CN1746289A - Microbial culture medium and use thereof - Google Patents
Microbial culture medium and use thereof Download PDFInfo
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- CN1746289A CN1746289A CN 200410074538 CN200410074538A CN1746289A CN 1746289 A CN1746289 A CN 1746289A CN 200410074538 CN200410074538 CN 200410074538 CN 200410074538 A CN200410074538 A CN 200410074538A CN 1746289 A CN1746289 A CN 1746289A
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Abstract
A microbial medium and its use are disclosed. The medium consists of pancreatic casein peptolysis 5-30 proportion, yeast extractive powder 0.5-20 proportion, grape sugar 0.5-20 proportion, sodium chloride 1-15 proportion, L-cystine 0.1-5 proportion and sulfur sodium ethylate or sulfur glycolic acid 0.1-8 proportion. It is characterized by adding agar into medium with content=50-500g/cm2. It can be used for CFU count of anaerobe or facultative anaerobe.
Description
Invention field
The present invention relates to a kind of microbiological culture media and uses thereof, particularly a kind of improved sulphur glycollate culture medium, this substratum is applicable to colony-forming unit (the Colony Forming Units of anerobe or facultative anaerobe, CFU) counting, particularly give birth to spore clostridium (being called for short clostridium sporogenes) colony-forming unit (ColonyForming Units, CFU) counting, and be used for the sterility test culture sensitivity test, belong to field of biological product.
Background technology
Microbiological culture media (abbreviation substratum) is being brought into play the irreplaceable effect of other means as the important support condition of scientific research and applied science.Except that special regulation, require the kind of sterility test all to need to carry out sterility test in medicine, biological products, dressing, suture line, aseptic utensil and the pharmacopeia.Sterility test culture is the important means of medicine, biological products steriling test.If there are quality problems sterility test culture in itself,, and, may cause very serious consequence clinically in case use contains medicine, the biological products of bacterium with the sterility test result of the tested medicine of influence, biological products.The detected medicine of good and bad direct relation of sterility test culture quality, the quality of biological products, and further be related to the security of medication.
The substratum that present China carries out aseptic detection is mainly the Pharmacopoeia of the People's Republic of China (version in 2000, be called for short " pharmacopeia ") and " Chinese biological goods rules " (version in 2000, abbreviation " rules ") in disclosed THIOGLYCOLLIC ACID salt fluid substratum I﹠amp; II, improvement Martin substratum, selective medium, nutrient broth medium, nutrient agar and improvement Martin nutrient agar.
The sterility test of the assorted bacterium of aerobism and anaerobism sulphur glycollate culture medium commonly used, disclosed sulphur glycollate culture medium all is a flow-like in the prior art, main component is:
Junket peptone (pancreatin hydrolysis) 15.0g yeast extract powder 5.0g
Glucose 5.0g sodium-chlor 2.5g
L-Gelucystine 0.5g agar 0.5～0.7g
0.1% resazurin solution 1.0ml of the new preparation of sodium thioglycollate 0.5g
(or THIOGLYCOLLIC ACID 0.3ml) (or 0.2% methylene blue solution 0.5ml of new preparation)
Water 1000ml
Think in the prior art, because above-mentioned substratum has certain viscosity, be not suitable for detecting some sample, as the thickness trial-product, in this case, remove agar and resazurin in the above-mentioned prescription usually, in the U.S., this sulphur glycollate culture medium that does not contain agar and resazurin also is called the selectivity sulphur glycollate culture medium, the domestic sulphur glycollate culture medium that does not contain agar that then is called.This substratum should under anaerobic be cultivated.
In the prior art, sterility test culture following test method is the means of Quality Control sterility test with substratum, its method mainly is disclosed in " pharmacopeia " and " rules " of version in 2000, the aerophil of regulation and anerobe sterility test culture sensitivity test method all comprise bacterium liquid in " pharmacopeia " preparation and inoculation, and the high dilution that presents growth more than 2/3 by inoculation culture parent tube number is the sensitivity of this substratum basically, in 3 tests, be criterion with the maximum sensitivity that reaches for 2 times.
Following sterility test culture sensitivity test method is disclosed in the exposure draft of 2005 editions " pharmacopeia ", the inspection method that wherein relates to aerophil, anerobe is: the fresh culture thing of a certain bacterial classification is inoculated in foster training certain hour in the tested substratum respectively, and above-mentioned culture is made every milliliter of bacteria suspension that contains bacterium 10～100 colony-forming units (cfu) with 0.9% aseptic sodium chloride solution.7 of the THIOGLYCOLLIC ACID salt fluid substratum that to get every pipe loading amount be 12ml are inoculated each 2 of bacterial classifications respectively, and every inoculum size is that 1ml (contains bacterium 10～100cfu), do not inoculate as blank for 1 in addition, cultivated 3 days, day by day observations.As if blank pipe asepsis growth, add the equal well-grown of cultivation parent tube of bacterium, then the following test of this substratum is up to specification.
