CN117589543A - Composition for acid-fast bacteria staining, staining method and kit - Google Patents
Composition for acid-fast bacteria staining, staining method and kit Download PDFInfo
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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
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- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/32—Mycobacterium
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- G—PHYSICS
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- 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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Abstract
The invention belongs to the technical field of medical detection, and discloses a composition, a dyeing method and a kit for acid-fast bacteria dyeing. The composition for acid fast bacteria staining comprises a primary staining solution, a decolorizing solution and a double staining solution; the primary dyeing liquid comprises the following components: basic fuchsin dye, ethanol, surfactant and cationic surfactant; and the primary dye liquor does not contain phenol. The invention adopts the surfactant and the cationic surfactant to replace phenol, and the effect of the surfactant for acid-fast bacteria staining is equal to or better than the effect of traditional phenol-containing staining agent; compared with phenol which can cause serious harm to human body and environment, the invention adopts the surfactant and the cationic surfactant to be safer, eliminates the toxic factor and carcinogenicity of phenol to human body, and greatly reduces the harm of dye liquor to environment and water body; according to the invention, the malachite green dye liquor or the brilliant green dye liquor is adopted, so that the coloring of the thalli is more prominent, the thalli are in strong contrast with the background, the acid-fast bacteria are easier to find, and the detection rate is improved.
Description
Technical Field
The invention belongs to the technical field of medical detection, and particularly relates to a composition, a dyeing method and a kit for acid-fast bacteria dyeing.
Background
Acid fast bacilli, also known as mycobacteria, are a class of elongated bacilli that are known for their tendency to grow in branching when propagated. The surface of the acid-fast bacillus has a layer of lipoid or lipoid film, which is not easy to be colored, but even an acidic alcohol solution is not easy to decolorize after the coloring. For example, a common mycobacterium tuberculosis is one of the mycobacteria, and when it infects the lung, it causes pulmonary tuberculosis, which poses a great threat to human health.
At present, two standard methods for acid fast bacteria staining recommended by World Health Organization (WHO) are available, one is an alkaline reddening method, which is also called a thermal staining method; the other is the fluorescent dye method, including the gold amine O method and the gold amine O-rhodamine B method, but the fluorescent dye method is limited by fluorescent equipment and technology, so the thermal dyeing method is the preferred method for current smear dyeing.
The thermal dyeing method mainly comprises an initial dyeing liquid (carbolic acid alkaline reddish dye liquor), a decolorizing liquid (acid alcohol solution) and a counterstain liquid (methylene blue dye liquor), wherein the carbolic acid alkaline reddish dye liquor contains phenol which not only plays a role in sterilization but also plays a role in penetrating agent and dyeing auxiliary, and is a key component for promoting alkaline reddish dye to enter thalli and enabling the dye to be difficult to decolorize by acid alcohol in the thalli. When the carbolic acid alkaline reddish dye solution is added and the dyeing is promoted by heating, the alkaline reddish dye-acid-fast bacteria form a firm compound which is red, the compound is decolorized by an acidic alcohol solution, other bacteria and tissue cells except the acid-fast bacteria and the background are removed to be colorless or light, finally the compound dyeing is carried out by using a methylene blue dye solution, other bacteria and tissue cells and the background are dyed with methylene blue, and the red acid-fast bacteria and the blue background are compared to identify the acid-fast bacteria.
Phenol (also called carbolic acid, hydroxy benzene) is the simplest phenolic organic matter, is weak acid, is colorless crystal at normal temperature, is toxic, has strong corrosion effect on skin and mucous membrane, can cause burn of human body, and can inhibit central nerve or damage liver and kidney functions by inhalation. Has serious harm to the environment and can pollute the water body and the atmosphere.
Therefore, it is necessary to develop a product which is free of phenol addition and which can be used for staining acid-fast bacteria.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. For this reason, the invention provides a composition for acid fast bacteria staining, a staining method and a staining kit, the composition does not need to add phenol, and the effect of the composition for acid fast bacteria staining is equal to or better than that of the traditional phenol-containing staining agent.
The first aspect of the invention provides a composition for acid fast staining, comprising a primary staining solution, a decolorizing solution and a counterstain solution; the primary dyeing liquid comprises the following components: basic fuchsin dye, ethanol, surfactant and cationic surfactant; and the primary dye liquor does not contain phenol.
