CN112210584A - Rapid lactic acid bacteria counting method - Google Patents
Rapid lactic acid bacteria counting method Download PDFInfo
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- CN112210584A CN112210584A CN202010854135.XA CN202010854135A CN112210584A CN 112210584 A CN112210584 A CN 112210584A CN 202010854135 A CN202010854135 A CN 202010854135A CN 112210584 A CN112210584 A CN 112210584A
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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 84
- 241000894006 Bacteria Species 0.000 title claims abstract description 67
- 239000004310 lactic acid Substances 0.000 title claims abstract description 42
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000000243 solution Substances 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000012192 staining solution Substances 0.000 claims abstract description 19
- 238000004043 dyeing Methods 0.000 claims abstract description 18
- 230000000007 visual effect Effects 0.000 claims abstract description 18
- 238000005406 washing Methods 0.000 claims abstract description 16
- 229910052740 iodine Inorganic materials 0.000 claims abstract description 12
- 239000011630 iodine Substances 0.000 claims abstract description 12
- 239000000523 sample Substances 0.000 claims abstract description 12
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000010790 dilution Methods 0.000 claims abstract description 10
- 239000012895 dilution Substances 0.000 claims abstract description 10
- 239000011521 glass Substances 0.000 claims abstract description 9
- 238000003235 crystal violet staining Methods 0.000 claims abstract description 6
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000007865 diluting Methods 0.000 claims abstract description 5
- 238000001035 drying Methods 0.000 claims abstract description 5
- 239000004576 sand Substances 0.000 claims abstract description 5
- 239000012470 diluted sample Substances 0.000 claims abstract description 4
- 238000010186 staining Methods 0.000 claims description 3
- 241000186660 Lactobacillus Species 0.000 abstract description 8
- 229940039696 lactobacillus Drugs 0.000 abstract description 8
- 230000007547 defect Effects 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 4
- 241000194017 Streptococcus Species 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 238000010561 standard procedure Methods 0.000 abstract description 3
- 241000192125 Firmicutes Species 0.000 description 6
- 239000013078 crystal Substances 0.000 description 6
- 210000002421 cell wall Anatomy 0.000 description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 241000194020 Streptococcus thermophilus Species 0.000 description 4
- 239000007788 liquid Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 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 description 2
- 241000192142 Proteobacteria Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 229960000907 methylthioninium chloride Drugs 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 108700009560 Bacteria Q Proteins 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- ZINJLDJMHCUBIP-UHFFFAOYSA-N ethametsulfuron-methyl Chemical compound CCOC1=NC(NC)=NC(NC(=O)NS(=O)(=O)C=2C(=CC=CC=2)C(=O)OC)=N1 ZINJLDJMHCUBIP-UHFFFAOYSA-N 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Toxicology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a rapid lactic acid bacteria counting method, which comprises the following steps: s1, diluting the sample to be detected by a set dilution gradient F; s2, absorbing the diluted sample to be detected, smearing the sample on a set area M on a glass slide, dyeing with crystal violet staining solution, washing with water, dyeing with gram iodine solution, washing with water again, decoloring with ethanol, washing with water again, adding yellow sand staining solution or safranin staining solution for counterdyeing, drying and performing microscopic examination; s3, examining a plurality of different visual fields in the set region M of the slide glass, recording the number of purple viable bacteria in each visual field, and obtaining the average number of viable bacteria n in each visual field; the invention can directly count the number of the lactobacillus and the streptococcus, shortens the detection time to 1 hour, solves the defect of long time consumption of the national standard method, uses less reagents and saves the cost for enterprises.
Description
Technical Field
The invention relates to the field of lactic acid bacteria counting, in particular to a rapid lactic acid bacteria counting method.