The main microorganism promotes growth test method(s) that adopts in developed country such as Europe, the U.S., Britain and the geographic pharmacopeia, a small amount of (10-100CFU) bacterium of inoculation in the liquid medium within observes apparent microorganism growth whether occurs after cultivating certain hour.For aerophil and anerobe, the substratum that is adopted also is thioglycollate medium or selectivity sulphur glycollate culture medium.
Existing studies have shown that, the accuracy of sterility test culture sensitivity test method is not good enough in " rules " (waits people's " to preliminary study of sterility test culture sensitivity test method accuracy " earlier referring to nobleness, " pharmaceutical analysis magazine ", 2004,24 (1): 52-58).And in " pharmacopeia ", adopt to prepare bacteria suspension than turbid dilution method, error is bigger, is difficult to accurately add the viable count of Quality Control bacterium.The CFU of disclosed Quality Control bacterium counting is dilution method in 2005 editions " pharmacopeia ", and fict CFU counting, and the CFU counting of clostridium sporogenes lacks operability.The external CFU method of counting of also not seeing clostridium sporogenes.
In addition, substratum also is one of important means of medical diagnosis on disease.Although molecule and immunology diagnosis technology constantly develop, adopt culture of microorganism to identify that some pathogenic bacterium remains vital means.
If substratum contains certain or various bacteria when growing needed nutritive ingredient, inoculation on substratum, is cultivated under suitable temperature, and thalline can be bred by cell fission many times in 1-2 days, form the colony of a visible cell colony, be called bacterium colony.The formed bacterium colony of each bacterium all has its characteristics, the size of bacterium colony for example, surface drying or moistening, protuberance or flat, coarse or smooth, neat in edge or irregular, bacterium colony is transparent or semitransparent or opaque, and color and quality are loose or tight etc.Therefore, in the prior art, cultivate by flat board or plate and to check number of bacteria and type.
Colony-forming unit (Colony Forming Units, CFU) counting is when viable bacteria is cultivated counting, by single thalline or assemble agglomerating a plurality of thalline growth and breeding on solid medium and form macroscopic bacterium colony, be called colony-forming unit, amount of viable bacteria is called as colony-forming unit counting (CFU) in this use colony-forming unit expression sample.That is to say that when adopting the CFU counting, viable bacteria content adds up to the colony-forming unit in the unit capacity under the certain culture condition in the sample, abbreviation can be expressed as: CFU/mL, CFU/25cm
2, the CFU/ ware; Rather than absolute bacterium number in the sample.At present, the colony-forming unit counting is widely used in adopting microbiological culture media to diagnose the illness, referring to Chinese patent application 01117809.4.
So exploitation can be used in the substratum of Quality Control bacterium CFU counting in the sterility test culture sensitivity test or the substratum of medical diagnosis on disease is very urgent and has practical significance.
Summary of the invention
The object of the invention is to provide a kind of microbiological culture media, this substratum is suitable for anaerobism or facultative anaerobe, particularly give birth to spore clostridium (being called for short clostridium sporogenes) colony-forming unit (Colony Forming Units, CFU) counting, its CFU counting can be near maximum viable count, improve the accuracy of Quality Control bacterium clostridium sporogenes CFU counting in the sterility test culture sensitivity test greatly, and the accuracy of medical diagnosis on disease, good operability had.
Another object of the present invention is to provide mentioned microorganism substratum purposes in clostridium sporogenes CFU counting and the medical diagnosis on disease in the sterility test culture sensitivity test.
To achieve these goals, the technical solution used in the present invention is: a kind of microbiological culture media, it contains in the water of 1000 weight parts: pancreas casein peptone 5-30 weight part, yeast extract powder 0.5-20 weight part, glucose 0.5-20 weight part, sodium-chlor 1-15 weight part, L-Gelucystine 0.1-5 weight part, sodium thioglycollate or THIOGLYCOLLIC ACID 0.1-8 weight part, it is characterized in that also containing agar in the described microbiological culture media, the content of agar is that to make the gel-strength of described microbiological culture media be 50-500g/cm
2
The content of preferred agar is that to make the gel-strength of described microbiological culture media be 100-400g/cm
2More preferably the content of agar is that to make the gel-strength of described microbiological culture media be 140-350g/cm
2
Preferred other components contents are: pancreas casein peptone 12-18 weight part, yeast extract powder 3-8 weight part, glucose 3-8 weight part, sodium-chlor 2-4 weight part, L-Gelucystine 0.2-0.8 weight part, sodium thioglycollate or THIOGLYCOLLIC ACID 0.2-0.8 weight part.
More preferably in 1000 weight parts waters, contain pancreas casein peptone 15.0 weight parts, yeast extract powder 5.0 weight parts, glucose 5.0 weight parts, sodium-chlor 2.5 weight parts, L-Gelucystine 0.5 weight part, sodium thioglycollate or THIOGLYCOLLIC ACID 0.5 weight part.
