CN104830956A - Method for rapidly detecting Candida albicans in textile - Google Patents
Method for rapidly detecting Candida albicans in textile Download PDFInfo
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- CN104830956A CN104830956A CN201510151308.0A CN201510151308A CN104830956A CN 104830956 A CN104830956 A CN 104830956A CN 201510151308 A CN201510151308 A CN 201510151308A CN 104830956 A CN104830956 A CN 104830956A
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Abstract
The invention relates to the field of textile microbe identification, and especially relates to a method for rapidly identifying Candida albicans in textile. The method for rapidly identifying Candida albicans in textile is provided against problems existing in the present identification process of Candida albicans in the textile. The method comprises the following steps: 1, acquiring a detection sample; 2, preparing a sample solution; 3, diluting the sample solution, and filtering; 4, carrying out detection culture on Candida albicans; 5, preparing a Biotyper system standard substance solution and a matrix solution; 6, extracting bacterium colony proteins through a formic acid extraction technology; 7, carrying out MALDI BioTyper automatic identification; and 8, calculating to obtain a result. The method for identifying Candida albicans in the textile considers the area and the thickness of various samples, and the collected sample is representative, so the detection result is accurate; and the method also has the advantages of high automation, experiment error reduction, no need of other additional experiments, and detection cost saving.
Description
Technical field
The present invention relates to textiles microorganism detection field, particularly a kind of method of Candida albicans bacterium in rapid detection textiles.
Background technology
The detection of tradition Candida albicans bacterium is generally Candida albicans bacterium and cultivates, and then do Germinative test, the biochemical test checkings such as sugar fermentating test, mainly comprise the following steps:
1, Zengjing Granule: get test liquid 10mL directly or be seeded in the Sabouraud dextrose broth culture of appropriate (being no less than 100mL) after process, cultivate 48h ~ 72h;
2, separation and Culture: get above-mentioned culture streak inoculation on Sabouraud glucose agar flat board, cultivates 24h ~ 48h (extending to 72h if desired).
The bacterium colony that Candida albicans grows on Sabouraud glucose agar is creamy white accidental faint yellow, and smooth surface has dense yeasty notes, and incubation time is then bacterium colony increase slightly for a long time, darkens, quality is hardening or have wrinkle folding.If flat board is not inconsistent without the bacterium colony of colony growth or growth and above-mentioned colony morphology characteristic, sentences trial-product and do not detect Candida albicans; As the bacterium colony of grow on plates to conform to above-mentioned colony morphology characteristic or doubtful, 2 ~ 3 bacterium colonies should be selected and be seeded on Candida chromogenic medium flat board respectively, cultivate 24h ~ 48h (extending to 72h if desired); If redgreen or emerald colony growth, sentence trial-product and do not detect Candida albicans on flat board; If the bacterium colony of grow on plates is green or emerald green, picking conform to or doubtful colony inoculation on 1% tween 80 corn agar medium, cultivate 24h ~ 48h.Get culture to dye, microscopy and Germinative test;
3, Germinative test: the culture on picking 1% tween 80 corn agar medium, be inoculated in and be added with on a slide glass of bleeding clearly, covered, put in moistening plate, put 35 DEG C ~ 37 DEG C, 1h ~ 3h, put basis of microscopic observation, can see and grow short and small germ tube by spore, if above-mentioned doubtful bacterium is non-gram positive organism, microscope has no chlamydospore, pseudohypha, germ tube, sentences trial-product and does not detect Candida albicans;
4, sugar-fermenting experiment: the test tube getting 18mm bore, put into the test tube of inverted 15mm bore, after tampon sealing, sterilizing is stand-by, first glucose, sucrose, maltose is made into sugar-fermenting substratum, often get 4mL to load in small test tube and to access thalline one ring, be inverted in after shaking up in Boiling tube, whether the rear 26 DEG C of cultivations of tampon sealing, observe substratum colour-change therebetween and have gas to produce;
5, the assimilation-liquid experiment of carbon-source cpd: select glucose, sucrose, maltose, Zulkovsky starch, glycerine, citric acid; The bacterium of activation is inoculated in the aseptic nitrogenous source liquid nutrient medium of 4mL, cultivates 2d, to consume unnecessary carbon source for 26 DEG C; In 15mm bore sterile test tube, often pipe adds 4mL liquid nutrient medium; Then use aseptic straw through the hungry bacteria suspension cultivated, instill in vitro ready, often pipe five, the rear 26 DEG C of quiescent culture 2d of tampon sealing, shake up rear observations, and it is positive that turbidity shows increase person, otherwise are then negative;
Above-mentioned traditional technique in measuring Candida albicans bacterium, incubation time is long, susceptibility is low, complex steps, easily cause living contaminants, affect the detection of net result, and be and report the result qualitatively, and Candida albicans bacterium is conditioned pathogen, extensively there is occurring in nature, not strong to the resistibility of heat, be heated to 60 DEG C, can be dead after 1h, but to drying, daylight, the resistibility such as ultraviolet and chemicals is stronger, when body's immunity or general phylactic power defensive power decline or the mutual restrictive function imbalance of normal microflora, then this bacterium amount reproduction change growth forms (blastogenesis Hyphal form) and invade cell and cause disease, its infective dose is higher, generally be greater than 10
5individual fungi, so the qualitative detection of dialogue candidiasis is very little to its pathogenic directive function, quantitative detection then has more meaning.
