inoculated in candy ferments pipe, put 36 DEG C ± 1 DEG C and cultivate 24h-48h, nutrient culture media is yellow person by purple stain look is positive reaction, and nondiscolouring person is negative reaction, judged result after feminine gender can extend at most 7 days;
2), mucic acid biochemistry differentiates pipe using method: get rearmounted 36 DEG C ± 1 DEG C of fresh cultured thing inoculation and cultivate 24h observations, nutrient culture media is yellow by blue-green variable color is positive reaction, and nondiscolouring person is negative reaction;
3), potassium tartrate biochemistry differentiates pipe using method: puncture potassium tartrate developmental tube to deep layer with oese or transfer needle picking pure culture, slowly add rearmounted 36 ± 1 DEG C of sterilizing paraffin oil 1ml along test tube wall and cultivate 24-48h observations, positive is in yellow, and negative patient color is constant;
4), H phase special induced soft agar using method: get culture to be checked with or 5 method inoculations at 3, put 36 DEG C ± 1 DEG C and cultivate 24h, with H phase Vi, c, 5 factor serums carry out aggegation, if c and 5 has any 1 phase not aggegation person, continuing preparation H phase induces the single-phase bacterium induction of soft agar flat board dull and stereotyped: the H phase namely adding liquefaction in the aseptic plastic plate of diameter 5cm induces soft agar, to be cooledly after less than 50 DEG C, add the H phase antiserum that 1 has occurred slide agglutination, after placing 20min in 4 DEG C after cooling after mixing gently, the Induced cultures still getting front 1 generation with or 5 methods at 3 is inoculated in the soft agar flat board of this single-phase inducing, the 2nd phase person is had not yet to see to 3 generations, single-phase bacterium can be reported as or get rid of for paratyphoid C or Salmonella choleraesuis,
5), the blood serum induced using method of the H phase b factor is with 4;
6) result judges:
When serotype meets 6,7:c:1,5, and Vi slide agglutination person, sulfuretted hydrogen is positive, potassium tartrate negative patient is judged as salmonella paratyphi C;
When serotype meets 6,7:c:1,5, Vi slide not aggegation person, and sulfuretted hydrogen is negative, potassium tartrate positive is judged as Salmonella choleraesuis;
When serotype meets 6,7:c:1,5, Vi slide not aggegation person, if sulfuretted hydrogen and potassium tartrate are the positive, otherwise then dulcitol, mucic acid, arabinose, ALPHA-D-glycopyranosyl-ALPHA-D-glycopyranoside simultaneously negative patient be judged as that if hog cholera hole becomes the judgement of doffer's mutation detection of Salmonella candy ferments total positives to be that hog cholera must be conjectured too mutation detection of Salmonella, candy ferments and has the moon to have positive person to be judged as pig Salmonella typhi.
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