CN101423863A - Inspection method of streptococcus in cosmetics - Google Patents
Inspection method of streptococcus in cosmetics Download PDFInfo
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- CN101423863A CN101423863A CNA2008100730102A CN200810073010A CN101423863A CN 101423863 A CN101423863 A CN 101423863A CN A2008100730102 A CNA2008100730102 A CN A2008100730102A CN 200810073010 A CN200810073010 A CN 200810073010A CN 101423863 A CN101423863 A CN 101423863A
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Abstract
The invention discloses a method for testing streptococcus in cosmetics, against that no method for testing the streptococcus in the cosmetics is reported at home or abroad. The method identifies and determines spoilage microorganisms of the streptococcus mainly existing in the cosmetics through adopting SCDLP as pre-enriched liquid, blood agar and brain heart infusion agar as an isolation medium through catalase tests and vancocin series biochemical tests. The method is significant to safety of the cosmetics.
Description
Invention field
The present invention relates to makeup microbial testing technology field.Specifically, the present invention relates to the inspection technology field of bacterium in a kind of makeup.
Background technology
Makeup use very general in daily life, and therefore detection and the assessment to its security seems particularly important.Makeup are because of existing numerous trophicity compositions such as grease, protein, starch, VITAMIN, moisture in the raw material, growing and breed the nutrient environment that provides good for microorganism.Though prevent that by adding sanitas makeup are rotten, in production and use, still can't avoid microbiological contamination.Microorganism is decomposed the nutritive ingredient metabolism in the makeup, causes makeup putrid and deteriorated, and the color of makeup and formulation are changed, and the health that brings the user causes serious harm.Therefore, some harmful microbes separation are the major issues of current cosmetics health quality control with detecting in the makeup.
At present relevant makeup Micro biological Tests standard only relates to total plate count, excrement colibacillus group, Pseudomonas aeruginosa, four projects of streptococcus aureus usually, and with regard to security, obviously there is hysteresis phenomenon in existing examination criteria.In fact, the microbe species that pollutes makeup is a lot, and bacterium, fungi and yeast etc. are arranged.The bacterium that detects from makeup the human body cause illness at present has Pseudomonas aeruginosa, streptococcus aureus, klepsiella pneumoniae, colibacillus of excrement, bacillus cereus and suis etc.; Fungi mainly contains Penicillium citrinum, bent fungi, interlinkage spore fungi etc.Wherein, suis can cause infectious diseases, is present in as suis then easily to cause phenomenons such as dermatitis, folliculitis and furuncle in the makeup, but does not have corresponding detecting method.For strengthening that constantly the security of importing and exporting makeup is detected, the method for inspection of setting up the makeup streptococcus intermedius is significant.
Summary of the invention
At the method for inspection of not setting up at present complete detection makeup streptococcus intermedius, do not see the domestic and foreign literature report yet.The makeup streptococcus intermedius causes some diseases reality perplexing people always.
The invention provides a kind of method of inspection of makeup streptococcus intermedius.
Technical scheme of the present invention: the present invention is directed to and have streptococcic possibility in the makeup, and, adopt multiple suis enrichment medium, multiple suis isolation medium that the different sorts makeup are carried out streptococcic separation according to streptococcic feature; And after adopting standard strains of streptococcus artificial contamination makeup simultaneously, multiple suis enrichment medium, the multiple suis isolation medium of selecting carried out confirmatory test.For this reason, the present invention has set up a kind of method of inspection of makeup streptococcus intermedius.
Known in the art, suis is distributed widely in the Nature, rounded or the ovate gram positive bacterium of this bacterium, no gemma, unpowered, the hydrogen peroxide feminine gender, thermo-labile, the present invention determines to set up certain culture condition by testing repeatedly, as medium component, culture temperature and time, PH etc., sets up the method for inspection system that is fit to detect the makeup streptococcus intermedius.
Common most of suis all adopts the dull and stereotyped and Ge Lunya agar plate of blood to carry out separation and Culture, need is carried out increasing early stage the suis of bacterium cultivation and all adopts enrichment liquids such as a Ke Shi enrichment liquid, pancreas peptone soybean broth (TSB), nutrient broth, serum broth.The streptococcus separating method of now having reported, be primarily aimed at a certain kind and carry out isolation identification, as streptococcus pneumoniae, swine streptococcus, thermophilus streptococcus, Hemolytic streptococcus etc., and in fact, for bacteria growing inhibiting has added certain sanitas, increased difficulty in the makeup to the makeup streptococcus intermedius.