What the sample collecting of traditional textiles was conventional has two kinds:
The first streak method, this method great advantage does not destroy sample, be applicable to sample texture thinner and intensive, the sample that bacteria content is higher, but in practice, because textiles has vesicular structure and water absorption character, and textiles has certain thickness, only the contaminated real situation of sample can not be detected completely with surface smear sampling, when running into the lower sample of bacteria content or the more abundant sample of quality, smear in process that to there will be sample collecting not thorough, the situation that sample not exclusively gathers, easily because recall rate is low, cause false negative result, thus misleading testing staff produces mistake judgement,
The second shreds 20g weighing method, this method destroys sample, be applicable to the small area textiles that bacteria content is low, but textiles kind varies in reality, size, variable thickness, if only weigh 20g as sample, under-represented, if sample water-absorbent is strong, made sample heterogeneity, more easily causes false negative result.
Summary of the invention
For problem existing in Candida albicans bacterium testing process in existing textiles, the object of the present invention is to provide the collection of Candida albicans bacterium, new detecting method in a kind of textiles, overcome the deficiency and defect that exist in the process that in existing textiles, Candida albicans bacterium detects.
Its technical scheme of dealing with problems is:
A method for Candida albicans bacterium in rapid detection textiles, comprises the following steps:
Step one: sample collection
Evenly to deploy to ensure effective monitoring and control of illegal activities 5 ~ 10 sampling points in the textiles surrounding of censorship and centre, measure with sterilizing stainless steel ruler, each sampling point presses 5cm × 5cm (25cm
2) areal extent cuts out, every 25cm
2sampling area is 1 part of sample, if test sample area is excessive or too small, sampling area can expand in proportion or reduce;
Step 2: prepared by sample liquid
Be collected 5 parts ~ 10 parts samples are put into the aseptic homogenizing bag of full filter screen filling 200mL sterile saline, 1min ~ 2min is patted with slap type homogenizer, obtain a physiological saline sample liquid, make sample liquid as stoste, if test sample absorb water in a large number and cause can not sucking-off enough sample liquid time, sterile saline amount can increase progressively by each 100mL, until energy extracting has the test sample liquid of enough 100mL ~ 120mL; In each sample liquid 30min, extracting detects complete;
Step 3: sample liquid dilutes, filters
The rear membrane filtration of sample liquid dilution step 2 prepared is for subsequent use, often opens membrane filtration 100mL sample liquid; Should the extension rate of pollution condition determination sample liquid per sample, be less than 100 bacterium colonies be advisable can produce after filtering a sterilised membrane filter, if sample contamination is serious, sample liquid stoste can be diluted 10 times, get 100mL diluent and filter;
Step 4: Candida albicans bacterium detects to be cultivated
Be 0.45 μm of micropore sterilizing filter membrane edge section with anodontia sterilizing tweezers gripping aperture, by uneven surface upwards, be placed with on sterilized filter bed, fix filter, the sample liquid after filtering is injected filter, opens filter valve, negative 0.5 × 10
5suction filtration under Pa, after sample liquid has been filtered, bleed about 5s again, shut filter valve, take off filter, with sterilizing tweezers gripping filter membrane edge section, move and be placed on rose bengal medium flat board, membrane retention bacterium is towards upper, filter membrane should be adjacent to completely with substratum, do not stay bubble between the two, then rose-bengal flat board is put into 28 DEG C ± 1 DEG C incubator and cultivate 24h ~ 48h, the bacterium colony that Candida albicans bacterium draws red substratum to raise at Meng Jia has following characteristics: the out-of-shape bacterium colony that lightpink is large, central concave, edge color is shallow irregular, bacterium colony surface drying, some bacterium colonies have mycelia, S. cervisiae draws the bacterium colony that red substratum is turned out to have following characteristics at Meng Jia: without mycelia, neat in edge, lightpink, the macrocolony of central concave, picking 5 suspicious bacterium colonies on each flat board, verify by microbial rapid detection system (MALDI BioTyper),
Step 5: the preparation of microbial rapid detection system (MALDI BioTyper) standard solution and matrix solution