Microbiological culture media of the present invention also comprises the clear or new 0.2% methylene blue solution 0.2---0.8 weight part of preparing in the sword sky of 0.0005-0.01 weight part.
In another embodiment of the invention, described microbiological culture media is for to contain in the water of 1000 weight parts: pancreas casein peptone 5-30 weight part, yeast extract powder 0.5-20 weight part, glucose 0.5-20 weight part, sodium-chlor 1-15 weight part, L-Gelucystine 0.1-5 weight part, sodium thioglycollate or THIOGLYCOLLIC ACID 0.1-8 weight part, it is characterized in that also containing the agar of 2-10 weight part in the described microbiological culture media.
Wherein the content of agar is preferably the 4-6 weight part; 5 weight parts more preferably.
Preferred other components contents are: in 1000 weight parts waters, and pancreas casein peptone 12-18 weight part, yeast extract powder 3-8 weight part, glucose 3-8 weight part, sodium-chlor 2-4 weight part, L-Gelucystine 0.2-0.8 weight part, sodium thioglycollate or THIOGLYCOLLIC ACID 0.2-0.8 weight part.
More preferably in 1000 weight parts waters, contain pancreas casein peptone 15.0 weight parts, yeast extract powder 5.0 weight parts, glucose 5.0 weight parts, sodium-chlor 2.5 weight parts, L-Gelucystine 0.5 weight part, sodium thioglycollate or THIOGLYCOLLIC ACID 0.5 weight part.
Also further comprise the clear or new 0.2% methylene blue solution 0.2-0.8ml for preparing in 0.001-0.003g sword sky in the above-mentioned substratum.
The preparation method of substratum of the present invention is: except that glucose, resazurin, getting mentioned component adds in the entry, be lower than 40 the degree under the dissolving after, adjusting PH is a weakly alkaline, boils, filter please, add glucose and/or resazurin, shake up, regulate PH6.5-7.5, sterilization and branch are filled in the suitable container, and the ratio of its loading amount and container height should meet cultivation end back substratum zone of oxidation (pink) and be no more than 1/2 of the substratum degree of depth.Sterilization.Before trial-product was inoculated, the color of substratum zone of oxidation must not surpass 1/3 of the substratum degree of depth, otherwise, must be through 100 ℃ of heating in water bath to pink color disappeared (being no more than 20 minutes), cooling is rapidly only limit heating once, and is prevented contaminated.
Substratum of the present invention can be used for colony-forming unit (the ColonyForming Units of anerobe or facultative anaerobe, CFU) counting, particularly give birth to spore clostridium (being called for short clostridium sporogenes) colony-forming unit (Colony Forming Units, CFU) counting, and the application on the sterility test culture sensitivity test.
Researchist of the present invention is surprised to find that in the research to the test of clostridium sporogenes CFU counting, opposite with instruction of the prior art, when the consumption of agar in the sulphur glycollate culture medium increases to the gel-strength that makes this substratum when being 50-500, under the comprehensive action of each component, substratum of the present invention is the soft solid shape and has good formability, high resilience and toughness after the moulding, the viable bacteria of anerobe or facultative anaerobe distribute and well-grown on this substratum; This makes that adopting this substratum to carry out clostridium sporogenes CFU counting becomes possibility.Discover that further adopt this substratum can make the approaching maximum viable count of CFU counting of this bacterium, colony diameter is big, form is good, be single colonies typical.Therefore, adopt substratum of the present invention to carry out clostridium sporogenes CFU counting, have higher sensitivity, accuracy and operability.
Colony-forming unit among the present invention (colony forming unit, CFU) counting is meant when viable bacteria is cultivated counting, by single thalline or assemble the agglomerating formed colony of a plurality of thalline growth and breeding on solid medium, be called colony-forming unit, express amount of viable bacteria with this.
A large amount of experimental results show that, the CFU count results of clostridium sporogenes in microbiological culture media of the present invention, obviously be better than disclosed other various substratum in the prior art, including but not limited to anaerobic agar substratum, THIOGLYCOLLIC ACID salt (1.4% agar) solid medium, nutrient agar, containing agar concentration is nutrient agar of 0.5% etc.Particularly gel-strength is 140-250g/cm
2Or agar content is the microbiological culture media of 4-8 weight part, and effect is better than disclosed any substratum in the prior art far away, and this may be owing to cross when hanging down when gel-strength, agar only plays the effect of thickening, and can not solidify, when gel-strength is too high, be unfavorable for the growth of bacterium.
Though the colony-forming unit count results of substratum of the present invention under aerobic conditions is better than other microbiological culture medias of the prior art, the result that anaerobic condition is cultivated down obviously is better than aerobic conditions.
