CN102134583A - Photosynthetic bacteria counting method - Google Patents
Photosynthetic bacteria counting method Download PDFInfo
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- CN102134583A CN102134583A CN2010106169462A CN201010616946A CN102134583A CN 102134583 A CN102134583 A CN 102134583A CN 2010106169462 A CN2010106169462 A CN 2010106169462A CN 201010616946 A CN201010616946 A CN 201010616946A CN 102134583 A CN102134583 A CN 102134583A
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Abstract
The invention discloses a photosynthetic bacteria counting method. A staining fluid is added on the basis of the traditional bacteria counting method, thus the sensitivity and the accuracy in bacteria counting are improved by using the method provided by the invention. The photosynthetic bacteria counting method is simple to operate, has good repeatability, and is applicable to production and operation in labs or industries.
Description
Technical field
The present invention relates to experimental implementation method field, be specifically related to the method for counting of a kind of photosynthetic bacterium.
Background technology
Photosynthetic bacterium (
Photosynthetic bacteria,Be called for short PBS) be the prokaryotic organism that occur having original luminous energy synthetic system on the earth the earliest, be the general name that a big class is not under anaerobic put the photosynthetic bacterium of oxygen.Because it has and can utilize the physiological property of organic molecule as carbon source, and the characteristics that thalline itself is nontoxic, nutritious again, therefore in industries such as aquaculture, wastewater treatment, obtained widespread use, and demonstrated wide application prospect in industries such as new energy development, plant husbandry, food, makeup, medicines and health protections.The photosynthetic bacterium of some kinds produces viscous substance under the certain culture condition, along with the prolongation viscous substance of incubation time increases, as: capsula Rhodopseudomonas, Rhodopseudomonas spheroides, but the nutrient solution of Rhodopseudomonas palustris does not possess adhesive characteristics, and this has just proposed the specific aim requirement for the method for counting of different bacterium.
The mensuration of photosynthetic bacterium number is the key index of production quality control always, the measuring method that photosynthetic bacterium bacterium number is commonly used has microscope count method (blood counting chamber) method at present, dilution-plate method, the double-layer plate method, the maximum most probable number (MPN) method of MPN(), nephelometer is counted the mensuration that method etc. is carried out photosynthetic bacterium bacterium number.These methods ubiquity complicated operation, incubation time in actual production be long, problem such as poor repeatability as a result.As: the blood counting chamber counting process, though it is simple to operate, but it is little because of the thalline of photosynthetic bacterium not to be suitable for photosynthetic bacterium, general 1 ~ 1.3 μ m * 2 ~ 4 μ m, blood counting chamber is thicker, be difficult to photosynthetic bacterium is counted, blood counting chamber generally is used for saccharomycetic counting more, and saccharomycetic size is generally 2.5 ~ 10 μ m * 4.5 ~ 21 μ m; It is few that dilution plate is used on photosynthetic bacterial technology, have only the bacterium that metabolic capacity is vigorous under dark, aerobic situation individually just to be suitable for, the growth of most of photosynthetic bacterium needs anaerobism, illumination, carry out anaerobism, illumination cultivation process middle plateform incubation time is long, and there is the globule to produce, pollutes easily; The double-layer plate counting process has solved the anaerobic problem, but the bacterium of cultivating is often less, light, even colourless, and the cycle of growth is long, the MPN method is accurate, and be the method that rower is recommended use, but technological operation is loaded down with trivial details, the time cycle is long, generally needed 12 ~ 15 days just can judge the result, and when carrying out the viscosity photosynthetic bacteria counting, result difference is bigger, is not suitable for the monitoring of production process quality; Turbidimetry is most of inviscid material photosynthetic bacteriums method of counting commonly used, but meetings such as substratum produce certain influence to the result.
Summary of the invention
The objective of the invention is to problems such as long according to the gate time of existing photosynthetic bacterium existence when counting, that result difference is bigger, a kind of detected result rapid counting method of photosynthetic bacterium accurately is provided.
The object of the invention is achieved by the following technical programs:
The method of counting of a kind of photosynthetic bacterium comprises the steps:
(1) preparation staining fluid;
(2) dilution of bacterium liquid: get photosynthetic bacterium 1 ~ 2ml, the sterilized water dilution;
(3) cleaning of bateria chamber: bateria chamber and cover glass are soaked 3 ~ 5min in 75% alcohol, take out the back and draw unnecessary alcohol with aseptic filter paper, standby;
(4) preparation of slide: draw the photosynthetic bacterium suspension, add to the count block of bateria chamber, drip staining fluid, covered;
(5) counting.
As a kind of preferred version, staining fluid comprises Viola crystallina 1 ~ 2.0g, ammonium oxalate 0.5 ~ 0.8g, alcohol 20ml and distilled water 80ml described in the step (1).
