CN101659982A - Rapid counting method of total viable count of lactic acid bacteria - Google Patents
Rapid counting method of total viable count of lactic acid bacteria Download PDFInfo
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- CN101659982A CN101659982A CN200910186079A CN200910186079A CN101659982A CN 101659982 A CN101659982 A CN 101659982A CN 200910186079 A CN200910186079 A CN 200910186079A CN 200910186079 A CN200910186079 A CN 200910186079A CN 101659982 A CN101659982 A CN 101659982A
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- lactic acid
- viable count
- acid bacteria
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Abstract
The invention relates to a rapid counting method of total viable count of lactic acid bacteria; 0.1-1g of methylene blue, 0.5-2g of sodium carbonate and 100ml of distilled water are mixed, and placedstill for 0.5-2 hours, and then filtration is carried out and then the mixture is placed still for 2 hours; 1g of bacterial powder or 1ml of lactic acid bacteria nutrient solution is weighted and is diluted with 10-100 times of bacteria free water, so as to prepare bacterial suspension; the bacterial suspension is used and is diluted with 10 times of gradients; 5mul of the bacterial suspension isplaced on a glass slide, 5mul of dye liquid is added, and then a cover glass is covered; 10-20 of visual fields are chosen to alculate the total viable count of lactic acid bacteria and the average value of the total viable count of lactic acid bacteria; the viable count of lactic acid bacteria, contained in every gram of bacterial powder / every milliliter of bacteria solution, is calculated according to the formula, wherein, C is the area of the cover glass, B is the area of the visual field and A is the average value of the total viable count of lactic acid bacteria. In the invention, the steps are simplified, the error is low, the repeatability is good, and long cultivation time is omitted, therefore, the method is a rapid and simple counting method of total viable count of lactic acidbacteria, so as to realize on-line detection of the viable count of lactic acid bacteria.
Description
Technical field
The online counting work of a large amount of total viable count of lactic acid bacteria that the total viable count of lactic acid bacteria fast counting is mainly used in that all kinds of milk-acid bacterias spread cultivation at bacterial classification, carried out in fermentation and the fungicide preparation process thereof.
Technical background
Milk-acid bacteria has good health care function and production characteristic, has been widely used in the modern industries productions such as milk fermentation, vegetable fermentation, agent of lactic acid bacteria preparation, pharmaceutical preparation, feed.The viable lactic acid bacteria number is the index that often needs tracking and measuring in research and the production, at present, viable lactic acid bacteria is counted measuring method plate dilution method commonly used, also has the deep agar of employing method, GB method, blood counting chamber counting process, specific stain counting process, nephelometer to count method etc. and carries out milk-acid bacteria bacterium number and measure.These methods respectively have relative merits: the dull and stereotyped dilution method of counting and GB method are classical viable lactic acid bacteria counting processs, but operating process is loaded down with trivial details, and incubation time is long, can not in time reflect the thalli growth situation, can't realize online detection; The deep agar method is loaded down with trivial details except operating process, and outside incubation time was long, operational requirement was higher, and it is small and dense to cultivate the bacterium colony that comes out, and is difficult for counting; The blood counting chamber counting process is unintelligible in when dyeing, makes the counting difficulty, detected result is obviously on the low side, and after use tally easy cleaning not, residue is easily arranged, influence is observed and count results; Toluidine blue specific stain counting process can not be distinguished cell anyway, and detected result is obviously higher; Though it is easier that nephelometer is counted method, can METHOD FOR CONTINUOUS DETERMINATION, error is very big, and its count results is subjected to the influence of medium component, meta-bolites very big.
The content of invention
The objective of the invention is to propose a kind of rapid counting method of total viable count of lactic acid bacteria, the online enumeration problem of a large amount of total viable count of lactic acid bacteria that solves that all kinds of milk-acid bacterias spread cultivation at bacterial classification, carried out in fermentation and the fungicide preparation process thereof.
The present invention is achieved by the following technical solutions.
1, preparation dye liquor.Methylene blue 0.1-1g, yellow soda ash 0.5-2g, distilled water 100ml.Mixing, leave standstill 0.5-2h after, filter, leave standstill behind the 2h standby;
2, bacteria suspension preparation and gradient dilution.Take by weighing 1 gram bacterium powder or pipette the 1ml lactic acid bacteria culture solution, carry out 10-100 with sterilized water and doubly dilute, concussion mixes makes bacteria suspension; Pipette bacteria suspension, with sterilized water bacteria suspension is carried out 10 times of gradient dilutions, concussion mixes, and total extension rate is 20-200 with thalline number in the field of microscope and is advisable;
3, stained preparation.Move pipe absorption 5 μ l bacteria suspensions with micro quantitative determination and put on the clean slide, add the above-mentioned dye liquor of 5 μ l, covered;
4, counting.Bacterium liquid is soaked sheet put the microscopically microscopy, total viable count of lactic acid bacteria is calculated in 10～20 visuals field selecting to soak 4 angles of sheet and central authorities, and asks for lactic acid viable count mean value in each visual field;
5, the lactic acid viable count calculation formula that contains in the every gram bacterium powder/every milliliter bacterium liquid is:
Wherein, C is the area of cover glass, and B is the area in a visual field, and A is a lactic acid viable count mean value in each visual field.
The present invention has the following advantages and effect:
The present invention can directly carry out online viable bacteria to milk-acid bacteria and detect, and can directly count viable count at microscopically, is a kind of quick, directly perceived, easy, the viable lactic acid bacteria method of counting that actual application value is higher.Methods such as detected result and plate dilution method, deep agar method are more approaching, special and GB method basically identical, and step is simplified, operate miss is little, favorable reproducibility as a result, saving very long incubation time, is a kind of quick, easy total viable count of lactic acid bacteria method of counting, can realize the online detection of viable lactic acid bacteria number.