Special standard substance Bacterial Test Standard (BTS) of microbial rapid detection system (MALDI BioTyper) is used to add standard solvent 50% acetonitrile+47.5% water+2.5% trifluoroacetic acid 50 μ l, use the mode of pipettor pressure-vaccum repeatedly, dissolve BTS powder (can not concuss dissolve); Room temperature places 5 minutes, repeats pressure-vaccum afterwards, the centrifugal 2min of 13000rpm, packing, and-20 DEG C of Refrigerator stores forbid the BTS that multigelation has dissolved, and each use gets a pipe;
Microbial rapid detection system (MALDI BioTyper) special matrix HCCAportioned is used to add 50% acetonitrile+47.5% water+2.5% trifluoroacetic acid 50 μ l) 250 μ l standard solvent (final concentration 10mg/mL), vibration mixing, until form settled solution;
Step 6: extract bacterium colony albumen by formic acid extraction method
In EP pipe (centrifuge tube), add 300 μ l deionized waters, get tested microorganism sample in centrifuge tube, use liquid-transfering gun pressure-vaccum or vortex oscillation fully to mix, add 900 μ l ethanol, fully mix; The centrifugal 2min of 13000rpm, removes supernatant, recentrifuge, and use liquid-transfering gun to remove remaining supernatant liquor completely, whole process should not touch precipitation; Pellet dried at room temperature 2min ~ 3min; Add 70% aqueous formic acid 1 μ l ~ 80 μ l, fully can mix according to biological sample amount and be as the criterion, fully be mixed by liquid-transfering gun pressure-vaccum or vortex oscillation, add acetonitrile pure with formic acid equal-volume, abundant mixing, the centrifugal 2min of 13000rpm, draws 1 μ l supernatant, is added drop-wise on MALDI target plate, and at room temperature dry, in 1h, cover above-mentioned sample spot with 1 μ l HCCA matrix solution, and at room temperature dry;
Step 7: microbial rapid detection system (MALDI BioTyper) is identified automatically
Target plate is put into instrument, automatic qualification result, contrast with standard diagram and determine Candida albicans bacterium;
Step 8: result calculates:
Then the result identified by step 7 carrys out direct census rose bengal medium flat board corresponds to Candida albicans bacterium feature bacterium colony, reports the result with the Candida albicans bacterium number in every square centimeter of sample, calculates by formula (1),
If sample stoste substratum grown without bacterium colony, report the test is 0CFU/cm
2; If 10 times of diluted sample liquid culture mediums grown without bacterium colony, report the test is < 10CFU/cm
2; If be detected as Candida albicans bacterium, then on Selective agar medium, colony number is less than the plate count of 100CFU, by formula (1) report calculated.
The present invention adopts the microorganism in physiological saline wash-out textiles, aperture is adopted to be 0.45 μm of microporous membrane Rapid extraction, this kind of filter membrane can filter large quantity of fluid, and bacterium contained in sample liquid is trapped on filter membrane, then filter membrane is attached on rose bengal medium, after cultivating, mould and yeast all can grow the bacterium colony with individual features on rose bengal medium, but yeast and mold has multiple classification from morphology and physiology, list is distinguished can't accurately judge from colonial morphology; The present invention can identify containing saccharomycetic classification in sample quickly and accurately, direct census same characteristic features colony number, calculates contained Candida albicans bacterium in every square centimeter of sample; In the present invention, in textiles, Candida albicans bacterium detection method considers size and the thickness of various sample area, the sample gathered is more representative, make detected result more accurate, more definite to the truth reflection of sample, MALDI BioTyper is utilized automatically to detect, according to detected result quantifying reporter, within two days, just result can be gone out, the present invention is increasingly automated, reduce experimental error, its result is clear and definite, unique, does not need other appended experimental, saving testing cost, is a kind of desirable Candida albicans bacterium method for quick.
Figure of description
Fig. 1: the method for the invention detects the feasibility result of Candida albicans bacterium.
Fig. 2 a:MALDI BioTyper qualification result.
Fig. 2 b:MALDI BioTyper qualification result.
Fig. 2 c:MALDI BioTyper qualification result.
Fig. 2 d:MALDI BioTyper qualification result.
Fig. 2 e:MALDI BioTyper qualification result.
Fig. 3: Meaning of Score Values (meaning of score value).
Fig. 4: Meaning of Consistency Categories (A-C) consistence.
Show in a width figure because Fig. 2 is larger and entirely existing Fig. 2 be split as Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d:, Fig. 2 e.