The invention still further relates to the method for a kind of anerobe or facultative anaerobe CFU counting, may further comprise the steps:
(1) the preparation bacterial content is 1 * 10
-6The anerobe of bacterium/ml or facultative anaerobe bacterium liquid;
(2) bacterium liquid is added in the sterile petri dish, with microbiological culture media cast of the present invention,
(3) plate CFU counting is carried out in 25-38 ℃ of cultivation under the anaerobic condition in 12-72 hour.
In the step (1), the preparation method of bacterium liquid is: with bacterial classification inoculation in the sulphur glycollate culture medium that does not contain agar, through 30-35 ℃ cultivate 12-72 hour after, be placed on be equipped with stroke-physiological saline solution in vitro and be diluted to even bacterium liquid, again bacterium liquid is diluted to the concentration identical, and doing 10 times, to be diluted to bacterial content be 1 * 10 with the standard opacity tube
-6Bacterium/mL.
Diluent wherein also can adopt 0.1% peptone water.
The preparation of bacterium liquid also can be adopted disclosed method in " rules " and " pharmacopeia ".
In the step (2), get above-mentioned bacterium liquid 1mL, put in the plate of the about 90mm of diameter, inject the about 15mL of substratum of the present invention after the sterilization about room temperature to 50 ℃, mixing.
Bacterial strain can adopt casting or L rod streak method to be inoculated on the microbiological culture media of the present invention.Preferred casting.
Treat to cultivate again after the culture medium solidifying in the step (2), be inverted and cultivate.Do contrast with nonvaccinated plate simultaneously.
In the step (3), respectively at 12,14,18,24,48,72 hours some meter colony numbers.
The present invention also further relates to the inspection method of a kind of anerobe or the sensitivity of facultative anaerobe sterility test culture, may further comprise the steps:
(1) the preparation bacterial content is equivalent to the bacterium liquid of 10-100 bacterium/mL approximately;
(2) bacterium liquid is inoculated in the tested substratum of at least two pipes, cultivates;
It is characterized in that simultaneously bacterium liquid being inoculated in plate carries out enumeration, when plate colony-forming unit count results is 10-100CFU/mL, judge according to whether all presenting growth in the inoculated tube after this inoculation whether this sterility test meets the requirements with the following test of substratum.
Wherein said plate enumeration is that bacterium liquid is placed on the plate or flat board that the corresponding substratum of suitable enumeration makes, and cultivates, counting.
Further, described plate enumeration is with each 1mL of bacterium liquid, puts in the plate of the about 90mm of diameter, the about 15mL of corresponding substratum that reinjects about 45 ℃.
Substratum wherein is that gel-strength is 50-500g/cm
2Microbiological culture media.Preferred microbiological culture media of the present invention and preferred composition the thereof.
When carrying out the plate enumeration, also comprise bacterium liquid and substratum mixing, solidify and be inverted cultivation.Culture temperature is 25-38 ℃, and incubation time is 12-72 hour, and respectively at 12,14,18,24,48,72 hours some meter colony numbers.Preferably under anaerobic cultivate the 18h counting, this moment, bacterium colony was single colonies typical, amixis and diffusion, and the CFU counting is maximum, and colonial morphology is good, the most approaching maximum viable count.
Anerobe among the present invention comprises clostridium tetani, Peptostreptococcus, peptone coccus, or facultative anaerobe comprises living spore clostridium (abbreviation clostridium sporogenes, Clostridium sporogenes, CMCC 64941 strains), diphtheria corynebacterium, short corynebacteria.
In the above-mentioned sterility test culture following test method, the preparation method of bacterium liquid is: with bacterial classification inoculation in tested substratum, through 30-35 ℃ cultivate 10-48 hour after, be placed on be equipped with stroke-physiological saline solution in vitro and be diluted to even bacterium liquid, again bacterium liquid is diluted to the concentration identical, makes 10 times of serial dilutions with physiological saline then or be diluted to the bacterium liquid of 10-100CFU/mL with the standard opacity tube.The preparation of bacterium liquid also can be adopted disclosed method in " rules " and " pharmacopeia ".
During inoculation, get above-mentioned bacterium liquid 1mL usually, be inoculated in the tested substratum of 9mL.Bacterial strain can adopt casting or L rod streak method to be inoculated on the microbiological culture media of the present invention.Preferred casting.
Substratum of the present invention can be used for the CFU counting of the facultative or anerobe of clostridium sporogenes and other etc., optimum clostridium sporogenes CFU counting.
Among the present invention, in bacterium liquid inoculation culture, also will be with contrasting under the nonvaccinated tested substratum same culture conditions, and write down the result day by day.When plate carries out enumeration, should make the counting of 2 plates at least simultaneously, and carry out the nonvaccinated contrast of plate simultaneously.
Be in the plate count result under the situation of 10-100CFU/mL, all present the following test that is grown to this substratum with 2 pipes behind the tested substratum of this inoculation and meet the requirements.If 2 pipes all do not present growth after this inoculation, undesirable for the following test of this substratum, if 1 pipe presents growth after this inoculation, 1 pipe does not present growth, retry as stated above.
