CN103555812A - Method for quickly counting content of viable bacteria in composite bacillus powder - Google Patents
Method for quickly counting content of viable bacteria in composite bacillus powder Download PDFInfo
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- CN103555812A CN103555812A CN201310572544.0A CN201310572544A CN103555812A CN 103555812 A CN103555812 A CN 103555812A CN 201310572544 A CN201310572544 A CN 201310572544A CN 103555812 A CN103555812 A CN 103555812A
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- dilution
- viable bacteria
- diluent
- aseptic
- nutrient broth
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Abstract
The invention provides a method for quickly counting the content of viable bacteria in composite bacillus powder. The method comprises the following steps: sufficiently dispersing bacillus into single cells after the sample to be detected is diluted properly, then enabling the single cells to grow and proliferate in sterile nutrient broth through incubation and cultivation so as to form a muddy solution which is visible to the naked eyes, further determining that the muddy solution with maximum dilution contains a large amount of spore through spore staining, and finally, determining the sum of viable bacteria of the sample to be detected. Compared with a direct counting method by adopting a microscope, the quick method can distinguish dead bacteria from viable bacteria so as to ensure that the determination result on the sum of the viable bacteria is more accurate; compared with a panel bacterial colony counting method, the quick method is simpler, easier and quicker in operation, and is extremely suitable for a large quantity of persons engaged in production of bacillus preparations to use.
Description
Technical field
The present invention relates to content of microorganisms method for measuring, be specifically related to a kind of rapid counting method of composite bacillus powder viable bacteria content.
Background technology
Bacillus preparation be a kind ofly have no side effect, the green product of drug residue free, the viable bacteria containing in its product is to exist with endosporic form, is conventional fodder additives and improver of water quality in beasts, birds and aquatic products cultivation.Yet lack quality standard, be that bacillus preparation is developing one of subject matter of middle existence, and one of maximum problem that causes quality standard shortage is exactly that viable count and label do not spread out, some is even difficult to detect viable bacteria.Therefore the method for counting of reliable and stable bacillus preparation has great importance to the normal production of bacillus preparation and quality control thereof undoubtedly.At present, traditional method of counting of the bacillus preparations such as ascites method, the method for plate culture count, owing to there being some defects, makes count results unstable.For example ascites method cannot be distinguished dead thalline and viable bacteria body, and count results is the summation of dead thalline and viable bacteria body; The method of plate culture count trivial operations, and count results is subject to the impact of many factors.
Summary of the invention
The object of the present invention is to provide a kind of rapid counting method of composite bacillus powder viable bacteria content.
The rapid counting method of a kind of composite bacillus powder viable bacteria content provided by the invention, comprise the steps: 1) preparation of sample: aseptic technique is competent composite bacillus powder l g at random, add in the 45mL tool plug centrifuge tube that fills the aseptic nutrient broth of 8mL, on vortex oscillation instrument, vibration mixes the dilution mother liquor of making 1: 10, establishes 3 Duplicate Samples;
2) dilution of sample: get 3 Duplicate Samples, be handled as follows respectively: aseptic technique is drawn the lmL dilution mother liquor of 1: 10 with lmL glass pipet, add in the 45mL tool plug centrifuge tube that fills the aseptic nutrient broth of 8mL the even one-tenth of tear 1: 10
2diluent, so successively dilution, obtain respectively 1: 10
3, 1: 10
4, 1: 10
5, 1: 10
6, 1: 10
7, 1: 10
8, 1: 10
8, 1: 10
10, 1: 10
11,1: 10
12, 1: 10
13dilution diluent must be changed lmL glass pipet while diluting at every turn;
3) cultivate: by the dilution mother liquor of 1: 10 and 1: 10
2, 1: 10
3,1: 10
4, 1: 10
5, 1: 10
6, 1: 10
7, 1: 10
8, 1: 10
8, 1: 10
10, 1: 10
11, 1: 10
12, 1: 10
13diluent to be placed in rotating speed be that 200r/min, temperature are the temperature control shaking table of 35 37 ℃, cultivate 18-24h; With the 45mL tool plug centrifuge tube that fills the aseptic nutrient broth of 8mL in contrast;
4) measure: with the 45mL tool plug centrifuge tube that fills the aseptic nutrient broth of 8mL in contrast, aseptic technique is drawn 0. lmL with lmL glass pipet and is cultivated 18 24h and become each muddy diluent and adopt Schaef f er Fu l ton Albert'stain Albert method to carry out spore staining, and microscopic examination has the gemma of redgreen; Microscopic examination has green gemma and becomes the viable bacteria content (cfu/ g) that the muddy corresponding dilution inverse of diluent is composite bacillus powder.