The present invention is according to makeup streptococcus intermedius characteristic, adopt SCDLP, brain heart infusion agar and azide glucose broth as the enrichment liquid in early stage first, blood agar, brain heart infusion agar, KF agar is as isolation medium, set up detect the makeup streptococcus intermediuses before increase bacterium and separating experiment whole detection technical system.And the SCDLP that the present invention selects to contain neutralizing agent as preceding enrichment liquid as gordian technique point of the present invention.
The present invention is concrete, at the separation that the makeup streptococcus intermedius belongs to, has adopted SCDLP to make up back mutually and the isolating complete detection method of makeup streptococcus intermedius that build up as preceding enrichment liquid, blood agar, brain heart infusion agar as isolation medium.
The present invention specifically provides a kind of checking procedure of makeup streptococcus intermedius as follows:
One, increasing bacterium early stage cultivates
Get 1:10 dilute sample 10mL and join the 90mLSCDLP liquid nutrient medium, put 36 ℃ ± 1 ℃, cultivated 24 hours.
Two, separation and Culture
Picking culture from nutrient solution, streak inoculation are put 36 ℃ ± 1 ℃ and are cultivated 24h on blood agar, brain heart infusion agar flat board.Suis colony characteristics on the blood agar: be canescence, translucent or opaque, smooth surface has opalescence, the about 0.5mm-2mm of colony diameter.On the brain heart infusion agar flat board, its bacterium colony is canescence, and is translucent or opaque, and smooth surface has opalescence, the about 0.5mm-2mm of colony diameter.
Three, dyeing microscopic examination
The bacterium colony that picking is suspicious, gramstaining, microscopy are the gram-positive cocci of chain or double row.
Four, catalase experiment
The white filter paper of getting a cleaning is placed in the sterilization plate, is coated on the filter paper with the suspicious bacterium colony of transfering loop picking suis, adds one 3% superoxol then, and within the 30s, the person is positive bubble to the occur, and the bubbling person is not negative.
Five, vancomycin experiment
The suspicious colony inoculation of picking is put 36 ℃ ± 1 ℃ and is cultivated 18-24h in the brain heart infusion agar substratum that adds vancomycin (0.5 μ g/L-4.0 μ g/L).Suis all can not grow.
Six, result's report: in conjunction with above-mentioned feature and corresponding data, can clearly judge, analytic sample is after increasing the bacterium separation and Culture, suspicious colony growth is arranged, bacterium colony characteristics in conjunction with corresponding thalline, verified is the coccus of Gram-positive chain or double row, and negative, the responsive person of vancomycin of catalase experiment can report to detect suis in the test sample.
Simultaneously, the present invention is by comparing its growth curve to determine that enrichment medium is SCDLP, brain heart infusion agar and azide glucose broth, best cultivation is streptococcic to be the SCDLP substratum, and its component is: 17.0g casein peptone, 3.0g soy peptone, 5.0g sodium-chlor, 2.5g dipotassium hydrogen phosphate, 2.5g glucose, 1.0g Yelkin TTS, 7.0g tween 80,1000ml distilled water.
Five kinds of bacterium selecting for use among the present invention: first type Streptococcus hemolyticus, Streptococcus constellatus, streptococcus pyogenes, streptococcus faecium and streptococcus acidi lactici, well known, five kinds of selected bacterial classifications all are to cause putrid main suis, it is streptococcic typical bacterial classification, all open report, those skilled in the art can obtain by the public.
The present invention has quoted makeup microbial standard method of inspection general provisions parts (GB7918.1-87) in the preparation sample preparation.
By implementing the concrete technical indicator of the present invention, realize content of the present invention, can reach following beneficial effect.
By having adopted SCDLP to make up the isolating complete detection method of makeup streptococcus intermedius that the back is built up mutually as isolation medium as preceding enrichment liquid, blood agar, brain heart infusion agar, verified is the coccus of Gram-positive chain or double row, negative, the responsive person of vancomycin of catalase experiment, can judge accurately has streptococcic existence in the makeup, and science is judged as the contaminated degree of makeup.