When adopting method of the present invention to carry out sterility test, should guarantee that the bacterial classification dispersity is good, if necessary, bacterium liquid preparation process of the present invention also comprises centrifugal and gets upper strata bacterium liquid.And with physiological saline as diluent.
The height of CFU counting and colonial morphology have reflected directly whether the suitable CFU that carries out corresponding Quality Control bacterium counts certain substratum.Adopt microbiological culture media of the present invention and in conjunction with described enumeration and sterility test culture following test method, CFU counting under cast mixing and anaerobism culture condition at most, colonial morphology is good, CFU counts the most approaching maximum viable count, make the CFU counting of clostridium sporogenes in the existing sterility test culture following test method that standard, assurance and operability arranged, guaranteed the quality of tested sterility test, and then guaranteed the quality of tested medicine or biological products with substratum.
Embodiment
Further describe the present invention below in conjunction with embodiment, but described embodiment is used to illustrate the present invention rather than restriction the present invention.
Embodiment 1
Pancreas casein peptone 15.0g, yeast extract powder 5.0g, sodium-chlor 2.5g, L-Gelucystine 0.5g, sodium thioglycollate or THIOGLYCOLLIC ACID 0.5g, glucose 8.0, agar 5g are added in the 1000ml water and mix, the tepor dissolving, adjusting pH is a weakly alkaline, boil, filtering adds glucose 5.0g, shake up, after adjusting pH value makes sterilization is 7.1 ± 0.2, sterilization, packing, and agar concentration is 0.5%.
Embodiment 2
With embodiment 1, different is, agar is 6g, and agar concentration is 0.6%.
Embodiment 3
With embodiment 1, different is, agar is 7g, and agar concentration is 0.7%.
Embodiment 4
With embodiment 1, different is to comprise that also 0.001g sword sky is clear.
Embodiment 5
With embodiment 1, different is to comprise in the substratum: pancreas casein peptone 18.0g, yeast extract powder 3.0g, glucose 8.0g, sodium-chlor 1g, L-Gelucystine 0.8g, sodium thioglycollate or THIOGLYCOLLIC ACID 0.8g, agar 5g, the clear or new 0.2% methylene blue solution 0.2-0.8ml for preparing in 0.003g sword sky, 1000ml water.
Embodiment 6
With embodiment 1, different is that substratum comprises: pancreas casein peptone, yeast extract powder g, glucose 3.0g, sodium-chlor 4g, L-Gelucystine 0.2g, sodium thioglycollate or THIOGLYCOLLIC ACID 0.4g, agar 6g, 1000ml water.
Embodiment 6-9
With reference to embodiment 1, prepare substratum according to the proportioning of table 1:
Table 1.
| Embodiment 6 | Embodiment 7 | Embodiment 8 | Embodiment 9 | |
| Pancreas casein peptone (g) | 12.0 | 5 | 30 | 25 |
| Yeast extract powder (g) | 8.0 | 20 | 16 | 0.5 |
| Glucose (g) | 3.0 | 20 | 0.5 | 15 |
| Sodium-chlor (g) | 4.0 | 8 | 10 | 15 |
| L-Gelucystine (g) | 0.2 | 2 | 3 | 5 |
| Sodium thioglycollate (g) or THIOGLYCOLLIC ACID | 0.4 | 8 | 0.1 | 4 |
| Agar (g) | 4.0 | 8 | 3 | 10 |
| 0.2% methylene blue solution (ml) of sword sky clear (g) or new preparation | 0.002 | 0 0.2 | 0 | 0 |
Experimental example 1
The gel-strength determinator that adopts Qingdao Haiyang institute of the Chinese Academy of Sciences to produce is measured gel-strength, the results are shown in Table 2.
The gel-strength of table 2. microbiological culture media CFU of the present invention
| Substratum | Gel-strength (n=10) g/cm 2 |
| x±s | |
| Embodiment 1 | 150±7.4 |
| Embodiment 2 | 230±8.7 |
| Embodiment 3 | 326±11.5 |
The result: the gel-strength of 3 substratum has significant difference (p＜0.05).
The gel-strength of other embodiment is: 50-500g/cm
2
Experimental example 2
This experimental example is to study the CFU counting and the colonial morphology of described microbiological culture media.
1. material and method
1.1 bacterial classification
Clostridium sporogenes (bacterial classification CMCC64941) is provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
1.2 substratum
Sulphur glycollate culture medium (not containing agar) is Beijing three medicine scientific and technological development company products, prepare agar concentration according to embodiment 1-3 is three microbiological culture medias of the present invention of 0.5%, 0.6% and 0.7%.Nutrient agar is produced by U.S. Difco company.Agar is produced by U.S. Difco company.