Because the cell wall of the acid-fast bacillus contains negatively charged fatty acid chains, the interaction of surface active substances (negatively charged) and cell membranes can induce the perforation of a lipid bilayer and form ion channels, so that alkaline fuchsin (cations) can enter bacteria conveniently, and meanwhile, the cationic surface active substances are positively charged and combined with negatively charged molecules or groups on the cell membranes, and the arrangement mode of the cell membrane bilayer, namely the tension thereof, is changed due to the action of electrostatic attraction and Van der Waals force, so that the permeability of the cell membranes is enhanced; according to the invention, through the combined action of the surfactant and the cationic surfactant, basic fuchsin can easily enter bacteria for coloring under the condition of no phenol.
The surfactant adopted by the invention is a biosurfactant and an amphiphilic cyclic lipopeptide, which is a cyclic lipopeptide formed by combining cyclic anion heptapeptide (L-Glu/L-Leu/D-Leu/L-Val/L-Asp/D-Leu/L-Leu) and beta-hydroxy fatty acid with carbon chain length of 12 in a lactone bond in bacillus subtilis.
According to some embodiments of the invention, the primary dye liquor consists of the following components: 1-5wt% of basic fuchsin dye, 20-30wt% of ethanol, 14-20 mu mol/L of surfactant, 2-6wt% of cationic surfactant and the balance of water. The adoption of the surfactant with proper concentration can better promote membrane separation and induce perforation of a phospholipid layer and formation of an ion channel; if the concentration is too low, the permeation effect is insufficient, and if the concentration is too high, the cell membrane is easily and completely destroyed, and the staining effect is poor.
According to some embodiments of the invention, the cationic surfactant comprises a quaternary ammonium salt surfactant.
According to some embodiments of the invention, the quaternary ammonium salt surfactant comprises at least one of cetyl ammonium bromide, dodecyl trimethyl ammonium chloride.
According to some embodiments of the invention, the decolorized solution is an acidic alcoholic solution.
According to some embodiments of the invention, the acidic alcoholic solution comprises the following components: 1-6wt% of hydrochloric acid, 74-84wt% of ethanol and 10-25wt% of deionized water.
According to some embodiments of the invention, the counterstain is a malachite green dye liquor or a brilliant green dye liquor. Because strong alkali is needed to be added when the double-dyeing liquid (methylene blue dyeing liquid) is prepared according to the traditional method, the blue color of the background dyeing is not pure, usually in gray blue, purple blue or deep blue, cannot form strong contrast with the purple red of acid-fast bacteria dyed by alkaline reddish brown, and even in the area of the thickness of the sputum film, the purple red thallus is covered by the purple blue background and is not easy to identify. According to the invention, the malachite green dye liquor or the bright green dye liquor is adopted, so that the background cells, other bacteria and the like are dyed green, the bright red acid-fast bacteria can be more set off by the bright green, and red acid-fast bacteria can not be covered in the places with thick phlegm, so that the background contrast is increased, the thallus identification degree is improved, compared with the traditional comparison of purplish red and purplish blue, the acid-fast bacteria can be more easily found, and the detection rate is further improved.
According to some embodiments of the invention, the malachite green dye liquor comprises the following components: 0.03-0.08wt% of malachite green dye, 8-15wt% of ethanol and the balance of water.
According to some embodiments of the invention, the bright green dye solution comprises the following components: 0.03-0.08wt% of bright green dye, 8-15wt% of ethanol and the balance of water.
The second aspect of the invention provides a staining method of acid-fast bacteria, which adopts the composition for acid-fast bacteria staining to stain, and comprises the following steps:
and coating a sample to be detected on a glass slide, and sequentially adopting the primary dyeing liquid for primary dyeing, decoloring the decoloring liquid and counterstaining the counterstaining liquid.
According to some embodiments of the invention, the method for staining acid-fast bacteria comprises the following steps:
(1) Coating a sample to be measured on a glass slide, naturally drying, heating and fixing, and then dripping a primary dye solution on the glass slide and heating to perform primary dyeing, wherein the primary dye solution covers or submerges all the samples to be measured;
(2) After the primary dye solution on the glass slide is washed by flowing water, a decolorizing solution is dripped on the glass slide for decolorizing until the sample to be measured has no visible color;
(3) Flushing the decolorizing liquid on the glass slide with flowing water, and then dripping counterstaining liquid on the glass slide for counterstaining;
(4) After washing off the counterstain on the slide with running water, observation was performed under a microscope.