The present invention can be fast, accurately, and directly Observe and measure goes out the viable bacteria content of composite bacillus powder.Its principle is to hold test sample product through after suitably diluting, genus bacillus wherein is fully dispersed into individual cells, then in aseptic nutrient broth, by hatching, cultivate and growth and breeding, thereby form macroscopic turbid solution, further by spore staining, determine in greatest dilution turbid solution and contain a large amount of gemma again, finally determine the total viable count of testing sample.It compare with ascites method because of can distinguish dead thalline and
tthalline and making
tthe measurement result of bacterium sum is more accurate, compares with the method for plate culture count, operates simplyr, and simple and fast more, is more suitable for producing practitioner without the vast bacillus preparation of very strong professional skill and adopts.
Embodiment
Embodiment 1
1 materials and methods
1. 1 experiment material
Composite bacillus powder I3CM, the viable bacteria content of measuring by colony counting method is 3 * 1011cf u bait, is purchased from Shanxi Province Wei Shi Yu Kang Industrial Co., Ltd.; LmL glass pipet, aseptic glass spreading rod, nutrient agar plate, nutrient broth, 45mL tool plug centrifuge tube, slide glass, the 5 % malachite green aqueous solution, the luxuriant red aqueous solution of 0. 5 %, be purchased from Chemical Reagent Co., Ltd., Sinopharm Group; LmL glass pipet, fill the 45mL tool plug centrifuge tube of the aseptic nutrient broth of 8mL, sterilizing 20min under 121 ℃ of conditions.
1. the preparation of 2 samples
Aseptic technique is competent composite bacillus powder BCM l g at random, adds in the 45mL tool plug centrifuge tube that fills the aseptic nutrient broth of 8mL the even sample of making of the tear that vibrates on vortex oscillation instrument, the dilution mother liquor of 1: 10.Experiment has 3 Duplicate Samples.
1. the dilution of 3 samples
Get 3 Duplicate Samples, be handled as follows respectively: aseptic technique is drawn the lmL dilution mother liquor of 1: 10 with lmL glass pipet, adds in the 45mL tool plug centrifuge tube that fills the aseptic nutrient broth of 8mL the even one-tenth of tear 1:10
2liquid, dilution, obtains respectively 1: 10 so successively
3, 1: 10
4, 1: 10
5, 1: 10
6, 1: 10
7, 1: 10
8, 1: 10
8, 1: 10
10, 1: 10
11, 1: 10
12, 1: 10
13etc. dilution diluent, while diluting, must change lmL glass pipet at every turn.
1. 4 cultivate
By the dilution mother liquor of 1: 10 and 1: 10
2, 1: 10
3, 1: 10
4, 1: 10
5, 1: 10
6, 1: 10
7,
1: 10
8, 1: 10
8, 1: 10
10, 1: 10
11, 1: 10
12, 1: 10
13deng diluent to be placed in rotating speed be the temperature control shaking table that 200r/min, temperature are 35-37 ℃, cultivate 18-24h.With the 45mL tool plug centrifuge tube that fills the aseptic nutrient broth of 8mL in contrast.
1. 5 measure
With the 45mL tool plug centrifuge tube that fills the aseptic nutrient broth of 8mL in contrast, aseptic technique is drawn each muddy diluent of 0. lmL cultivation 18-24h change with lmL glass pipet and is adopted Schaef f er Fu l ton Albert'stain Albert method to carry out spore staining, and microscopic examination has the gemma of redgreen.Microscopic examination has green gemma and becomes the muddy corresponding dilution inverse of diluent and is composite bacillus powder BCM's
tbacterial content (cfu/ g).
2 experimental results
Experimental result is in Table 1.Experimental result shows, the composite bacillus powder BCM that extent of dilution is 1: 1011 is muddy after cultivating 18-24h, and after spore staining, under microscope, can be observed green gemma; And extent of dilution is respectively 1: 10
12with 1: 10
13composite bacillus powder BCM all muddy after cultivating 18-24h, and after spore staining, under microscope, do not observe green gemma yet.Therefore, the viable bacteria content of composite bacillus powder BCM is 10
11-10
12cfu/g, compares with the viable bacteria content of measuring by colony counting method, and the order of magnitude is identical, can be used as the composite bacillus powder BCM t bacterium sum method of freight volume fast.