1.3 method
1.3.1 clostridium sporogenes is inoculated in sulphur glycollate culture medium (not containing agar), puts 35 ℃ and cultivate 24-48h; The fresh culture thing of clostridium sporogenes is put in the aseptic centrifuge tube the centrifugal 5min of 500rpm.Get upper strata bacterium liquid, be diluted to and standard opacity tube same concentrations, and make 10 times of serial dilutions to about 1 * 10 with 0.9% aseptic sodium chloride solution
-6Standby.
1.3.2 the preparation agar concentration is 3 three microbiological culture medias of the present invention of 0.5%, 0.6% and 0.7%, 3 microbiological culture medias of the present invention after the sterilization are chilled to about 45 ℃, it is in the sterile petri dish of 90mm that the clostridium sporogenes bacterium liquid 1mL that dilution is good adds diameter, again with about 20mL substratum cast, mixing 10s, treat culture medium solidifying after (abbreviation casting) put the following 35 ℃ of cultivations of anaerobic condition.Carry out the CFU/mL counting in 12h, 14h, 16h, 18h, 24h, 30h, 48h and 72h, and observe colonial morphology.
2 results
Referring to table 3, agar concentration is 0.5% and 0.6% microbiological culture media CFU count results comparison there was no significant difference (P＞0.05) of the present invention, but its colony diameter obvious difference (P＜0.05), be on 0.5% the microbiological culture media of the present invention at agar concentration, the colony diameter of clostridium sporogenes is bigger, and form is good; Agar concentration is that 0.5% and 0.7% CFU Counting Culture Medium for Clostridium sporogenes CFU count results relatively has significant difference (P＜0.05); Agar concentration is that 0.6% and 0.7% CFU Counting Culture Medium for Clostridium sporogenes CFU count results relatively has significant difference (P＜0.05).During 12h, bacterium colony is very little and few.During 18h, be single colonies typical, amixis and diffusion.During 24h, obvious fusion and diffusion are arranged, should not count, and do not see that newborn bacterium colony forms.After the 30h, be large stretch of and merge, can't count, so optimum CFU counts when clostridium sporogenes is cultivated 18h on agar concentration is 0.5% microbiological culture media of the present invention.
Three microbiological culture media CFU countings of the present invention of table 3 and colonial morphology (18h)
| Substratum | Test number (TN) | CFU/mL(n＝10) | Colony diameter (mm) (n=30) |
| x±s | x±s | ||
| 1 # | 1 | 150.3±13.6 | 1.2±0.27 |
| 2 | 139.3±15.4 | 1.2±0.37 | |
| 3 | 134.5±10.0 | 1.2±0.24 | |
| 2 # | 1 | 143.9±10.3 ■ | 0.5±0.12 △ |
| 2 | 134.2±10.8 ■ | 0.5±0.17 △ | |
| 3 | 126.9±7.7 ■ | 0.6±0.23 △ | |
| 3 # | 1 | 126.2±11.0 △▲ | 0.4±0.10 △ |
| 2 | 115.9±9.4 △▲ | 0.5±0.14 △ | |
| 3 | 114.0±10.6 △▲ | 0.4±0.25 △ |
Annotate: 1
#: microbiological culture media of the present invention (0.5% agar), 2
#: microbiological culture media of the present invention (0.6% agar), 3
#: microbiological culture media of the present invention (0.7% agar).
△ P＜0.05,2
#, 3
#With 1
#Relatively
■ P＞0.05,2
#With 1
#Relatively
▲ P＜0.05,3
#With 2
#Relatively
Experimental example 3
The purpose of this experimental example is to study clostridium sporogenes in the CFU of different substratum, different culture condition and time count results, to determine the purposes of microbiological culture media of the present invention in CFU counting and definite culture medium for asepsis test sensitivity.
1. material and method
1.1 bacterial classification:
Clostridium sporogenes (bacterial classification CMCC64941) is provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
1.2 substratum
The anaerobic agar substratum is (referring to " Chinese medical check pandect " in August, 97 version, the Li Yinglin chief editor, People's Health Publisher's publication), sulphur glycollate culture medium (does not contain agar, see the 32nd page of Chinese biological goods rules version in 2000), THIOGLYCOLLIC ACID salt (1.4% agar) solid medium (with above-mentioned sulphur glycollate culture medium, but agar content 1.4%), nutrient agar (referring to two appendix P89 of Pharmacopoeia of People's Republic of China version in 2000), to contain agar concentration be that 0.5% nutrient agar prepares by Beijing three medicine scientific and technological development companies.It according to embodiment 1 preparation agar concentration 0.6% microbiological culture media of the present invention.
1.3 method
1.3.1 clostridium sporogenes is inoculated in sulphur glycollate culture medium (not containing agar), puts 35 ℃ and cultivate 24-48h; The fresh culture thing of clostridium sporogenes is put in the aseptic centrifuge tube the centrifugal 5min of 500rpm.Get upper strata bacterium liquid, be diluted to and standard opacity tube same concentrations, and do 10 times of serial dilution to 1 * 10 with 0.9% aseptic sodium chloride solution
-6Standby.