Embodiment
Be described in detail below in conjunction with the content of specific embodiment to invention
Embodiment one
A method for Candida albicans bacterium in rapid detection textiles, comprises the following steps:
Step one: sample collection
Evenly to deploy to ensure effective monitoring and control of illegal activities 5 ~ 10 sampling points in the silk goods surrounding of censorship and centre, measure with sterilizing stainless steel ruler, each sampling point presses 5cm × 5cm (25cm
2) areal extent cuts out, every 25cm
2sampling area is 1 part of sample, if test sample area is excessive or too small, sampling area can expand in proportion or reduce;
Step 2: prepared by sample liquid
Be collected 5 parts ~ 10 parts samples are put into the aseptic homogenizing bag of full filter screen filling 200mL sterile saline, 1min ~ 2min is patted with slap type homogenizer, obtain a physiological saline sample liquid, make sample liquid as stoste, if test sample absorb water in a large number and cause can not sucking-off enough sample liquid time, sterile saline amount can increase progressively by each 100mL, until energy extracting has the test sample liquid of enough 100mL ~ 120mL; In each sample liquid 30min, extracting detects complete;
Step 3: sample liquid dilutes, filters
The rear membrane filtration of sample liquid dilution step 2 prepared is for subsequent use, often opens membrane filtration 100mL sample liquid; Should the extension rate of pollution condition determination sample liquid per sample, be less than 100 bacterium colonies be advisable can produce after filtering a sterilised membrane filter, if sample contamination is serious, sample liquid stoste can be diluted 10 times, get 100mL diluent and filter;
Step 4: Candida albicans bacterium detects to be cultivated
Be 0.45 μm of micropore sterilizing filter membrane edge section with anodontia sterilizing tweezers gripping aperture, by uneven surface upwards, be placed with on sterilized filter bed, fix filter, the sample liquid after filtering is injected filter, opens filter valve, negative 0.5 × 10
5pa suction filtration, after sample liquid has been filtered, bleed about 5s again, shut filter valve, take off filter, with sterilizing tweezers gripping filter membrane edge section, move and be placed on rose bengal medium flat board, membrane retention bacterium is towards upper, and filter membrane should be adjacent to completely with substratum, do not stay bubble between the two, then rose-bengal flat board is put into 28 DEG C ± 1 DEG C incubator and cultivate 24h ~ 48h, the bacterium colony that Candida albicans bacterium draws red substratum to raise at Meng Jia has following characteristics: the out-of-shape bacterium colony that lightpink is large, central concave, edge color is shallow irregular, and some bacterium colonies have mycelia; S. cervisiae draws red bacterium colony of turning out to have following characteristics at Meng Jia: without mycelia, neat in edge, lightpink, the macrocolony of central concave; Picking 5 suspicious bacterium colonies on each flat board, verify by microbial rapid detection system (MALDI BioTyper);
Step 5: the preparation of microbial rapid detection system (MALDI BioTyper) special standard solution and matrix solution
Special standard substance Bacterial Test Standard (BTS) of microbial rapid detection system (MALDI BioTyper) is used to add standard solvent 50% acetonitrile+47.5% water+2.5% trifluoroacetic acid 50 μ l, use the mode of pipettor pressure-vaccum repeatedly, dissolve BTS powder (can not concuss dissolve); Room temperature places 5 minutes, repeats pressure-vaccum afterwards, centrifugal 2 minutes of 13000rpm, packing, and-20 DEG C of Refrigerator stores, forbid the BTS that multigelation has dissolved, and each use gets a pipe;
Microbial rapid detection system (MALDI BioTyper) special matrix HCCAportioned is used to add 50% acetonitrile+47.5% water+2.5% trifluoroacetic acid 50 μ l) 250 μ l standard solvent (final concentration 10mg/mL), vibration mixing, until form settled solution;
Step 6: extract bacterium colony albumen by formic acid extraction method
In EP pipe (centrifuge tube), add 300 μ l deionized waters, get tested microorganism sample in centrifuge tube, use liquid-transfering gun pressure-vaccum or vortex oscillation fully to mix, add 900 μ l ethanol, fully mix; Centrifugal 2 minutes of 13000rpm, removes supernatant, recentrifuge, and use liquid-transfering gun to remove remaining supernatant liquor completely, whole process should not touch precipitation; Pellet dried at room temperature 2min ~ 3min; Add 70% aqueous formic acid 1 μ l ~ 80 μ l, fully can mix according to biological sample amount and be as the criterion, fully be mixed by liquid-transfering gun pressure-vaccum or vortex oscillation, add acetonitrile pure with formic acid equal-volume, abundant mixing, centrifugal 2 minutes of 13000rpm, draws 1 μ l supernatant, is added drop-wise on MALDI target plate, and at room temperature dry, in 1 hour, cover above-mentioned sample spot with 1 μ l HCCA matrix solution, and at room temperature dry;
Step 7: microbial rapid detection system (MALDI BioTyper) is identified automatically
Target plate is put into instrument, automatic qualification result, contrast with standard diagram and determine Candida albicans bacterium;
Step 8: result calculates:
Then the result identified by step 7 carrys out direct census rose bengal medium flat board corresponds to Candida albicans bacterium feature bacterium colony, reports the result with the Candida albicans bacterium number in every square centimeter of sample.
