CN101659982A - Rapid counting method of total viable count of lactic acid bacteria - Google Patents
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 90
- 241000894006 Bacteria Species 0.000 title claims abstract description 59
- 239000004310 lactic acid Substances 0.000 title claims abstract description 45
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 28
- 230000001580 bacterial effect Effects 0.000 claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000000725 suspension Substances 0.000 claims abstract description 15
- 239000006059 cover glass Substances 0.000 claims abstract description 13
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims abstract description 12
- 239000008223 sterile water Substances 0.000 claims abstract description 10
- 239000000843 powder Substances 0.000 claims abstract description 8
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000012153 distilled water Substances 0.000 claims abstract description 6
- 229960000907 methylthioninium chloride Drugs 0.000 claims abstract description 6
- 229910000029 sodium carbonate Inorganic materials 0.000 claims abstract description 6
- 239000011521 glass Substances 0.000 claims abstract description 5
- 238000002156 mixing Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 14
- 239000012895 dilution Substances 0.000 claims description 13
- 238000010790 dilution Methods 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 6
- 230000000007 visual effect Effects 0.000 claims description 4
- 238000001514 detection method Methods 0.000 abstract description 4
- 238000001914 filtration Methods 0.000 abstract 1
- 238000005303 weighing Methods 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- 239000012192 staining solution Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000012733 comparative method Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000003670 easy-to-clean Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000036449 good health Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229950003937 tolonium Drugs 0.000 description 1
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
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Abstract
一种乳酸菌活菌总数快速计数方法，取次甲基蓝0.1－1g，碳酸钠0.5－2g，蒸馏水100ml，混匀，静置0.5－2h后，过滤，静置2h；称取1克菌粉或移取1ml乳酸菌培养液，用无菌水进行10－100倍稀释，制成菌悬液；移取菌悬液，用无菌水进行10倍梯度稀释；吸取5μl菌悬液置于载玻片上，加5μl上述染液，盖上盖玻片；选择10～20个视野计算乳酸菌活菌总数，并乳酸活菌数平均值；计算每克菌粉/每毫升菌液中含有的乳酸活菌数为上述公式，其中，C为盖玻片的面积，B为一个视野的面积，A为每个视野中乳酸活菌数平均值。本发明步骤简化，误差小，重现性好，省去漫长的培养时间，是一种快速、简便的乳酸菌活菌总数计数方法，可实现乳酸菌活菌数在线检测。
A rapid counting method for the total number of viable lactic acid bacteria, taking 0.1-1g of methylene blue, 0.5-2g of sodium carbonate, and 100ml of distilled water, mixing evenly, after standing for 0.5-2h, filtering, and standing for 2h; weighing 1g of bacterial powder or Pipette 1ml of lactic acid bacteria culture solution and dilute it 10-100 times with sterile water to make a bacterial suspension; pipette the bacterial suspension and dilute it 10 times with sterile water; draw 5μl of the bacterial suspension and place it on a glass slide , add 5 μl of the above dye solution, and cover with a cover glass; select 10 to 20 fields of view to calculate the total number of viable lactic acid bacteria, and calculate the average number of viable lactic acid bacteria; calculate the number of viable lactic acid bacteria per gram of bacteria powder/ml of bacterial solution For the above formula, wherein, C is the area of the cover glass, B is the area of a field of view, and A is the average number of lactic acid viable bacteria in each field of view. The invention has simplified steps, small error, good reproducibility, saves long cultivation time, is a fast and simple method for counting the total number of live lactic acid bacteria, and can realize online detection of the number of live lactic acid bacteria.
Description
技术领域 technical field
乳酸菌活菌总数快速计数法主要应用于各类乳酸菌在菌种扩培、发酵及其菌剂制备过程中所进行的大量乳酸菌活菌总数在线计数工作。The rapid counting method of the total number of live lactic acid bacteria is mainly used in the online counting of the total number of live lactic acid bacteria in a large number of lactic acid bacteria in the process of strain expansion, fermentation and preparation of bacterial agents.