Description of drawings:
Figure 1 shows that the growth curve of first type Streptococcus hemolyticus in brain heart infusion agar, azide glucose broth and SCDLP, wherein SCDLP-◆, brain heart infusion agar-■, azide glucose broth-▲.
Figure 2 shows that the growth curve of Streptococcus constellatus, wherein SCDLP-at brain heart infusion agar, azide glucose broth and SCDLP ◆, brain heart infusion agar-■, azide glucose broth-▲.
Figure 3 shows that the growth curve of streptococcus pyogenes in brain heart infusion, azide glucose broth and SCDLP, wherein SCDLP-◆, brain heart infusion agar-■, azide glucose broth-▲.
Figure 4 shows that the growth curve of streptococcus faecium, wherein SCDLP-at brain heart infusion, azide glucose broth and SCDLP ◆, brain heart infusion agar-■, azide glucose broth-▲.
Figure 5 shows that the growth curve of streptococcus acidi lactici, wherein SCDLP-at brain heart infusion, azide glucose broth and SCDLP ◆, brain heart infusion agar-■, azide glucose broth-▲.
Figure 6 shows that the inspection process figure of makeup streptococcus intermedius.
Embodiment
Below, for embodiment the present invention is described, still, the present invention is not limited to following embodiment.In addition, in following explanation, if no special instructions, % all refers to volume weight per-cent meter.
Equipment and material have among the present invention: 36 ℃ ± 1 ℃ constant incubator, autoclaving pot, 0 ℃～4 ℃ refrigerators, 10 *～100 * microscope, maximums are weighed and are reached 2000g, the sterilization test tube of sterilization suction pipe, 16mm * 160mm and the 18mm * 180mm of the balance of sensibility reciprocal 0.1g, 1ml (tool 0.01ml scale), 5ml (tool 0.1ml scale) and 10ml (tool 0.1ml scale), sterilization culture dish, pH meter, pH test paper, spirit lamp, sterilization scissors, homogenizer and the tweezers etc. of diameter 90mm.
Embodiment one:
For determining whether SCDLP, brain heart infusion agar and azide glucose broth can be used as enrichment liquid in streptococcic early stage, before 5 kinds of reference cultures are inserted above-mentioned three kinds in the enrichment medium, 37 ℃ of cultivations, from each enrichment liquid, drew the 1mL nutrient solution every 4 hours, behind ten times of gradient dilutions of stroke-physiological saline solution, select suitable extent of dilution to coat brain heart infusion agar, carry out enumeration, carry out 48 hours experiment continuously, by comparing its growth curve to determine best enrichment medium.
Select for use Streptococcus constellatus CMCC (B) 32116, streptococcus pyogenes CMCC (B) 32002, streptococcus faecium CMCC (B) 32223, alpha hemolytic streptococcus CMCC (B) 32205 and streptococcus acidi lactici as the standard bacterium, be made into the bacterium liquid of 0.5 Maxwell concentration respectively, with ten times of gradient dilutions of stroke-physiological saline solution, get respectively in 1ml to SCDLP, brain heart infusion agar and the azide glucose broth, 37 ℃ of cultivations, carry out enumeration every 4 hours, carry out 48 hours countings continuously.The results are shown in accompanying drawing 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5.
By accompanying drawing 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5 as seen, except that streptococcus acidi lactici in the SCDLP nutrient solution growth phase to more weak, other streptococcus growth all better.Illustrate that three kinds of substratum all can be used as preceding enrichment liquid, but best still SCDLP substratum.
Can find out also that from accompanying drawing several reference cultures are basic climax for growth during cultivating 20～30 hours.Therefore standard determine incubation time be 24 hours be suitable.
Embodiment two:
For determining whether blood agar, brain heart infusion agar and KF agar are suitable as streptococcic isolation medium.5 kinds of reference cultures of equivalent suitable concn are inserted respectively in blood agar, brain heart infusion agar and the KF agar, cultivate counting after 24 hours for 37 ℃, and the growth characteristics of observation in three kinds of nutrient agars, growth population etc. are to determine best streptococcic isolation medium.