Background
In the prior art, for example, chinese patent application with publication number CN101659982A discloses a method for rapidly counting total viable bacteria of lactic acid bacteria, which comprises the steps of: taking 0.1-1g of methylene blue, 0.5-2g of sodium carbonate and 100ml of distilled water, uniformly mixing, standing for 0.5-2h, filtering, and standing for 2 h; weighing 1 gram of bacteria powder or transferring 1ml of lactic acid bacteria culture solution, diluting with 10-100 times of sterile water to obtain bacteria suspension; transferring the bacterial suspension, and performing 10-fold gradient dilution by using sterile water; sucking 5 mul of bacterial suspension, placing on a glass slide, adding 5 mul of the dye solution, and covering the glass slide; selecting 10-20 visual fields to calculate the total viable count of the lactic acid bacteria, and calculating the average value of the viable count of the lactic acid bacteria; and calculating the number of the viable lactic acid bacteria contained in each gram of bacteria powder/each milliliter of bacteria liquid to be the formula, wherein C is the area of the cover glass, B is the area of one visual field, A is the average value of the number of the viable lactic acid bacteria in each visual field, and the like. According to the technical scheme, the dyeing solution prepared from the methylene blue and the sodium carbonate is adopted for dyeing in a ratio of 1:1, and as the lactic acid bacteria are gram-positive bacteria, when purple bacteria are observed to be counted, the problem that the negative bacteria are mistakenly counted as the lactic acid bacteria possibly exists, and the counting accuracy is influenced.
The existing national standard GB 4789.35-2016 lactic acid bacteria requires about 72h +/-2 h for counting lactic acid bacteria, because one lactic acid bacteria absorbs nutrition and proliferates, and finally, a colony visible to naked eyes needs a long time for counting. Moreover, lactobacillus is an anaerobic bacterium, streptococcus thermophilus is a facultative anaerobic bacterium, and the culture conditions of the lactobacillus and the streptococcus thermophilus are different, so that the lactobacillus and the streptococcus thermophilus can only be separately detected, and manpower and culture media are consumed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a rapid lactic acid bacteria counting method, which can directly count the number of lactobacillus and streptococcus, shortens the detection time to 1 hour, overcomes the defect of long time consumption of the national standard method, uses fewer reagents, saves the cost for enterprises and the like.
The purpose of the invention is realized by the following scheme:
a rapid lactic acid bacteria counting method comprises the following steps:
s1, diluting the sample to be detected by a set dilution gradient F;
s2, absorbing the diluted sample to be detected, smearing the sample on a set area M on a glass slide, dyeing with crystal violet staining solution, washing with water, dyeing with gram iodine solution, washing with water again, decoloring with ethanol, washing with water again, adding yellow sand staining solution or safranin staining solution for counterdyeing, drying and performing microscopic examination;
s3, a plurality of different visual fields are examined in the slide setting region M, the number of purple viable bacteria in each visual field is recorded, and the average number of viable bacteria n in each visual field is determined.
Further, comprising the steps of:
s4, the area S1 of the objective lens is measured by a micrometer, and the size N of the objective lens in the set region M is calculated by setting the area M/S1 to N.
Further, comprising the steps of:
s5, the total number q of live lactic acid bacteria in the set region M is calculated to be N × N.
Further, comprising the steps of:
and S6, calculating the total number Q of the live lactic acid bacteria before the sample to be detected is diluted to be Q multiplied by F.
Further, the dilution gradient F is at 104～106In the meantime.
Further, the dye is dyed by crystal violet dyeing liquid for 1-2 mins.
Further, staining was performed again with gram iodine solution for 1 min.
Further, decoloring for 20-30 s by using ethanol.
Further, counterdyeing for 1-2 mins.
Further, 10 different fields of view are microscopically examined within the slide setting region M.
The invention has the beneficial effects that:
(1) according to the invention, lactic acid bacteria are counted by a microscope, and the lactobacillus is dyed by using a crystal violet dyeing solution, washed by water, dyed by using a gram iodine solution, washed by water, decolorized by using ethanol, and counterdyed by adding a yellow sand dyeing solution or a safranin dyeing solution, so that the number of lactobacillus and streptococcus can be directly counted, the detection time is shortened to 1 hour, the defect of long time consumption of a national standard method is solved, meanwhile, fewer reagents are used, and the cost is saved for enterprises.