According to some embodiments of the invention, the microscopic observations are: acid-fast bacteria are bright red or bright red, and other bacteria, tissue cells and background are green.
According to some embodiments of the invention, after the primary stain is added dropwise to the slide, the stain is maintained for 5-10 minutes by heating until vapor appears on the slide.
According to some embodiments of the invention, the counterstaining is performed for a time period of 30-60s.
The third aspect of the invention provides a kit for acid fast staining, comprising the composition for acid fast staining.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts the surfactant and the cationic surfactant to replace phenol, and the effect of the surfactant for acid-fast bacteria staining is equal to or better than the effect of the traditional phenol-containing staining agent, so that the thalli are bright red or bright red (the traditional phenol-containing staining is purple); compared with phenol which can cause serious harm to human body and environment, the invention adopts the surfactant and the cationic surfactant, which is safer, eliminates the toxic factor and carcinogenicity of phenol to human body, and greatly reduces the harm of dye liquor to environment and water body.
The counterstain liquid adopts the malachite green dye liquid or the brilliant green dye liquid to replace the methylene blue dye liquid, so that the thallus is more prominently colored, and is strongly compared with the background, and compared with the traditional purple red and purple blue contrast, the counterstain liquid is easier to find acid-fast bacteria, and the detection rate is further improved.
Drawings
The invention is further described below with reference to the drawings and examples.
FIG. 1 is a graph showing the dyeing effect of example 1;
FIG. 2 is a graph showing the dyeing effect of example 2;
FIG. 3 is a graph showing the dyeing effect of example 3;
FIG. 4 is a graph showing the dyeing effect of example 4;
FIG. 5 is a graph showing the dyeing effect of example 5;
FIG. 6 is a graph showing the dyeing effect of comparative example 1;
FIG. 7 is a graph showing the dyeing effect of comparative example 2;
FIG. 8 is a graph showing the dyeing effect of comparative example 3.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
Example 1
The present example provides a composition for acid fast staining consisting essentially of a primary staining solution, a decolorizing solution and a counterstain solution, wherein:
primary dyeing liquid: 4wt% of basic fuchsin dye, 24wt% of ethanol, 16 mu mol/L of surfactant, 4wt% of cetyl ammonium bromide and the balance of distilled water;
decolorization liquid: 5wt% of hydrochloric acid, 85wt% of ethanol and 10wt% of deionized water;
and (3) a double-dyeing solution: 0.06wt% of malachite green dye, 10wt% of ethanol and the balance of distilled water.
A staining method of acid-fast bacteria adopts the composition for acid-fast bacteria staining to stain, and comprises the following steps:
(1) Coating a sample to be measured on a glass slide, naturally drying, and then placing the sample on a dyeing rack, wherein the distance between the glass slides is kept to be more than 10 mm; heating and fixing (the slide is passed back and forth through flame for 4 times within 5 seconds), and dripping the primary dyeing liquid on the slide for primary dyeing (by heating until steam appears on the slide, keeping dyeing for 4 minutes), wherein the primary dyeing liquid covers or submerges all samples to be detected;
(2) Washing from one end of the glass slide with running water, draining after washing the primary dye liquor on the glass slide, and adding a decolorizing liquid dropwise on the glass slide for decolorizing for 1.5min; if the decolorization is not thorough, washing off the decolorization liquid by using running water, and then carrying out the decolorization step again until the sample to be detected has no visible color;
(3) Washing the glass slide for 15s by using running water from one end of the glass slide, draining after washing off the decolorized solution on the glass slide, and dripping a counterstain solution on the glass slide for counterstaining for 45s;
(4) Washing with flowing water from one end of the slide, flushing to remove counterstain liquid on the slide, and observing under a microscope.
The results observed under the microscope were: acid-fast bacteria are bright red or bright red, and other bacteria, tissue cells and background are green.
Example 2
The present example provides a composition for acid fast staining consisting essentially of a primary staining solution, a decolorizing solution and a counterstain solution, wherein:
primary dyeing liquid: 1wt% of basic fuchsin dye, 20wt% of ethanol, 14 mu mol/L of surfactant, 2wt% of cetyl ammonium bromide and the balance of distilled water;
decolorization liquid: 3wt% of hydrochloric acid, 87wt% of ethanol and 10wt% of deionized water;
and (3) a double-dyeing solution: 0.03 weight percent of malachite green dye, 8 weight percent of ethanol and the balance of distilled water.