Select for use Streptococcus constellatus CMCC (B) 32116, streptococcus pyogenes CMCC (B) 32002, streptococcus faecium CMCC (B) 32223, alpha hemolytic streptococcus CMCC (B) 32205 and streptococcus acidi lactici as the standard bacterium, be made into the bacterium liquid of 0.5 Maxwell concentration respectively, with ten times of gradient dilutions of stroke-physiological saline solution, get 1mL and coat blood agar, brain heart infusion agar and KF agar, cultivated 24 hours and counting, add up the bacterium colony number (CFU/mL) of each reference culture on three kinds of agar for 37 ℃.The results are shown in Table 1
The growing state of table 1.5 kind of suis on blood agar, brain heart infusion agar and KF agar
By table 1 as seen, 5 kinds of reference cultures all can be grown on blood agar and brain heart infusion agar, a little less than growth on the KF nutrient agar.Blood agar and brain heart infusion agar separating resulting are more or less the same.
Embodiment three: artificial contamination's makeup disengaging latch coccus
Select for use Streptococcus constellatus CMCC (B) 32116, streptococcus pyogenes CMCC (B) 32002, streptococcus faecium CMCC (B) 32223, alpha hemolytic streptococcus CMCC (B) 32205 and streptococcus acidi lactici as the standard bacterium, be made into respectively and be adjusted to 0.5 Maxwell concentration, amount by 1% is mixed (tender skin water (liquid), smoothing toner (liquid), face cleaning milk (cream class), moisturizer (emulsion semisolid), nourishing cream (oiliness), perfume (aerosol and organic solvent)) in 6 kinds of makeup respectively, fully mixing.Samples such as the liquid of abundant mixing, semisolid are carried out specimen preparation with reference to GB7918.1-87 (makeup microbial standard method of inspection general provisions).The sample 10mL that gets the 1:10 dilution is inoculated in the 90mLSCDLP liquid nutrient medium, puts 37 ℃ of incubators, cultivates 24h.In above-mentioned enrichment culture medium, get 1～2 transfering loop, streak inoculation is put 37 ℃ of incubators and is cultivated at blood agar and brain heart infusion agar, adds up separation case behind the 24h, referring to accompanying drawing 6.The results are shown in Table 2, table 3, table 4.
Line blood agar, brain heart infusion agar separating resulting when showing 2:SCDLP as enrichment liquid
Annotate :+expression is isolated;-expression is not isolated
Table 3: brain heart infusion agar lines blood agar, brain heart infusion agar separating resulting during as enrichment liquid
Annotate :+expression is isolated;-expression is not isolated
Table 4: azide glucose broth lines blood agar, brain heart infusion agar separating resulting during as enrichment liquid
Annotate :+expression is isolated;-expression is not isolated
By table 2, table 3, table 4 as seen, 6 kinds of makeup by 5 kinds of reference cultures of artificial contamination after, increase bacterium with SCDLP, brain heart infusion agar and azide glucose broth respectively, on blood agar, brain heart infusion agar, separate.The result shows usefulness SCDLP as enrichment liquid, the isolated probability maximum of makeup streptococcus intermedius.
Embodiment four: the preparation of enrichment medium
1, SCDLP liquid nutrient medium component:
17.0g casein peptone, 3.0g soy peptone, 5.0g sodium-chlor, 2.5g dipotassium hydrogen phosphate, 2.5g glucose, 1.0g Yelkin TTS, 7.0g tween 80,1000ml distilled water
2, method for making: after the mentioned component mixing, heating for dissolving, transferring pH is 7.2～7.4 packing, 121 ℃ of autoclaving 20min.Note vibration, make the tween 80 thorough mixing that is deposited in bottom, be cooled to about 25 ℃ and use.Can replace casein peptone and soy peptone with the many peptones of Japan.
Embodiment five: the preparation of isolation medium
One, brain heart leach liquor substratum:
1, composition
10g peptone, 12.5g ox brain soak powder, 2.0g glucose, 5.0g beef heart infusion, 5.0g sodium-chlor, 2.5g Sodium phosphate dibasic, 1000ml distilled water
2, method for making: after the mentioned component mixing, heating for dissolving, transferring pH is 7.2～7.4 packing, 121 ℃ of autoclaving 20min.Be cooled to about 25 ℃ and use.