Calculate by formula (1),
If sample stoste substratum grown without bacterium colony, report the test is 0CFU/cm
2; If 10 times of diluted sample liquid culture mediums grown without bacterium colony, report the test is < 10CFU/cm
2; If be detected as Candida albicans bacterium, then on Selective agar medium, colony number is less than the plate count of 100CFU, by formula (1) report calculated.
Embodiment two
A method for Candida albicans bacterium in rapid detection textiles, comprises the following steps:
Step one: sample collection
Evenly to deploy to ensure effective monitoring and control of illegal activities 5 ~ 10 sampling points in the cotton goods surrounding of censorship and centre, measure with sterilizing stainless steel ruler, each sampling point presses 5cm × 5cm (25cm
2) areal extent cuts out, every 25cm
2sampling area is 1 part of sample, if test sample area is excessive or too small, sampling area can expand in proportion or reduce;
Step 2: prepared by sample liquid
Be collected 5 parts ~ 10 parts samples are put into the aseptic homogenizing bag of full filter screen filling 200mL sterile saline, 1min ~ 2min is patted with slap type homogenizer, obtain a physiological saline sample liquid, make sample liquid as stoste, if test sample absorb water in a large number and cause can not sucking-off enough sample liquid time, sterile saline amount can increase progressively by each 100mL, until energy extracting has the test sample liquid of enough 100mL ~ 120mL; In each sample liquid 30min, extracting detects complete;
Step 3: sample liquid dilutes, filters
The rear membrane filtration of sample liquid dilution step 2 prepared is for subsequent use, often opens membrane filtration 100mL sample liquid; Should the extension rate of pollution condition determination sample liquid per sample, be less than 100 bacterium colonies be advisable can produce after filtering a sterilised membrane filter, if sample contamination is serious, sample liquid stoste can be diluted 10 times, get 100mL diluent and filter;
Step 4: Candida albicans bacterium detects to be cultivated
Be 0.45 μm of micropore sterilizing filter membrane edge section with anodontia sterilizing tweezers gripping aperture, by uneven surface upwards, be placed with on sterilized filter bed, fix filter, the sample liquid after filtering is injected filter, opens filter valve, negative 0.5 × 10
5pa suction filtration, after sample liquid has been filtered, bleed about 5s again, shut filter valve, take off filter, with sterilizing tweezers gripping filter membrane edge section, move and be placed on rose bengal medium flat board, membrane retention bacterium is towards upper, and filter membrane should be adjacent to completely with substratum, do not stay bubble between the two, then rose-bengal flat board is put into 28 DEG C ± 1 DEG C incubator and cultivate 24h ~ 48h, the bacterium colony that Candida albicans bacterium draws red substratum to raise at Meng Jia has following characteristics: the out-of-shape bacterium colony that lightpink is large, central concave, edge color is shallow irregular, and some bacterium colonies have mycelia; S. cervisiae draws red bacterium colony of turning out to have following characteristics at Meng Jia: without mycelia, neat in edge, lightpink, the macrocolony of central concave; Picking 5 suspicious bacterium colonies on each flat board, verify by microbial rapid detection system (MALDI BioTyper);
Step 5: the preparation of microbial rapid detection system (MALDI BioTyper) special standard solution and matrix solution
Special standard substance Bacterial Test Standard (BTS) of microbial rapid detection system (MALDI BioTyper) is used to add standard solvent 50% acetonitrile+47.5% water+2.5% trifluoroacetic acid 50 μ l, use the mode of pipettor pressure-vaccum repeatedly, dissolve BTS powder (can not concuss dissolve); Room temperature places 5 minutes, repeats pressure-vaccum afterwards, the centrifugal 2min of 13000rpm, packing, and-20 DEG C of Refrigerator stores forbid the BTS that multigelation has dissolved, and each use gets a pipe;
Microbial rapid detection system (MALDI BioTyper) special matrix HCCAportioned is used to add 50% acetonitrile+47.5% water+2.5% trifluoroacetic acid 50 μ l) 250 μ l standard solvent (final concentration 10mg/mL), vibration mixing, until form settled solution;