BCM is effective for table 1 composite bacillus powder
tthe measurement result of bacterium sum
| Extent of dilution | Whether muddy | Have or not gemma |
| 1：10 | Be | Have |
| 1：10 2 | Be | Have |
| 1：10 3 | Be | Have |
| 1：10 4 | Be | Have |
| 1：10 5 | Be | Have |
| 1：10 6 | Be | Have |
| 1：10 7 | Be | Have |
| 1：10 8 | Be | Have |
| 1：10 9 | Be | Have |
| 1：10 10 | Be | Have |
| 1：10 11 | Be | Have |
| 1：10 12 | No | Nothing |
| 1：10 13 | No | Nothing |
3 conclusions
Fast, accurately, directly observation can determine the viable bacteria content of composite bacillus powder BCM to the quantivative approach of this total viable count.Its principle is to hold test sample product through after suitably diluting, genus bacillus wherein is fully dispersed into individual cells, then in aseptic nutrient broth, by hatching, cultivate and growth and breeding, thereby form macroscopic turbid solution, further by spore staining, determine that in greatest dilution turbid solution, containing a large amount of buds runs again, finally determine testing sample
tbacterium sum.
It is compared with ascites method because distinguishing dead thalline and viable bacteria body makes
tthe measurement result of bacterium sum is more accurate, compares with the method for plate culture count, operates simplyr, and simple and fast more, is more suitable for producing practitioner without the vast bacillus preparation of very strong professional skill and adopts.
Claims (1)
1. a rapid counting method for composite bacillus powder viable bacteria content, is characterized in that, comprises the steps:
1) preparation of sample: aseptic technique takes composite bacillus powder l g at random, adds in the 45mL tool plug centrifuge tube that fills the aseptic nutrient broth of 8mL, and on vortex oscillation instrument, vibration mixes the dilution mother liquor of making 1: 10;
2) dilution of sample: aseptic technique is drawn the lmL dilution mother liquor of 1: 10 with lmL glass pipet, add in the 45mL tool plug centrifuge tube that fills the aseptic nutrient broth of 8mL, be mixed into the diluent of 1: 102, dilution, obtains respectively so successively
1?:?10
3、1?:?10
4、1?:?10
5、1?:?10
6、1?:?10
7、1?:?10
8、1?:?10
8、1?:?10
10、1?:?10
11、1?:?
10?、
1: 10
13dilution diluent;
3) cultivate: by the dilution mother liquor of 1: 10 and 1: 10
2, 1: 10
3, 1: 10
4, 1: 10
5, 1: 10
6, 1: 10
7,
1: 10
8, 1: 10
8, 1: 10
10, 1: 10
11, 1: 10
12, 1: 10
13diluent to be placed in rotating speed be the temperature control shaking table that 200r/min, temperature are 35-37 ℃, cultivate 18-24h; With the 45mL tool plug centrifuge tube that fills the aseptic nutrient broth of 8mL in contrast;
4) measure: with the 45mL tool plug centrifuge tube that fills the aseptic nutrient broth of 8mL in contrast, with the aseptic technique of lmL glass pipet, draw each muddy diluent of 0. lmL cultivation 18-24h change and adopt Schaef f er-Fu l ton Albert'stain Albert method to carry out spore staining, microscopic examination has the gemma of redgreen; Microscopic examination has green gemma and becomes the viable bacteria content that the muddy corresponding dilution inverse of diluent is composite bacillus powder.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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|---|---|---|---|
| CN201310572544.0A CN103555812A (en) | 2013-11-18 | 2013-11-18 | Method for quickly counting content of viable bacteria in composite bacillus powder |
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| CN103555812A true CN103555812A (en) | 2014-02-05 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350570A (en) * | 2016-10-27 | 2017-01-25 | 广东省微生物研究所 | Method for real-time observation of single cells of spore generating bacteria |
-
2013
- 2013-11-18 CN CN201310572544.0A patent/CN103555812A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350570A (en) * | 2016-10-27 | 2017-01-25 | 广东省微生物研究所 | Method for real-time observation of single cells of spore generating bacteria |
| CN106350570B (en) * | 2016-10-27 | 2020-04-10 | 广东省微生物研究所 | Real-time observation method for single cell of spore-forming bacteria |
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| C06 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
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Application publication date: 20140205 |