技术背景 technical background
乳酸菌具有良好的保健功能和生产特性，已被广泛用于牛奶发酵、蔬菜发酵、乳酸菌剂制备、药物制剂、饲料等现代工业生产中。乳酸菌活菌数是研究和生产中经常需要跟踪测定的指标，目前，乳酸菌活菌数测定方法常用平板稀释法，也有采用高层琼脂法、GB法、血球计数板计数法、特殊染色计数法、比浊计数法等进行乳酸菌菌数测定。这些方法各有优缺点：平板稀释计数法和GB法是经典的乳酸菌活菌计数法，但操作过程繁琐，培养时间长，不能及时反映菌体生长情况，无法实现在线检测；高层琼脂法除了操作过程繁琐，培养时间长外，操作要求较高，培养出来的菌落小而密，不易计数；血球计数板计数法在染色时不清晰，使计数困难、检测结果明显偏低，而且在使用后计数板不易清洗，易有残留物，影响观察和计数结果；甲苯胺蓝特殊染色计数法不能区分细胞死活，检测结果明显偏高；比浊计数法虽然比较简便，可以连续测定，但误差很大，其计数结果受培养基成分、代谢产物的影响很大。Lactic acid bacteria have good health care functions and production characteristics, and have been widely used in modern industrial production such as milk fermentation, vegetable fermentation, preparation of lactic acid bacteria preparations, pharmaceutical preparations, and feed. The number of live lactic acid bacteria is an index that often needs to be tracked and determined in research and production. At present, the method of determining the number of live lactic acid bacteria is commonly used by plate dilution method, and there are also high-layer agar method, GB method, hemocytometer counting method, special staining counting method, comparative method, etc. The number of lactic acid bacteria was measured by turbidity counting method. These methods have their own advantages and disadvantages: the plate dilution counting method and the GB method are classic lactic acid bacteria counting methods, but the operation process is cumbersome, the cultivation time is long, and the growth of the bacteria cannot be reflected in time, and online detection cannot be realized; The process is cumbersome, the cultivation time is long, the operation requirements are high, and the cultured colonies are small and dense, which is not easy to count; the hemocytometer counting method is not clear during staining, which makes counting difficult and the test results are obviously low, and counting after use The plate is not easy to clean, and there are residues, which will affect the observation and counting results; the special toluidine blue staining counting method cannot distinguish between dead and alive cells, and the detection results are obviously high; although the turbidimetric counting method is relatively simple and can be continuously measured, the error is large. The counting results are greatly affected by the medium components and metabolites.
发明的内容content of the invention
本发明的目的是提出一种乳酸菌活菌总数快速计数方法，解决各类乳酸菌在菌种扩培、发酵及其菌剂制备过程中所进行的大量乳酸菌活菌总数在线计数问题。The purpose of the present invention is to propose a method for quickly counting the total number of live lactic acid bacteria, which solves the problem of online counting of the total number of live lactic acid bacteria in a large number of lactic acid bacteria in the process of strain expansion, fermentation and bacterial agent preparation.
本发明是通过以下技术方案实现的。The present invention is achieved through the following technical solutions.
1、制备染液。次甲基蓝0.1-1g，碳酸钠0.5-2g，蒸馏水100ml。混匀，静置0.5-2h后，过滤，静置2h后备用；1. Prepare the dye solution. Methylene blue 0.1-1g, sodium carbonate 0.5-2g, distilled water 100ml. Mix well, let stand for 0.5-2h, filter, stand for 2h and set aside;
2、菌悬液制备与梯度稀释。称取1克菌粉或移取1ml乳酸菌培养液，用无菌水进行10-100倍稀释，震荡混合均匀制成菌悬液；移取菌悬液，用无菌水对菌悬液进行10倍梯度稀释，震荡混合均匀，总稀释倍数以显微镜视野中菌体数为20-200个为宜；2. Bacterial suspension preparation and gradient dilution. Weigh 1 gram of bacterial powder or pipette 1ml of lactic acid bacteria culture solution, dilute it 10-100 times with sterile water, shake and mix evenly to make a bacterial suspension; pipette the bacterial suspension, and dilute the bacterial suspension with sterile water for 10 2-fold gradient dilution, shake and mix evenly, the total dilution factor should be 20-200 bacteria in the field of view of the microscope;
3、染色制片。用微量定量移管吸取5μl菌悬液置干净载玻片上，加5μl的上述染液，盖上盖玻片；3. Dyeing film production. Use a micro-quantitative pipette to draw 5 μl of the bacterial suspension and place it on a clean slide, add 5 μl of the above staining solution, and cover with a cover glass;
4、计数。将菌液浸片置显微镜下镜检，选择浸片4个角和中央的10～20个视野计算乳酸菌活菌总数，并求取每个视野中乳酸活菌数平均值；4. Count. Place the dipped piece of bacteria solution under a microscope for microscopic inspection, select 10 to 20 fields of vision in the four corners and the center of the dipped piece to calculate the total number of viable lactic acid bacteria, and calculate the average number of viable lactic acid bacteria in each field of view;
5、每克菌粉/每毫升菌液中含有的乳酸活菌数计算公式为：5. The formula for calculating the number of live lactic acid bacteria per gram of bacteria powder/ml of bacteria solution is:
其中，C为盖玻片的面积，B为一个视野的面积，A为每个视野中乳酸活菌数平均值。Among them, C is the area of the cover glass, B is the area of a visual field, and A is the average number of viable lactic acid bacteria in each visual field.