Step 6: extract bacterium colony albumen by formic acid extraction method
In EP pipe (centrifuge tube), add 300 μ l deionized waters, get tested microorganism sample in centrifuge tube, use liquid-transfering gun pressure-vaccum or vortex oscillation fully to mix, add 900 μ l ethanol, fully mix; Centrifugal 2 minutes of 13000rpm, removes supernatant, recentrifuge, and use liquid-transfering gun to remove remaining supernatant liquor completely, whole process should not touch precipitation; Pellet dried at room temperature 2min ~ 3min; Add 70% aqueous formic acid 1 μ l ~ 80 μ l, fully can mix according to biological sample amount and be as the criterion, fully be mixed by liquid-transfering gun pressure-vaccum or vortex oscillation, add acetonitrile pure with formic acid equal-volume, abundant mixing, centrifugal 2 minutes of 13000rpm, draws 1 μ l supernatant, is added drop-wise on MALDI target plate, and at room temperature dry, in 1 hour, cover above-mentioned sample spot with 1 μ l HCCA matrix solution, and at room temperature dry;
Step 7: microbial rapid detection system (MALDI BioTyper) is identified automatically
Target plate is put into instrument, automatic qualification result, contrast with standard diagram and determine Candida albicans bacterium;
Step 8: result calculates:
Then the result identified by step 7 carrys out direct census rose bengal medium flat board corresponds to Candida albicans bacterium feature bacterium colony, reports the result with the Candida albicans bacterium number in every square centimeter of sample,
Calculate by formula (1),
If sample stoste substratum grown without bacterium colony, report the test is 0CFU/cm
2; If 10 times of diluted sample liquid culture mediums grown without bacterium colony, report the test is < 10CFU/cm
2; If be detected as Candida albicans bacterium, then on Selective agar medium, colony number is less than the plate count of 100CFU, by formula (1) report calculated.
Embodiment three
A method for Candida albicans bacterium in rapid detection textiles, comprises the following steps:
Step one: sample collection
Evenly to deploy to ensure effective monitoring and control of illegal activities 5 ~ 10 sampling points in the woolen knitwear surrounding of censorship and centre, measure with sterilizing stainless steel ruler, each sampling point presses 5cm × 5cm (25cm
2) areal extent cuts out, every 25cm
2sampling area is 1 part of sample, if test sample area is excessive or too small, sampling area can expand in proportion or reduce;
Step 2: prepared by sample liquid
Be collected 5 parts ~ 10 parts samples are put into the aseptic homogenizing bag of full filter screen filling 200mL sterile saline, 1min ~ 2min is patted with slap type homogenizer, obtain a physiological saline sample liquid, make sample liquid as stoste, if test sample absorb water in a large number and cause can not sucking-off enough sample liquid time, sterile saline amount can increase progressively by each 100mL, until energy extracting has the test sample liquid of enough 100mL ~ 120mL; In each sample liquid 30min, extracting detects complete;
Step 3: sample liquid dilutes, filters
The rear membrane filtration of sample liquid dilution step 2 prepared is for subsequent use, often opens membrane filtration 100mL sample liquid; Should the extension rate of pollution condition determination sample liquid per sample, be less than 100 bacterium colonies be advisable can produce after filtering a sterilised membrane filter, if sample contamination is serious, sample liquid stoste can be diluted 10 times, get 100mL diluent and filter;
Step 4: Candida albicans bacterium detects to be cultivated
Be 0.45 μm of micropore sterilizing filter membrane edge section with anodontia sterilizing tweezers gripping aperture, by uneven surface upwards, be placed with on sterilized filter bed, fix filter, the sample liquid after filtering is injected filter, opens filter valve, negative 0.5 × 10