Further, described staining fluid comprises Viola crystallina 2.0g, ammonium oxalate 0.8g, 95% alcohol 20ml, distilled water 80ml.
As a kind of preferred version, the compound method of staining fluid of the present invention is: earlier Viola crystallina is dissolved in alcohol, ammonium oxalate is dissolved in the distilled water, then two kinds of solution is mixed, and filters, and leaves standstill, and time of repose is preferably 48h.
As a kind of preferred version, the dilution of sterilized water described in the step (2) is to carry out the 1:100 dilution with sterilized water earlier, and whirlpool concussion 30 ~ 60s in dilution back gets diluent again and carries out the 1:10 dilution.
As a kind of preferred version, described in the step (4) viscosity photosynthetic bacterium suspension added to the count block of bateria chamber after, bacterial count of each counting lattice is 5 ~ 10.
As a kind of preferred version, in the step (4), described dropping staining fluid is the staining fluid that drips 3 μ L in the count block of bateria chamber.
As a kind of preferred version, described photosynthetic bacterium is the viscosity photosynthetic bacterium.
Compared with prior art, the present invention has following beneficial effect:
The used time of the inventive method is short, utilize the bateria chamber counting of improvement, added staining fluid, can finish a sample counting about general 15min very clearly on microscopically direct viewing bacterium, increased the sensitivity of counting, reduce error, good reproducibility is fit to use in the scale operation very much, the maximum most probable number (MPN) of present method and MPN() method has been carried out the count results contrast, and difference is not obvious.Present method step is simple, and is easy to operate, less demanding to laboratory condition, be a kind of simply, photosynthetic bacterial technology method efficiently.
Embodiment
Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
The used microscope of following examples be eyepiece 16 *, objective lens 40 *, the bateria chamber count block length of side is 1mm, the area of count block is 1mm
2, the area of each lattice is 1/400mm
2After the covered, the height of count block is 0.1mm, so the volume of each count block is 0.1mm
3, the volume of each lattice is 1/4000mm
31mm
3It is 1000mm that volume should contain the lattice number
3/ 1/4000mm
3=4 * 10
6Individual lattice, i.e. COEFFICIENT K=4 * 10
6
The counting of embodiment 1 viscosity photosynthetic bacterium
1, get Viola crystallina 2g, ammonium oxalate 0.8g, 95% alcohol 20ml, distilled water 80ml is dissolved in alcohol with Viola crystallina earlier, and ammonium oxalate is dissolved in the distilled water, then two liquid is mixed, and filters, and leaves standstill 48h.
2, get capsula Rhodopseudomonas culture 1ml, put in the 99ml sterilized water and dilute, whirlpool concussion 60 seconds, extension rate is 100 times.Each counts 8 of bacterium several months of little lattice.
3, bateria chamber and cover glass are soaked in 75% alcohol about 3min, take out the back and remove unnecessary alcohol with the aseptic filter paper suction, standby.
4, draw the technical area that 3 μ L diluents place bateria chamber with the micropipette rifle, drip the staining fluid of 3 μ L, on the covered, avoid the generation of bubble after the left side, bacterium liquid avoids overflowing cover glass as far as possible.
5, bateria chamber is examined under a microscope, selected to count greater than the quantity of 20 counting lattice, " bow " type route is generally adopted in the path of counting, guarantees the accurate of counting, amounts to 24 lattice, totally 180 of bacterium numbers.
6, calculation result:
The photosynthetic bacterium sum=
* K * bacterium liquid extension rate=
* 4 * 10
6* 100=3 * 10
9
Embodiment 2
The counting of viscosity photosynthetic bacterium
1, get Viola crystallina 1g, ammonium oxalate 0.5g, 95% alcohol 20ml, distilled water 80ml is dissolved in alcohol with Viola crystallina earlier, and ammonium oxalate is dissolved in the distilled water, then two liquid is mixed, and filters, and leaves standstill 48h.
2, get spissated Rhodopseudomonas spheroides culture 1ml, put in the 99ml sterilized water and dilute, whirlpool concussion 60 seconds, get in the sterilized water of diluent 1ml adding 9ml, whirlpool concussion 30 seconds, after getting diluent 1ml adding 9ml sterile diluent again, whirlpool concussion 30 seconds, this extension rate is 10000 times.Each counts about 9 of the bacterium number of little lattice.
3, bateria chamber and cover glass are soaked in 75% alcohol about 3min, take out the back and remove unnecessary alcohol with the aseptic filter paper suction, standby.
4, draw the technical area that 3 μ L diluents place bateria chamber with the micropipette rifle, drip the staining fluid of 3 μ L, on the covered, avoid the generation of bubble after the left side, bacterium liquid avoids overflowing cover glass as far as possible.