A staining method of acid-fast bacteria adopts the composition for acid-fast bacteria staining to stain, and comprises the following steps:
(1) Coating a sample to be measured on a glass slide, naturally drying, and then placing the sample on a dyeing rack, wherein the distance between the glass slides is kept to be more than 10 mm; heating and fixing (the slide is passed back and forth through flame for 4 times within 5 seconds), and dripping the primary dyeing liquid on the slide for primary dyeing (by heating until steam appears on the slide, keeping dyeing for 6 minutes), wherein the primary dyeing liquid covers or submerges all samples to be detected;
(2) Washing the glass slide from one end of the glass slide with running water, draining after washing the primary dye solution on the glass slide, and adding a decolorizing solution dropwise on the glass slide for decolorizing for 2min; if the decolorization is not thorough, washing off the decolorization liquid by using running water, and then carrying out the decolorization step again until the sample to be detected has no visible color;
(3) Washing the glass slide for 20s by using running water from one end of the glass slide, draining after washing off the decolorized solution on the glass slide, and dripping a counterstain solution on the glass slide for counterstaining for 60s;
(4) Washing with flowing water from one end of the slide, flushing to remove counterstain liquid on the slide, and observing under a microscope.
The results observed under the microscope were: acid-fast bacteria are bright red or bright red, and other bacteria, tissue cells and background are green.
Example 3
The present example provides a composition for acid fast staining consisting essentially of a primary staining solution, a decolorizing solution and a counterstain solution, wherein:
primary dyeing liquid: 5wt% of basic fuchsin dye, 30wt% of ethanol, 20 mu mol/L of surfactant, 6wt% of dodecyl trimethyl ammonium chloride and the balance of distilled water;
decolorization liquid: 6wt% of hydrochloric acid, 74wt% of ethanol and 20wt% of deionized water;
and (3) a double-dyeing solution: 0.08wt% of malachite green dye, 15wt% of ethanol and the balance of distilled water.
A staining method of acid-fast bacteria adopts the composition for acid-fast bacteria staining to stain, and comprises the following steps:
(1) Coating a sample to be measured on a glass slide, naturally drying, and then placing the sample on a dyeing rack, wherein the distance between the glass slides is kept to be more than 10 mm; heating and fixing (the slide is passed back and forth through flame for 4 times within 5 seconds), and dripping the primary dyeing liquid on the slide for primary dyeing (by heating until steam appears on the slide, keeping dyeing for 2 minutes), wherein the primary dyeing liquid covers or submerges all samples to be detected;
(2) Washing from one end of the glass slide with running water, draining after washing the primary dye solution on the glass slide, and adding a decolorizing solution dropwise on the glass slide for decolorizing for 1min; if the decolorization is not thorough, washing off the decolorization liquid by using running water, and then carrying out the decolorization step again until the sample to be detected has no visible color;
(3) Washing the glass slide from one end of the glass slide for 10s by using running water, draining after washing off the decolorized solution on the glass slide, and dripping a counterstain solution on the glass slide for counterstaining for 30s;
(4) Washing with flowing water from one end of the slide, flushing to remove counterstain liquid on the slide, and observing under a microscope.
The results observed under the microscope were: acid-fast bacteria are bright red or bright red, and other bacteria, tissue cells and background are green.
Example 4
The present example provides a composition for acid fast staining consisting essentially of a primary staining solution, a decolorizing solution and a counterstain solution, wherein:
primary dyeing liquid: 5wt% of basic fuchsin dye, 30wt% of ethanol, 20 mu mol/L of surfactant, 6wt% of dodecyl trimethyl ammonium chloride and the balance of distilled water;
decolorization liquid: 6wt% of hydrochloric acid, 74wt% of ethanol and 20wt% of deionized water;
and (3) a double-dyeing solution: bright green dye 0.08wt%, ethanol 15wt% and distilled water for the rest.