5pa suction filtration, after sample liquid has been filtered, bleed about 5s again, shut filter valve, take off filter, with sterilizing tweezers gripping filter membrane edge section, move and be placed on rose bengal medium flat board, membrane retention bacterium is towards upper, and filter membrane should be adjacent to completely with substratum, do not stay bubble between the two, then rose-bengal flat board is put into 28 DEG C ± 1 DEG C incubator and cultivate 24h ~ 48h, the bacterium colony that Candida albicans bacterium draws red substratum to raise at Meng Jia has following characteristics: the out-of-shape bacterium colony that lightpink is large, central concave, edge color is shallow irregular, and some bacterium colonies have mycelia; S. cervisiae draws red bacterium colony of turning out to have following characteristics at Meng Jia: without mycelia, neat in edge, lightpink, the macrocolony of central concave;
Step 5: the preparation of microbial rapid detection system (MALDI BioTyper) special standard solution and matrix solution
Special standard substance Bacterial Test Standard (BTS) of microbial rapid detection system (MALDI BioTyper) is used to add standard solvent 50% acetonitrile+47.5% water+2.5% trifluoroacetic acid 50 μ l, use the mode of pipettor pressure-vaccum repeatedly, dissolve BTS powder (can not concuss dissolve); Room temperature places 5 minutes, repeats pressure-vaccum afterwards, centrifugal 2 minutes of 13000rpm, packing, and-20 DEG C of Refrigerator stores, forbid the BTS that multigelation has dissolved, and each use gets a pipe;
Microbial rapid detection system (MALDI BioTyper) special matrix HCCAportioned is used to add 50% acetonitrile+47.5% water+2.5% trifluoroacetic acid 50 μ l) 250 μ l standard solvent (final concentration 10mg/mL), vibration mixing, until form settled solution;
Step 6: extract bacterium colony albumen by formic acid extraction method
In EP pipe (centrifuge tube), add 300 μ l deionized waters, get tested microorganism sample in centrifuge tube, use liquid-transfering gun pressure-vaccum or vortex oscillation fully to mix, add 900 μ l ethanol, fully mix; Centrifugal 2 minutes of 13000rpm, removes supernatant, recentrifuge, and use liquid-transfering gun to remove remaining supernatant liquor completely, whole process should not touch precipitation; Pellet dried at room temperature 2 ~ 3 minutes; Add 70% aqueous formic acid 1-80 μ l, fully can mix according to biological sample amount and be as the criterion, fully be mixed by liquid-transfering gun pressure-vaccum or vortex oscillation, add acetonitrile pure with formic acid equal-volume, abundant mixing, centrifugal 2 minutes of 13000rpm, draws 1 μ l supernatant, is added drop-wise on MALDI target plate, and at room temperature dry, in 1 hour, cover above-mentioned sample spot with 1 μ l HCCA matrix solution, and at room temperature dry;
Step 7: microbial rapid detection system (MALDI BioTyper) is identified automatically
Target plate is put into instrument, automatic qualification result, contrast with standard diagram and determine Candida albicans bacterium;
Step 8: result calculates:
Then the result identified by step 7 carrys out direct census rose bengal medium flat board corresponds to Candida albicans bacterium feature bacterium colony, reports the result with the Candida albicans bacterium number in every square centimeter of sample,
Calculate by formula (1),
If sample stoste substratum grown without bacterium colony, report the test is 0CFU/cm
2; If 10 times of diluted sample liquid culture mediums grown without bacterium colony, report the test is < 10CFU/cm
2; If be detected as Candida albicans bacterium, then on Selective agar medium, colony number is less than the plate count of 100CFU, by formula (1) report calculated.
Result test experience
10 are made after cultivating with reference culture
-6bacteria suspension, every 1mL is 95CFU containing bacterium number, then with this bacteria suspension artificial contamination 10 samples, place detection in 6 hours, rose bengal medium grows suspicious bacterium colony, the several suspicious bacterium colony of picking utilizes MALDI BioTyper instrument to verify, counts according to the result.
The results are shown in Figure 1, Fig. 2, Fig. 3, Fig. 4
Being 89.6CFU with the bacterium colony mean number of the inventive method gained, be mean number 95CFU relative result is 94.3% relative to contrast bacterium colony.Coincidence rate is more than 70%, and therefore rebuilding method result is very accurate, and result that this experiment goes out is all parallel result, show that this rebuilding method is applicable to Candida albicans bacterium in textiles and detects, and method is very stable.