(2) The present invention uses gram staining because lactic acid bacteria are gram positive bacteria, the color is purple, and the negative bacteria are red. During observation, only the purple bacteria need to be counted, so that the negative bacteria can not be counted as lactic acid bacteria by mistake, and the detection accuracy is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a flow chart of the method steps of the present invention;
FIG. 2 is a schematic front view of a slide according to the present invention;
FIG. 3 is a schematic side view of a slide glass according to the present invention.
Detailed Description
All of the features disclosed in the specification for all of the embodiments (including any accompanying claims, abstract and drawings), or all of the steps of a method or process so disclosed, may be combined or substituted in any combination or manner, except for mutually exclusive features and/or steps.
As shown in fig. 1 to 3, a method for rapidly counting lactic acid bacteria comprises the steps of:
s1, diluting the sample to be detected by a set dilution gradient F;
s2, absorbing the diluted sample to be detected, smearing the sample on a set area M on a glass slide, dyeing with crystal violet staining solution, washing with water, dyeing with gram iodine solution, washing with water again, decoloring with ethanol, washing with water again, adding yellow sand staining solution or safranin staining solution for counterdyeing, drying and performing microscopic examination;
the cell wall of gram-positive bacteria is thicker, and is washed by ethanol after being dyed by crystal violet and iodine solution, so that the cell wall of the gram-positive bacteria can be dehydrated and shrunk, and the dye solution is retained in the cell wall; the cell wall of gram-negative bacteria is thin, large gaps appear on the cell wall when the cells are washed by ethanol, crystal violet and iodine staining solution can flow out, the cells are colorless, and the negative bacteria are redly stained by salsa staining solution. The positive bacteria are purple, so that the positive bacteria and the negative bacteria can be distinguished.
S3, a plurality of different visual fields are examined in the slide setting region M, the number of purple viable bacteria in each visual field is recorded, and the average number of viable bacteria n in each visual field is determined.
Further, comprising the steps of:
s4, the area S1 of the objective lens is measured by a micrometer, and the size N of the objective lens in the set region M is calculated by setting the area M/S1 to N.
Further, comprising the steps of:
s5, the total number q of live lactic acid bacteria in the set region M is calculated to be N × N.
Further, comprising the steps of:
and S6, calculating the total number Q of the live lactic acid bacteria before the sample to be detected is diluted to be Q multiplied by F.
Further, the dilution gradient F is at 104～106In the meantime.
Further, the dye is dyed by crystal violet dyeing liquid for 1-2 mins.
Further, staining was performed again with gram iodine solution for 1 min.
Further, decoloring for 20-30 s by using ethanol.
Further, counterdyeing for 1-2 mins.
Further, 10 different fields of view are microscopically examined within the slide setting region M.
Each washing was to rinse off crystal violet, iodine staining solution and ethanol.
Examples
1. For a sample to be tested 25g, 10 was selected4Performing smear observation by using the dilution gradient F; the dilution gradient for the observation was set at 104～106Can facilitate counting.
2. Sucking 10ul of the uniform solution, uniformly smearing the uniform solution in a 10 × 10mm area of a glass slide, namely setting an area M as the 10 × 10mm area shown in figure 2, dyeing for 1min by using crystal violet staining solution, washing with water, dyeing for 1min by using gram iodine solution, washing with water, decoloring for 30s by using 95% ethanol, washing with water, adding yellow staining solution or safranin staining solution, re-dyeing for 1min, drying, and performing microscopic examination;
3. 10 different visual fields are microscopically examined in a 10 x 10mm area, the number of purple viable bacteria in each visual field is recorded, purple is gram-positive bacteria, red is gram-negative bacteria, lactobacillus and streptococcus thermophilus are gram-positive bacteria, and the average number of viable bacteria n in each visual field can be quickly obtained according to the color.