本发明具有以下优点和效果：The present invention has the following advantages and effects:
本发明能直接对乳酸菌进行在线活菌检测，能在显微镜下直接数出活菌数，是一种快速，直观、简便，实际应用价值较高的乳酸菌活菌计数方法。检测结果与平板稀释法、高层琼脂法等方法比较接近，特别与GB法基本一致，且步骤简化，操作误差小，结果重现性好，省去漫长的培养时间，是一种快速、简便的乳酸菌活菌总数计数方法，可实现乳酸菌活菌数在线检测。The invention can directly detect live bacteria on-line for lactic acid bacteria, and can directly count the number of viable bacteria under a microscope, which is a fast, intuitive, simple and practical method for counting viable bacteria of lactic acid bacteria with high practical application value. The test results are relatively close to the plate dilution method, high-layer agar method, etc., especially basically consistent with the GB method, and the steps are simplified, the operation error is small, the reproducibility of the results is good, and the long incubation time is saved. It is a fast and simple method. The method for counting the total number of live lactic acid bacteria can realize online detection of the number of live lactic acid bacteria.
具体实施方式 Detailed ways
本发明将通过以下实施例作进一步说明。The invention will be further illustrated by the following examples.
以下实施例所用显微镜取目镜16×，接物镜40×，一个视野的直径d为0.27mm，一个视野的面积B＝πr2＝π(d/2)2＝3.14×(0.27/2mm)2＝0.0572265mm2，盖玻片的面积C＝(18mm)2＝324mm2。The microscope used in the following examples has an eyepiece of 16× and an objective lens of 40×. The diameter d of a field of view is 0.27 mm, and the area of a field of view B=πr 2 =π(d/2) 2 =3.14×(0.27/2mm) 2 = 0.0572265mm 2 , the area of the cover glass C=(18mm) 2 =324mm 2 .
实施例1。Example 1.
1、取次甲基蓝0.1g，碳酸钠0.5g，蒸馏水100ml。混匀，静置1h后，过滤，静置2h后备用；1. Take 0.1g of methylene blue, 0.5g of sodium carbonate, and 100ml of distilled water. Mix well, let stand for 1 hour, filter, let stand for 2 hours, and set aside;
2、称取1g菌粉，并将其加入到99ml无菌水中，震荡混合均匀；将菌悬液移取1ml加入装有9ml无菌水的试管中，震荡混匀后取出1ml加入下一支装有9ml无菌水的试管中，对菌悬液作两次10倍梯度稀释，即总稀释倍数为10000；2. Weigh 1g of bacterial powder, add it to 99ml of sterile water, shake and mix evenly; pipette 1ml of the bacterial suspension into a test tube filled with 9ml of sterile water, take out 1ml and add it to the next tube after shaking and mixing In a test tube filled with 9ml of sterile water, make two 10-fold gradient dilutions of the bacterial suspension, that is, the total dilution factor is 10,000;
3、用微量定量移管吸取5μl菌悬液置干净载玻片上，加5μl的上述染液，盖上盖玻片，注意不可有气泡，也不可将菌液溢出盖玻片以外；3. Use a micro-quantitative pipette to draw 5 μl of the bacterial suspension and place it on a clean glass slide, add 5 μl of the above-mentioned staining solution, and cover with a cover glass. Be careful not to have air bubbles, and do not spill the bacterial solution beyond the cover glass;
4、计数。将菌液浸片置显微镜下镜检，选择浸片4个角和中央的10个视野计算活菌体总数，并求出其乳酸活菌平均值A为：43.3；4. Count. Place the dipped piece of bacterial liquid under a microscope for microscopic examination, select the 4 corners of the dipped piece and 10 central fields of view to calculate the total number of viable bacteria, and calculate the average value A of the viable lactic acid bacteria: 43.3;
5、将C、B、A、总稀释倍数的值代入下式，每克乳酸菌粉中含有的乳酸活菌数为：5. Substitute the values of C, B, A, and the total dilution factor into the following formula, and the number of viable lactic acid bacteria contained in each gram of lactic acid bacteria powder is:
实施例2。Example 2.