Claims (1)
1. the method for Candida albicans bacterium in rapid detection textiles, comprises the following steps:
Step one: sample collection
Evenly to deploy to ensure effective monitoring and control of illegal activities 5 ~ 10 sampling points in the textiles surrounding of censorship and centre, measure with sterilizing stainless steel ruler, according to textiles size, every part textiles gathers 5 parts ~ 10 parts samples altogether; Sampling area by the textiles area expanded in size of censorship or can reduce;
Step 2: prepared by sample liquid
Be collected 5 parts ~ 10 parts samples are put into the aseptic homogenizing bag of full filter screen filling 200mL sterile saline, 1min ~ 2min is patted with slap type homogenizer, obtain a physiological saline sample liquid, make sample liquid as stoste, if test sample absorb water in a large number and cause can not sucking-off enough sample liquid time, sterile saline amount can increase progressively by each 100mL, until energy extracting has the test sample liquid of enough 100mL ~ 120mL; In each sample liquid 30min, extracting detects complete;
Step 3: sample liquid dilutes, filters
The rear membrane filtration of sample liquid dilution step 2 prepared is for subsequent use, often open membrane filtration 100mL sample liquid, should the extension rate of pollution condition determination sample liquid per sample, be less than 100 bacterium colonies be advisable can produce after filtering a sterilised membrane filter, if sample contamination is serious, sample liquid stoste can be increased progressively dilution by 10 times, get 100mL diluent and filter;
Step 4: Candida albicans bacterium detects to be cultivated
Be 0.45 μm of micropore sterilizing filter membrane edge section with anodontia sterilizing tweezers gripping aperture, by uneven surface upwards, be placed with on sterilized filter bed, fix filter, the sample liquid after filtering is injected filter, opens filter valve, negative 0.5 × 10
5suction filtration under Pa, after sample liquid has been filtered, bleed about 5s again, shut filter valve, take off filter, with sterilizing tweezers gripping filter membrane edge section, move and be placed on rose bengal medium flat board, membrane retention bacterium is towards upper, filter membrane should be adjacent to completely with substratum, do not stay bubble between the two, then rose-bengal flat board is put into 28 DEG C ± 1 DEG C incubator and cultivate 24h ~ 48h, the bacterium colony that Candida albicans bacterium draws red substratum to raise at Meng Jia has following characteristics, the out-of-shape bacterium colony that lightpink is large, central concave, edge color is shallow irregular, surface drying, some bacterium colonies have obvious mycelia, the bacterium colony that S. cervisiae draws red culture medium culturing to go out at Meng Jia has following characteristics: without mycelia, neat in edge, lightpink, the macrocolony of central concave, picking 5 suspicious bacterium colonies on each flat board, verify by microbial rapid detection system (MALDI BioTyper),
Step 5: the preparation of microbial rapid detection system (MALDI BioTyper) special standard solution and matrix solution
The special standard substance Bacterial TestStandard (BTS) of microbial rapid detection system (MALDI BioTyper) is used to add standard solvent 50% acetonitrile+47.5% water+2.5% trifluoroacetic acid 50 μ l, use the mode of pipettor pressure-vaccum repeatedly, dissolve BTS powder (can not concuss dissolve); Room temperature places 5min, repeats pressure-vaccum afterwards, the centrifugal 2min of 13000rpm, packing ,-20 DEG C of Refrigerator stores, forbids the BTS that multigelation has dissolved, and each use gets a pipe;
Microbial rapid detection system (MALDI BioTyper) special matrix HCCAportioned is used to add 50% acetonitrile+47.5% water+2.5% trifluoroacetic acid 50 μ l) 250 μ l standard solvent (final concentration 10mg/mL), vibration mixing, until form settled solution;
Step 6: extract bacterium colony albumen by formic acid extraction method
In EP pipe, add 300 μ l deionized waters, get tested microorganism sample in centrifuge tube, use liquid-transfering gun pressure-vaccum or vortex oscillation fully to mix, add 900 μ l ethanol, fully mix; The centrifugal 2min of 13000rpm, removes supernatant, recentrifuge, and use liquid-transfering gun to remove remaining supernatant liquor completely, whole process should not touch precipitation; Pellet dried at room temperature 2min ~ 3min; Add 70% aqueous formic acid 1 μ l ~ 80 μ l, fully can mix according to biological sample amount and be as the criterion, fully be mixed by liquid-transfering gun pressure-vaccum or vortex oscillation, add acetonitrile pure with formic acid equal-volume, abundant mixing, the centrifugal 2min of 13000rpm, draws 1 μ l supernatant, is added drop-wise on MALDI target plate, and at room temperature dry, in 1h, cover above-mentioned sample liquid point with 1 μ l HCCA matrix solution, and at room temperature dry;
Step 7: microbial rapid detection system (MALDI BioTyper) is identified automatically
Target plate is put into instrument, automatic qualification result, contrast with standard diagram and determine Candida albicans bacterium;
Step 8: result calculates:
Then the result identified by step 7 carrys out direct census rose bengal medium flat board corresponds to Candida albicans bacterium feature bacterium colony, reports the result with the Candida albicans bacterium number in every square centimeter of sample liquid,
Calculate by formula (1),
If sample stoste substratum grown without bacterium colony, report the test is 0CFU/cm
2; If 10 times of diluted sample liquid culture mediums grown without bacterium colony, report the test is < 10 CFU/cm
2; If be detected as Candida albicans bacterium, then on Selective agar medium, colony number is less than the plate count of 100 CFU, by formula (1) report calculated.
The method of Candida albicans bacterium in a kind of rapid detection textiles according to claim 1, to is characterized in that in step one that sample gathers each sampling point and cuts out by 5cm × 5cm areal extent, every 25cm
2sampling area is 1 part of sample.
The method of Candida albicans bacterium in a kind of rapid detection textiles according to claim 1, it is characterized in that in step 7 that microorganism detects instrument is automatically microbial rapid detection system (MALDIBioTyper).
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