5, bateria chamber is examined under a microscope, selected to count greater than the quantity of 20 counting lattice, " bow " type route is generally adopted in the path of counting, guarantees the accurate of counting, amounts to 30 lattice, totally 240 of bacterium numbers.
6, calculation result:
The photosynthetic bacterium sum=
* K * bacterium liquid extension rate=
4 * 10
6* 10000=3.2 * 10
11
The counting of embodiment 3 non-sticky photosynthetic bacteriums
1, get Viola crystallina 1g, ammonium oxalate 0.5g, 95% alcohol 20ml, distilled water 80ml is dissolved in alcohol with Viola crystallina earlier, and ammonium oxalate is dissolved in the distilled water, then two liquid is mixed, and filters, and leaves standstill 48h.
2, get Rhodopseudomonas palustris culture 1ml, put in the 99ml sterilized water and dilute, whirlpool concussion 60 seconds, whirlpool concussion 30 seconds, this extension rate is 100 times.Each counts about 7 of the bacterium number of little lattice.
3, bateria chamber and cover glass are soaked in 75% alcohol about 3min, take out the back and remove unnecessary alcohol with the aseptic filter paper suction, standby.
4, draw the technical area that 3 μ L diluents place bateria chamber with the micropipette rifle, drip the staining fluid of 3 μ L, on the covered, avoid the generation of bubble after the left side, bacterium liquid avoids overflowing cover glass as far as possible.
5, bateria chamber is examined under a microscope, selected to count greater than the quantity of 20 counting lattice, " bow " type route is generally adopted in the path of counting, guarantees the accurate of counting, amounts to 35 lattice, totally 245 of bacterium numbers.
6, calculation result:
The photosynthetic bacterium sum=
* K * bacterium liquid extension rate=
4 * 10
6* 100=2.8 * 10
9
Claims (9)
1. the method for counting of a photosynthetic bacterium is characterized in that comprising the steps:
(1) preparation staining fluid;
(2) dilution of bacterium liquid: get photosynthetic bacterium 1 ~ 2ml, the sterilized water dilution;
(3) cleaning of bateria chamber: bateria chamber and cover glass are soaked 3 ~ 5min in 75% alcohol, take out the back and draw unnecessary alcohol with aseptic filter paper, standby;
(4) preparation of slide: draw the photosynthetic bacterium suspension, add to the count block of bateria chamber, drip staining fluid, covered;
(5) counting.
2. the method for counting of photosynthetic bacterium according to claim 1 is characterized in that staining fluid comprises Viola crystallina 1 ~ 2.0g, ammonium oxalate 0.5 ~ 0.8g, alcohol 20ml and distilled water 80ml described in the step (1).
3. the method for counting of photosynthetic bacterium according to claim 2 is characterized in that described staining fluid comprises Viola crystallina 2.0g, ammonium oxalate 0.8g, 95% alcohol 20ml, distilled water 80ml.
4. according to the method for counting of any described photosynthetic bacterium of claim in the claim 1 ~ 3, the compound method that it is characterized in that described staining fluid is: earlier Viola crystallina is dissolved in alcohol, ammonium oxalate is dissolved in the distilled water, then two kinds of solution is mixed, filter, leave standstill.
5. the method for counting of photosynthetic bacterium according to claim 4 is characterized in that described time of repose is 48h.
6. the method for counting of photosynthetic bacterium according to claim 1 is characterized in that the dilution of sterilized water described in the step (2) is to carry out the 1:100 dilution with sterilized water earlier, and whirlpool concussion 30 ~ 60s in dilution back gets diluent again and carries out the 1:10 dilution.
7. the method for counting of photosynthetic bacterium according to claim 1 is characterized in that in the step (4), described viscosity photosynthetic bacterium suspension is added to the count block of bateria chamber after, each the counting lattice bacterial count be 5 ~ 10.
8. the method for counting of photosynthetic bacterium according to claim 1 is characterized in that in the step (4), and described dropping staining fluid is the staining fluid that drips 3 μ L in the count block of bateria chamber.
9. the method for counting of photosynthetic bacterium according to claim 1 is characterized in that described photosynthetic bacterium is the viscosity photosynthetic bacterium.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104099244A (en) * | 2014-06-26 | 2014-10-15 | 上海市园林设计院有限公司 | Plate culture dish capable of quickly counting bacterial colonies |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1392266A (en) * | 2001-06-19 | 2003-01-22 | 张华� | Quick gram staining liquid and method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1392266A (en) * | 2001-06-19 | 2003-01-22 | 张华� | Quick gram staining liquid and method |
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| CN104099244A (en) * | 2014-06-26 | 2014-10-15 | 上海市园林设计院有限公司 | Plate culture dish capable of quickly counting bacterial colonies |
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Application publication date: 20110727 |