4. Measuring the area s1 of the objective lens by a micrometer, and calculating the size N of the objective lens in the 10 multiplied by 10mm area;
5. calculating the total number q of the live lactic acid bacteria in the set region M as Nxn;
6. the total number of live lactic acid bacteria Q (pieces/ml) was calculated as N × F.
Other embodiments than the above examples may be devised by those skilled in the art based on the foregoing disclosure, or by adapting and using knowledge or techniques of the relevant art, and features of various embodiments may be interchanged or substituted and such modifications and variations that may be made by those skilled in the art without departing from the spirit and scope of the present invention are intended to be within the scope of the following claims.
Claims (10)
1. A rapid lactic acid bacteria counting method is characterized by comprising the following steps:
s1, diluting the sample to be detected by a set dilution gradient F;
s2, absorbing the diluted sample to be detected, smearing the sample on a set area M on a glass slide, dyeing with crystal violet staining solution, washing with water, dyeing with gram iodine solution, washing with water again, decoloring with ethanol, washing with water again, adding yellow sand staining solution or safranin staining solution for counterdyeing, drying and performing microscopic examination;
s3, a plurality of different visual fields are examined in the slide setting region M, the number of purple viable bacteria in each visual field is recorded, and the average number of viable bacteria n in each visual field is determined.
2. The method for rapidly counting lactic acid bacteria according to claim 1, comprising the steps of:
s4, the area S1 of the objective lens is measured by a micrometer, and the size N of the objective lens in the set region M is calculated by setting the area M/S1 to N.
3. The method for rapidly counting lactic acid bacteria according to claim 2, comprising the steps of:
s5, the total number q of live lactic acid bacteria in the set region M is calculated to be N × N.
4. The method for rapidly counting lactic acid bacteria according to claim 1, comprising the steps of:
and S6, calculating the total number Q of the live lactic acid bacteria before the sample to be detected is diluted to be Q multiplied by F.
5. The method for rapidly counting lactic acid bacteria according to claim 1, wherein the dilution gradient F is 104～106In the meantime.
6. The method for rapidly counting lactic acid bacteria according to claim 1, wherein the lactic acid bacteria are stained with a crystal violet staining solution for 1 to 2 mins.
7. The method for rapidly counting lactic acid bacteria according to claim 1, wherein the staining is performed for 1min with a gram-iodine solution.
8. The method for rapidly counting lactic acid bacteria according to claim 1, wherein the lactic acid bacteria are decolorized with ethanol for 20 to 30 seconds.
9. The method for rapidly counting lactic acid bacteria according to claim 1, wherein the counterstaining is performed for 1 to 2 mins.
10. The method for rapidly counting lactic acid bacteria according to claim 1, wherein 10 different fields of view are microscopically examined in a set region M of the slide.
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| CN202010854135.XA CN112210584A (en) | 2020-08-24 | 2020-08-24 | Rapid lactic acid bacteria counting method |
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| CN202010854135.XA CN112210584A (en) | 2020-08-24 | 2020-08-24 | Rapid lactic acid bacteria counting method |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851664A (en) * | 2009-12-30 | 2010-10-06 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting bifidobacterium in sour milk |
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2020
- 2020-08-24 CN CN202010854135.XA patent/CN112210584A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851664A (en) * | 2009-12-30 | 2010-10-06 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting bifidobacterium in sour milk |
Non-Patent Citations (3)
| Title |
|---|
| 孟晓华;: "市售酸奶中乳酸菌的分离与鉴定", 农产品加工 * |
| 李文斌;唐中伟;宋敏丽;李增波;温艳珍;吴晓丽;: "农家泡菜发酵液中乳酸菌的研究", 食品工程 * |
| 辛若竹: "活性酸乳中乳酸菌的直接镜检计数法", 《中国卫生检验杂志》 * |
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