A staining method of acid-fast bacteria adopts the composition for acid-fast bacteria staining to stain, and comprises the following steps:
(1) Coating a sample to be measured on a glass slide, naturally drying, and then placing the sample on a dyeing rack, wherein the distance between the glass slides is kept to be more than 10 mm; heating and fixing (the slide is passed back and forth through flame for 4 times within 5 seconds), and dripping the primary dyeing liquid on the slide for primary dyeing (by heating until steam appears on the slide, keeping dyeing for 2 minutes), wherein the primary dyeing liquid covers or submerges all samples to be detected;
(2) Washing from one end of the glass slide with running water, draining after washing the primary dye solution on the glass slide, and adding a decolorizing solution dropwise on the glass slide for decolorizing for 1min; if the decolorization is not thorough, washing off the decolorization liquid by using running water, and then carrying out the decolorization step again until the sample to be detected has no visible color;
(3) Washing the glass slide from one end of the glass slide for 10s by using running water, draining after washing off the decolorized solution on the glass slide, and dripping a counterstain solution on the glass slide for counterstaining for 30s;
(4) Washing with flowing water from one end of the slide, flushing to remove counterstain liquid on the slide, and observing under a microscope.
The results observed under the microscope were: acid-fast bacteria are bright red or bright red, and other bacteria, tissue cells and background are green.
Example 5 (differing from example 1 in that methylene blue was used instead of malachite green)
The present example provides a composition for acid fast staining consisting essentially of a primary staining solution, a decolorizing solution and a counterstain solution, wherein:
4wt% of basic fuchsin dye, 24wt% of ethanol, 16 mu mol/L of surfactant, 4wt% of cetyl ammonium bromide and the balance of distilled water;
decolorization liquid: 5wt% of hydrochloric acid, 85wt% of ethanol and 10wt% of deionized water;
and (3) a double-dyeing solution: 0.06wt% of methylene blue dye, 10wt% of ethanol and the balance of distilled water.
A staining method for acid-fast bacteria was the same as that of example 1, except that staining was performed using the composition for acid-fast bacteria staining provided in example 5.
Comparative example 1 (the difference from example 1 is that no surfactant was added)
The comparative example provides a composition for acid fast staining, consisting essentially of a primary staining solution, a decolorizing solution and a counterstain solution, wherein:
primary dyeing liquid: 4wt% of basic fuchsin dye, 24wt% of ethanol, 4wt% of cetyl ammonium bromide and the balance of distilled water;
decolorization liquid: 5wt% of hydrochloric acid, 85wt% of ethanol and 10wt% of deionized water;
and (3) a double-dyeing solution: 0.06wt% of malachite green dye, 10wt% of ethanol and the balance of distilled water.
A staining method for acid-fast bacteria was the same as that of example 1, except that staining was performed using the composition for acid-fast bacteria staining provided in comparative example 1.
Comparative example 2 (differing from example 1 in that no hexadecyl ammonium bromide was added)
The comparative example provides a composition for acid fast staining, consisting essentially of a primary staining solution, a decolorizing solution and a counterstain solution, wherein:
primary dyeing liquid: 4wt% of basic fuchsin dye, 24wt% of ethanol, 16 mu mol/L of surfactant and the balance of distilled water;
decolorization liquid: 5wt% of hydrochloric acid, 85wt% of ethanol and 10wt% of deionized water;
and (3) a double-dyeing solution: 0.06wt% of malachite green dye, 10wt% of ethanol and the balance of distilled water.
A staining method for acid-fast bacteria was the same as that of example 1, except that staining was performed using the composition for acid-fast bacteria staining provided in comparative example 2.
Comparative example 3 (differing from example 1 in that the nonionic surfactant Tergitol-TMN-3 was used instead of cetylammonium bromide)
The comparative example provides a composition for acid fast staining, consisting essentially of a primary staining solution, a decolorizing solution and a counterstain solution, wherein:
primary dyeing liquid: 4wt% of basic fuchsin dye, 24wt% of ethanol, 16 mu mol/L, tergitol-TMN-3-4 wt% of surfactant and the balance of distilled water;
decolorization liquid: 5wt% of hydrochloric acid, 85wt% of ethanol and 10wt% of deionized water;
and (3) a double-dyeing solution: 0.06wt% of malachite green dye, 10wt% of ethanol and the balance of distilled water.
A staining method for acid-fast bacteria was the same as that of example 1, except that staining was performed using the composition for acid-fast bacteria staining provided in comparative example 3.
Dyeing effect test
Feasibility test
The staining methods of examples 1 to 5 of the present invention were used to stain smears of Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 25922), respectively, and the results showed that both bacteria were acid-fast negative, demonstrating that the staining results of the composition for acid-fast staining of the present invention did not produce false positives.
Then, accidental mycobacteria (ATCC 6841) were sampled and stained with smears by the staining methods of examples 1 to 5 of the present invention, respectively, and the results showed that the bacteria were positive.