1.3.2 get the good clostridium sporogenes bacterium liquid 1mL of dilution,
(1) being inoculated in substratum of the present invention respectively and containing agar concentration with casting is 0.5% nutrient agar,
(2) smear inoculation (0.25mL bacterium liquid/plate) respectively (calling L rod streak method in the following text) anaerobic agar substratum, THIOGLYCOLLIC ACID salt (1.4% agar) solid medium, nutrient agar with the L rod.With anaerobic agar substratum, substratum of the present invention, THIOGLYCOLLIC ACID salt (1.4% agar) solid medium, nutrient agar, to contain agar concentration be that 0.5% nutrient agar is put under the anaerobic condition, nutrient agar and substratum of the present invention are equipped with under the oxygen condition 35 ℃ of cultivations simultaneously.Carry out the CFU/mL counting and observe colonial morphology in 12h, 14h, 16h, 18h, 24h, 30h, 48h and 72h.Every kind of substratum while 10 parts of repeated inoculation is also established the substratum blank.Repeat 3 tests.
2. result
Clostridium sporogenes anaerobic agar substratum, substratum of the present invention, THIOGLYCOLLIC ACID salt (1.4% agar) solid medium, nutrient agar, contain agar concentration be on 0.5% the nutrient agar anaerobism cultivate and on substratum of the present invention and nutrient agar aerobic cultivate, carry out the CFU/mL counting and observe colonial morphology in 12h, 14h, 16h, 18h, 24h, 27h, 30h, 48h and 72h.
When 12h, 14h, bacterium colony is very little and few.During 16h, bacterium colony increases to some extent, but the bacterium colony number during than 18h for few, be single colonies typical during 18h, amixis and diffusion, and compare colony number with 24h and do not see increase, and during 24h and after, obvious fusion and diffusion are arranged, should not count.So 18h is best gate time, table 3 is the count results of 18h.Show: with substratum of the present invention clostridium sporogenes is carried out the CFU/mL counting, its result obviously is better than other substratum (P＜0.05) and the anaerobic condition cultivation obviously is better than aerobic conditions (P＜0.05).
Table 3 clostridium sporogenes is in the CFU/mL of 5 different substratum and different culture condition count results
| Substratum | CFU/mL(n＝10) | ||
| Test number (TN) | |||
| 1 | 2 | 3 | |
| x±s | x±s | x±s | |
| 1 | 1.4±1.6 △ | 0.4±0.8 △ | 0.0±0.0 △ |
| 2 | 5.0±1.8 △ | 6.0±5.1 △ | 3.2±2.9 △ |
| 3 | 12.4±9.7 △ | 6.8±2.6 △ | 8.2±4.8 △ |
| 4 | 6.0±2.8 △ | 7.8±4.7 △ | 12.2±5.5 △ |
| 5 | 76.5±4.9 | 97.0±4.6 | 127.2±8.4 |
| 3* | 5.4±2.0 △▲ | 2.8±1.8 △▲ | 1.8±1.9 △▲ |
| 5* | 65.8±7.1 ■ | 75.9±6.4 ■ | 36.4±7.2 ■ |
Annotate: 1: anaerobic agar substratum, 2: nutrient agar medium (Difco), 3: nutrient agar medium, 4: THIOGLYCOLLIC ACID salt solid medium, 5: substratum of the present invention.
Test according to the method described above, the clostridium sporogenes CFU/mL counting of other embodiment of the present invention (2-9) substratum and the result who also all is higher than prior art for the CFU/mL count results of other anerobes or facultative anaerobe.
* represent under the aerobic conditions and cultivate, other is all under anaerobic cultivated.
▲ P＜0.05 and 3 relatively
■ P＜0.05 and 6 relatively
The The above results explanation, substratum of the present invention can be used for colony-forming unit (the Colony Forming Units of anerobe or facultative anaerobe, CFU) counting is particularly useful for clostridium sporogenes CFU counting, and is further used for the test of sterility test culture sensitivity.
Claims (10)
1. microbiological culture media, it contains in the water of 1000 weight parts: pancreas casein peptone 5-30 weight part, yeast extract powder 0.5-20 weight part, glucose 0.5-20 weight part, sodium-chlor 1-15 weight part, L-Gelucystine 0.1-5 weight part, sodium thioglycollate 0.1-8 weight part, it is characterized in that also containing agar in the described microbiological culture media, the content of agar is that to make the gel-strength of described microbiological culture media be 50-500g/cm
2
2. substratum according to claim 1 is characterized in that, the content of preferred agar is that to make the gel-strength of described microbiological culture media be 100-400g/cm
2, more preferably gel-strength is 140-350g/cm
2
3. microbiological culture media, described microbiological culture media is for to contain in the water of 1000 weight parts: pancreas casein peptone 5-30 weight part, yeast extract powder 0.5-20 weight part, glucose 0.5-20 weight part, sodium-chlor 1-15 weight part, L-Gelucystine 0.1-5 weight part, sodium thioglycollate or THIOGLYCOLLIC ACID 0.1-8 weight part is characterized in that also containing in the described microbiological culture media agar of 2-10 weight part.