Embodiment
The present invention will be described further by following examples.
The used microscope of following examples get eyepiece 16 *, objective lens 40 *, the diameter d in a visual field is 0.27mm, the area B in a visual field=π r
2=π (d/2)
2=3.14 * (0.27/2mm)
2=0.0572265mm
2, the area C=(18mm) of cover glass
2=324mm
2
Embodiment 1.
1, gets methylene blue 0.1g, yellow soda ash 0.5g, distilled water 100ml.Mixing, leave standstill 1h after, filter, leave standstill behind the 2h standby;
2, take by weighing 1g bacterium powder, and it is joined in the 99ml sterilized water, concussion mixes; Bacteria suspension is pipetted the 1ml adding be equipped with in the test tube of 9ml sterilized water, take out 1ml behind the concussion mixing and add in next test tube that the 9ml sterilized water is housed, bacteria suspension is made 10 times of gradient dilutions twice, promptly total extension rate is 10000;
3, move pipe absorption 5 μ l bacteria suspensions with micro quantitative determination and put on the clean slide, add the above-mentioned dye liquor of 5 μ l, covered, attention can not have bubble, also the bacterium hydrorrhea can not be gone out beyond the cover glass;
4, counting.Bacterium liquid is soaked sheet put the microscopically microscopy, viable bacteria body sums are calculated in 10 visuals field selecting to soak 4 angles of sheet and central authorities, and obtain its lactic acid viable bacteria mean value A and be: 43.3;
5, with the value substitution following formula of C, B, A, total extension rate, the lactic acid viable count that contains in every gram lactobacillus powder is:
Embodiment 2.
1, gets methylene blue 0.1g, yellow soda ash 2g, distilled water 100ml.Mixing, leave standstill 2h after, filter, leave standstill behind the 2h standby;
2, pipette the 1ml lactic acid bacteria culture solution, and it is joined in the 99ml sterilized water, concussion mixes, and promptly total extension rate is 100;
3, move pipe absorption 5 μ l bacteria suspensions with micro quantitative determination and put on the clean slide, add the above-mentioned dye liquor of 5 μ l, covered, attention can not have bubble, also the bacterium hydrorrhea can not be gone out beyond the cover glass;
4, counting.Bacterium liquid is soaked sheet put the microscopically microscopy, viable bacteria body sums are calculated in 20 visuals field selecting to soak 4 angles of sheet and central authorities, and obtain its lactic acid viable bacteria mean value A and be: 85.5;
5, with the value substitution following formula of C, B, A, total extension rate, the lactic acid viable count that contains in every milliliter of bacterium liquid is:
Embodiment 3.
1, gets methylene blue 0.5g, yellow soda ash 0.5g, distilled water 100ml.Mixing, leave standstill 0.5h after, filter, leave standstill behind the 2h standby;
2, pipette the 1ml lactic acid bacteria culture solution, and it is joined in the 99ml sterilized water, concussion mixes, and promptly total extension rate is 100;
3, move pipe absorption 5 μ l bacteria suspensions with micro quantitative determination and put on the clean slide, add the above-mentioned dye liquor of 5 μ l, covered, attention can not have bubble, also the bacterium hydrorrhea can not be gone out beyond the cover glass;
4, counting.Bacterium liquid is soaked sheet put the microscopically microscopy, viable bacteria body sums are calculated in 15 visuals field selecting to soak 4 angles of sheet and central authorities, and obtain its lactic acid viable bacteria mean value A and be: 135.6;
5, with the value substitution following formula of C, B, A, total extension rate, the lactic acid viable count that contains in every milliliter of bacterium liquid is:
Claims (1)
1, a kind of rapid counting method of total viable count of lactic acid bacteria is characterized in that
(1) get methylene blue 0.1-1g, yellow soda ash 0.5-2g, distilled water 100ml, mixing, leave standstill 0.5-2h after, filter, leave standstill 2h;
(2) take by weighing 1 and restrain the bacterium powder or pipette the 1ml lactic acid bacteria culture solution, carry out 10-100 with sterilized water and doubly dilute, concussion mixes makes bacteria suspension; Pipette bacteria suspension, with sterilized water bacteria suspension is carried out 10 times of gradient dilutions, concussion mixes, and total extension rate is 20-200 with thalline number in the field of microscope and is advisable;
(3) move pipe absorption 5 μ l bacteria suspensions with micro quantitative determination and put on the clean slide, add the above-mentioned dye liquor of 5 μ l, covered;
(4) bacterium liquid is soaked sheet and put the microscopically microscopy, total viable count of lactic acid bacteria is calculated in 10～20 visuals field selecting to soak 4 angles of sheet and central authorities, and asks for lactic acid viable count mean value in each visual field;
(5) calculating the lactic acid viable count that contains in every gram bacterium powder/every milliliter bacterium liquid is:
Wherein, C is the area of cover glass, and B is the area in a visual field, and A is a lactic acid viable count mean value in each visual field.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200910186079A CN101659982A (en) | 2009-09-22 | 2009-09-22 | Rapid counting method of total viable count of lactic acid bacteria |
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| CN200910186079A CN101659982A (en) | 2009-09-22 | 2009-09-22 | Rapid counting method of total viable count of lactic acid bacteria |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148478A (en) * | 2015-03-26 | 2016-11-23 | 北京大北农科技集团股份有限公司 | A kind of antibacterial viable count method for quick |
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2009
- 2009-09-22 CN CN200910186079A patent/CN101659982A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148478A (en) * | 2015-03-26 | 2016-11-23 | 北京大北农科技集团股份有限公司 | A kind of antibacterial viable count method for quick |
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Open date: 20100303 |