The results show that the composition for staining acid-fast bacteria can be used for staining acid-fast bacteria and detecting and identifying, and the feasibility of the composition is proved.
Detection rate test
200 parts of sputum specimens of tuberculosis patients and suspected tuberculosis patients in a laboratory were selected by cooperating with a national tuberculosis reference laboratory of a national tuberculosis prevention control center for Chinese disease control, and stained by using conventional acid fast staining methods (thermal staining method), examples 1 to 5 and comparative examples 1 to 3, respectively, and the acid fast staining methods, compositions for acid fast staining provided in examples 1 to 5 and compositions for acid fast staining provided in comparative examples 1 to 3 were compared with each other under the same microscope magnification for the detection rate of acid bacteria. Wherein the detection standard is shown in table 1, and the detection result is shown in table 2; the graphs of staining effect under the microscope of examples 1 to 5 and comparative examples 1 to 3 are shown in FIGS. 1 to 8, respectively.
TABLE 1
| Acid fast bacteria negative (-) | The 300 different visual fields were observed continuously, and no acid-fast bacteria were found |
| Reporting the acid fast bacteria count (< 1+) | 1-8/300 field of view |
| Acid fast bacteria positive (1+) | 3-9/100 visual fields, 300 visual fields are observed continuously |
| Acid fast bacteria positive (2+) | 1-9/10 visual fields, and 100 visual fields are observed continuously |
| Acid fast bacteria positive (3+) | 1-9 strips per field of view |
| Acid fast bacteria positive (4+) | Not less than 10 pieces per field of view |
TABLE 2
As can be seen from the data in Table 2 and the dyeing effect graphs of FIGS. 1 to 8, the composition for acid fast fungus dyeing of the present invention has a dyeing effect substantially equal to or slightly better than that of a conventional dye liquor containing phenol, and has a good detection effect. Comparative examples 1 and 2 were poor in detection effect due to the lack of surfactant and cetyl ammonium bromide, respectively; the comparative example 3 adopts a nonionic surfactant, and the detection effect is not as good as that of the invention, which shows that the composition for acid fast fungus staining of the invention can well promote alkaline reddish brown to enter thalli under the combined action of the surfactant and the cationic surfactant, has good staining effect, and can completely replace the traditional phenol. In addition, in example 5, the methylene blue counterstain background was used to slightly lower the detection effect than in example 1, which indicates that the invention can make the bacterial cells more prominently colored and more easily found by using malachite green or bright green counterstain liquid with better contrast.
Claims (10)
1. A composition for acid fast staining, comprising an initial staining solution, a decolorizing solution and a counterstain solution; the primary dyeing liquid comprises the following components: basic fuchsin dye, ethanol, surfactant and cationic surfactant; and the primary dye liquor does not contain phenol.
2. The composition for acid fast staining according to claim 1 wherein the primary staining solution consists of: 1-5wt% of basic fuchsin dye, 20-30wt% of ethanol, 14-20 mu mol/L of surfactant, 2-6wt% of cationic surfactant and the balance of water.
3. The composition for acid fast staining according to claim 1 or 2, wherein the cationic surfactant comprises a quaternary ammonium salt type surfactant.
4. The composition for acid fast staining according to claim 3, wherein the quaternary ammonium salt surfactant comprises at least one of cetylammonium bromide and dodecyltrimethylammonium chloride.
5. The composition for acid fast staining according to claim 1 wherein the decolorizing solution is an acidic alcoholic solution.
6. The composition for acid fast staining according to claim 1 wherein the counterstain is a malachite green dye liquor or a brilliant green dye liquor.
7. A staining method of acid-fast bacteria, characterized by using the composition for acid-fast bacteria staining according to any one of claims 1 to 6, comprising the steps of:
and coating a sample to be detected on a glass slide, and sequentially adopting the primary dyeing liquid for primary dyeing, decoloring the decoloring liquid and counterstaining the counterstaining liquid.
8. The method according to claim 7, wherein the initial dyeing liquid is dropped onto the slide, and then heated until vapor is generated on the slide, and the slide is kept dyed for 2 to 6 minutes.
9. Dyeing process according to claim 7, characterized in that the counterstaining is carried out for a time of 30-60s.
10. A kit for acid fast staining, comprising the composition for acid fast staining according to any one of claims 1 to 6.
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