Two, azide glucose broth
1, component: 15g Tryptones, 4.5g extractum carnis powder, 7.5g glucose, 0.2g sodium azide, 7.5g sodium-chlor, 1000ml distilled water
2, method for making: after the mentioned component mixing, heating for dissolving, transferring pH is 7.2～7.4 packing, 115 ℃ of autoclaving 15min.Be cooled to about 25 ℃ and use.
Three, blood agar
1, composition: 100mL bean powder agar, 5mL-10mL defiber sheep blood (or rabbit blood)
2, method for making: dissolve agar in pH7.4～7.6 heating, be as cold as 50 ℃, add defiber sheep blood, shake up pour plate with the sterilization formality.Also but packing sterilization test tube is set to the inclined-plane.Also available other nutritious basic medium preparation blood agar.
Embodiment six:
Select Shanghai check point, Fujian check point, Kuerle check point, Ala Mountain pass check point, Urumchi, Xinjiang check point to verify.
Experimental technique: Streptococcus constellatus, streptococcus pyogenes, streptococcus faecium, alpha hemolytic streptococcus and 5 kinds of standard bacterium of streptococcus acidi lactici are made be adjusted to 0.5 Maxwell concentration respectively, amount by 1% is mixed 6 kinds of makeup respectively: tender skin water (liquid), smoothing toner (liquid), face cleaning milk (cream class), moisturizer (emulsion semisolid), nourishing cream (oiliness), perfume (aerosol and organic solvent), fully mixing.Samples such as the liquid of abundant mixing, semisolid are carried out specimen preparation with reference to GB7918.1-87 (the makeup microbial standard method of inspection).The sample 10mL that gets the 1:10 dilution is inoculated in the 90mLSCDLP liquid nutrient medium, puts 37 ℃ of incubators, cultivates 24h.In above-mentioned enrichment culture medium, get 1～2 transfering loop, streak inoculation is put 37 ℃ of incubators and is cultivated at blood agar and brain heart infusion agar, adds up separation case behind the 24h, referring to accompanying drawing 6.Checking the results are shown in Table 5～table 10 between the chamber.
The result is verified between the check point chamber in table 5 Shanghai
Line blood agar separating resulting when showing 5.a.SCDLP as enrichment liquid
Annotate :+expression is isolated;-expression is not isolated
Line brain heart infusion agar separating resulting when showing 5.b.SCDLP as enrichment liquid
Annotate :+expression is isolated;-expression is not isolated
The result is verified between the check point chamber in table 6 Fujian
Line blood agar separating resulting when showing 6.a.SCDLP as enrichment liquid
Annotate :+expression is isolated;-expression is not isolated
Line brain heart infusion agar separating resulting when showing 6.b.SCDLP as enrichment liquid
Annotate :+expression is isolated;-expression is not isolated
The result is verified between the check point chamber in Kuerle, table 7 Xinjiang
Line blood agar separating resulting when showing 7.a SCDLP as enrichment liquid
Annotate :+expression is isolated;-expression is not isolated
Line brain heart infusion agar separating resulting when showing 7.b.SCDLP as enrichment liquid
Annotate :+expression is isolated;-expression is not isolated
The result is verified between the check point chamber in Ala Mountain pass, table 8 Xinjiang
Line blood agar separating resulting when showing 8.a SCDLP as enrichment liquid
Annotate :+expression is isolated;-expression is not isolated
Line brain heart infusion agar separating resulting when showing 8.b.SCDLP as enrichment liquid
Annotate :+expression is isolated;-expression is not isolated
The result is verified between the check point chamber in Urumchi, table 9 Xinjiang
Line blood agar separating resulting when showing 9.a SCDLP as enrichment liquid:
Annotate :+expression is isolated;-expression is not isolated
Line brain heart infusion agar separating resulting when showing 9.b.SCDLP as enrichment liquid:
Annotate :+expression is isolated;-expression is not isolated
The confirmatory experiment result shows between the chamber: six kinds of makeup are behind 5 kinds of reference cultures of artificial contamination, at 5 tame breadboard test result basically identicals.SCDLP is as enrichment liquid, and suis can be cultivated and isolate to blood agar, brain heart infusion agar all as isolation medium from makeup.