1、取次甲基蓝0.1g，碳酸钠2g，蒸馏水100ml。混匀，静置2h后，过滤，静置2h后备用；1. Take 0.1g of methylene blue, 2g of sodium carbonate, and 100ml of distilled water. Mix well, let stand for 2 hours, filter, stand for 2 hours and set aside;
2、移取1ml乳酸菌培养液，并将其加入到99ml无菌水中，震荡混合均匀，即总稀释倍数为100；2. Pipette 1ml of lactic acid bacteria culture solution, and add it to 99ml of sterile water, shake and mix evenly, that is, the total dilution factor is 100;
3、用微量定量移管吸取5μl菌悬液置干净载玻片上，加5μl的上述染液，盖上盖玻片，注意不可有气泡，也不可将菌液溢出盖玻片以外；3. Use a micro-quantitative pipette to draw 5 μl of the bacterial suspension and place it on a clean glass slide, add 5 μl of the above-mentioned staining solution, and cover with a cover glass. Be careful not to have air bubbles, and do not spill the bacterial solution beyond the cover glass;
4、计数。将菌液浸片置显微镜下镜检，选择浸片4个角和中央的20个视野计算活菌体总数，并求出其乳酸活菌平均值A为：85.5；4. Count. Place the dipped piece of bacterial liquid under a microscope for microscopic examination, select 20 fields of vision in the 4 corners and the center of the dipped piece to calculate the total number of viable bacteria, and calculate the average value A of the viable lactic acid bacteria: 85.5;
5、将C、B、A、总稀释倍数的值代入下式，每毫升菌液中含有的乳酸活菌数为：5. Substitute the values of C, B, A, and the total dilution factor into the following formula, and the number of viable lactic acid bacteria contained in each milliliter of bacterial liquid is:
实施例3。Example 3.
1、取次甲基蓝0.5g，碳酸钠0.5g，蒸馏水100ml。混匀，静置0.5h后，过滤，静置2h后备用；1. Take 0.5g of methylene blue, 0.5g of sodium carbonate, and 100ml of distilled water. Mix well, let stand for 0.5h, filter, stand for 2h and set aside;
2、移取1ml乳酸菌培养液，并将其加入到99ml无菌水中，震荡混合均匀，即总稀释倍数为100；2. Pipette 1ml of lactic acid bacteria culture solution, and add it to 99ml of sterile water, shake and mix evenly, that is, the total dilution factor is 100;
3、用微量定量移管吸取5μl菌悬液置干净载玻片上，加5μl的上述染液，盖上盖玻片，注意不可有气泡，也不可将菌液溢出盖玻片以外；3. Use a micro-quantitative pipette to draw 5 μl of the bacterial suspension and place it on a clean glass slide, add 5 μl of the above-mentioned staining solution, and cover with a cover glass. Be careful not to have air bubbles, and do not spill the bacterial solution beyond the cover glass;
4、计数。将菌液浸片置显微镜下镜检，选择浸片4个角和中央的15个视野计算活菌体总数，并求出其乳酸活菌平均值A为：135.6；4. Count. Place the dipped piece of bacterial liquid under a microscope for microscopic inspection, select 15 fields of vision in the 4 corners and the center of the dipped piece to calculate the total number of viable bacteria, and calculate the average value A of the viable lactic acid bacteria: 135.6;
5、将C、B、A、总稀释倍数的值代入下式，每毫升菌液中含有的乳酸活菌数为：5. Substitute the values of C, B, A, and the total dilution factor into the following formula, and the number of viable lactic acid bacteria contained in each milliliter of bacterial liquid is:
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