4. substratum according to claim 3 is characterized in that the content of agar is preferably the 3-7 weight part; More preferably 4-6 weight part, more preferably 5 weight parts.
5. according to any one described substratum of claim 1-4, it is characterized in that containing pancreas casein peptone 12-18 weight part, yeast extract powder 3-8 weight part, glucose 3-8 weight part, sodium-chlor 2-4 weight part, L-Gelucystine 0.2-0.8 weight part, sodium thioglycollate 0.2-0.8 weight part.
6 according to any one described substratum of claim 1-4, it is characterized in that pancreas casein peptone 15.0 weight parts, yeast extract powder 5.0 weight parts, glucose 5.0 weight parts, sodium-chlor 2.5 weight parts, L-Gelucystine 0.5 weight part, sodium thioglycollate or THIOGLYCOLLIC ACID 0.5 weight part.
7. according to any one described substratum of claim 1-4, it is characterized in that also further comprising in the described substratum the clear or new 0.2% methylene blue solution 0.2-0.8 weight part of preparing in sword sky of 0.001-0.003 weight part.
8. the preparation method of the described substratum of claim 1-7, comprise: except that glucose, resazurin, in proportion the composition of substratum is added in the entry and dissolve, adjusting PH is a weakly alkaline, boils filtering, add glucose and/or resazurin again, shake up, regulate PH6.5-7.5, sterilization and branch are filled in the suitable container.
9. preparation method according to claim 8, wherein its pH value of sterilization back is 6.9-7.4.
10. (ColonyForming Units, CFU) counting is especially in the enumerative purposes of clostridium sporogenes CFU, and the purposes on the sterility test culture sensitivity test at the colony-forming unit of anerobe or facultative anaerobe for the described substratum of claim 1-7.
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Cited By (7)
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| CN101851676A (en) * | 2010-04-21 | 2010-10-06 | 吉林农业大学 | Composite enrichment medium of salmonella, shigella and staphylococcus aureus, preparation method and application thereof |
| CN101491307B (en) * | 2009-02-20 | 2011-07-20 | 扬州绿源生物化工有限公司 | Rice leaf roller artificial feedstuff and preparation method and artificial feeding method applied on rice leaf roller |
| CN101092643B (en) * | 2006-06-23 | 2011-09-14 | 中国药品生物制品检定所 | Culture medium for asepsis test in medication of quinolone category, and application |
| CN104313110A (en) * | 2014-09-30 | 2015-01-28 | 青岛康合伟业商贸有限公司 | Fluid thioglycollate medium and application thereof |
| CN110317852A (en) * | 2019-06-04 | 2019-10-11 | 广州赛莱拉生物基因工程有限公司 | It is a kind of for detecting the culture medium and its preparation method and application of microorganism in cell product |
| CN111808787A (en) * | 2020-08-12 | 2020-10-23 | 河南农业大学 | Method for improving germination rate of spores of clostridium sporogenes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US4330622A (en) * | 1979-09-12 | 1982-05-18 | Becton Dickinson & Co. | Elimination of non-microbial turbidity in culture media |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101092643B (en) * | 2006-06-23 | 2011-09-14 | 中国药品生物制品检定所 | Culture medium for asepsis test in medication of quinolone category, and application |
| CN101491307B (en) * | 2009-02-20 | 2011-07-20 | 扬州绿源生物化工有限公司 | Rice leaf roller artificial feedstuff and preparation method and artificial feeding method applied on rice leaf roller |
| CN101851676A (en) * | 2010-04-21 | 2010-10-06 | 吉林农业大学 | Composite enrichment medium of salmonella, shigella and staphylococcus aureus, preparation method and application thereof |
| CN101851676B (en) * | 2010-04-21 | 2013-07-24 | 吉林农业大学 | Composite enrichment medium of salmonella, shigella and staphylococcus aureus, preparation method and application thereof |
| CN104313110A (en) * | 2014-09-30 | 2015-01-28 | 青岛康合伟业商贸有限公司 | Fluid thioglycollate medium and application thereof |
| CN110317852A (en) * | 2019-06-04 | 2019-10-11 | 广州赛莱拉生物基因工程有限公司 | It is a kind of for detecting the culture medium and its preparation method and application of microorganism in cell product |
| CN111808787A (en) * | 2020-08-12 | 2020-10-23 | 河南农业大学 | Method for improving germination rate of spores of clostridium sporogenes |
| CN111808787B (en) * | 2020-08-12 | 2023-07-25 | 河南农业大学 | Method for improving germination rate of clostridium sporogenes |
| CN113308510A (en) * | 2021-07-12 | 2021-08-27 | 阿谷巴(杭州)生物科技发展有限公司 | Culture medium for rapidly detecting fungi in cell product and preparation method and application thereof |
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