Claims (4)
1, a kind of method of inspection of makeup streptococcus intermedius, cultivate by selecting substratum or nutrient solution, separation and Culture, dyeing microscopic examination, through catalase, vancomycin experiment, identify the method that obtains concrete spoilage microorganisms, it is characterized in that, adopted SCDLP to cultivate as preceding enrichment liquid, with blood agar, brain heart infusion agar as isolation medium.
2, the method for inspection of makeup streptococcus intermedius as claimed in claim 1, it is characterized in that described SCDLP is as in the preceding enrichment liquid, by volume or the weight ratio meter, get 1: 10 dilution cosmetic sample 10mL and join the 90mLSCDLP liquid nutrient medium, put 36 ℃ ± 1 ℃, cultivated 24 hours.
3, the method for inspection of makeup streptococcus intermedius as claimed in claim 1, it is characterized in that described blood agar, brain heart infusion agar be as isolation medium, picking culture from nutrient solution, streak inoculation is put 36 ℃ ± 1 ℃ and is cultivated 24h on blood agar, brain heart infusion agar flat board.
4, the method for inspection of makeup streptococcus intermedius as claimed in claim 1, it is characterized in that, in described catalase, the vancomycin experiment, the white filter paper of getting a cleaning is placed in the sterilization plate, be coated on the filter paper with the transfering loop picking colony, by W/V, add 3% superoxol, within the 30s, it is positive the bubble person to occur, and the bubbling person is not negative, and verified is the coccus of Gram-positive chain or double row, negative, the responsive person of vancomycin of catalase experiment detects the makeup streptococcus intermedius.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602717A (en) * | 2013-11-18 | 2014-02-26 | 珠海迪尔生物工程有限公司 | Chromogenic catalase reaction solution and application thereof |
| CN106399454A (en) * | 2015-07-27 | 2017-02-15 | 上海市质量监督检验技术研究院 | Detection method for Streptococcus faecalis in down and feather |
| CN111423997A (en) * | 2020-03-31 | 2020-07-17 | 广州市妇女儿童医疗中心（广州市妇幼保健院、广州市儿童医院、广州市妇婴医院、广州市妇幼保健计划生育服务中心） | Group B streptococcus enriched broth and application thereof |
| CN111662952A (en) * | 2020-07-13 | 2020-09-15 | 北京海关技术中心 | Sample for verifying qualitative detection capability of hemolytic streptococcus in mask and preparation method thereof |
| CN111705105A (en) * | 2020-07-13 | 2020-09-25 | 北京海关技术中心 | Qualitative detection capability verification sample for hemolytic streptococcus in cotton swab and preparation method thereof |
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2008
- 2008-11-27 CN CNA2008100730102A patent/CN101423863A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602717A (en) * | 2013-11-18 | 2014-02-26 | 珠海迪尔生物工程有限公司 | Chromogenic catalase reaction solution and application thereof |
| CN103602717B (en) * | 2013-11-18 | 2016-04-06 | 珠海迪尔生物工程有限公司 | A kind of colour developing catalase reaction solution and application thereof |
| CN106399454A (en) * | 2015-07-27 | 2017-02-15 | 上海市质量监督检验技术研究院 | Detection method for Streptococcus faecalis in down and feather |
| CN111423997A (en) * | 2020-03-31 | 2020-07-17 | 广州市妇女儿童医疗中心（广州市妇幼保健院、广州市儿童医院、广州市妇婴医院、广州市妇幼保健计划生育服务中心） | Group B streptococcus enriched broth and application thereof |
| CN111423997B (en) * | 2020-03-31 | 2022-02-11 | 广州市妇女儿童医疗中心（广州市妇幼保健院、广州市儿童医院、广州市妇婴医院、广州市妇幼保健计划生育服务中心） | Group B streptococcus enriched broth and application thereof |
| CN111662952A (en) * | 2020-07-13 | 2020-09-15 | 北京海关技术中心 | Sample for verifying qualitative detection capability of hemolytic streptococcus in mask and preparation method thereof |
| CN111705105A (en) * | 2020-07-13 | 2020-09-25 | 北京海关技术中心 | Qualitative detection capability verification sample for hemolytic streptococcus in cotton swab and preparation method thereof |
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