Some standard content:
Agricultural Industry Standard of the People's Republic of China
NY411--2000
Nitrogen-fixing bacteria fertilizer
Azotobacter fertilizer
Published on 2000-12-22
Published by the Ministry of Agriculture of the People's Republic of China
Implemented on 2001-04-01
Agricultural Industry Standard of the People's Republic of China
Ammonia-fixing bacteria fertilizer
NY411-2000
Published by China Standards Press
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NY411-2000
This standard is based on the original standard NY/T227-1994 "Microbial Fertilizer", and modifies the nitrogen-fixing bacteria fertilizer part. The main modifications are as follows:
1. Delete the harmless index of finished products in the technical requirements; 1. Add the allowable difference in the inspection method, and add the sampling and judgment rules. This standard shall replace the nitrogen-fixing bacteria fertilizer part in NY/T227-1994 from the date of implementation. Appendix A, Appendix B and Appendix C of this standard are all appendices of the standard. This standard is proposed by the Ministry of Agriculture of the People's Republic of China. The drafting units of this standard are: Microbial Fertilizer Quality Supervision, Inspection and Testing Center of the Ministry of Agriculture, Institute of Soil and Fertilizer of the Chinese Academy of Agricultural Sciences. The main drafters of this standard are: Li Yuanfang, Wu Guanyi, Li Huiquan, Ge Cheng, Shen Delong. 1 Scope
Agricultural Industry Standard of the People's Republic of China
Nitrogen-fixing bacteria fertilizer
Azotobacter fertilizer
NY411-2000
This standard specifies the classification, technical requirements, inspection methods, inspection rules, packaging, labeling, transportation, storage, etc. of nitrogen-fixing bacteria fertilizer products. This standard applies to living products containing beneficial nitrogen-fixing bacteria that can fix nitrogen in the air in the soil and the rhizosphere of various crops to provide nitrogen nutrition for crops and can secrete hormones to stimulate crop growth. This standard also applies to compound nitrogen-fixing fertilizers containing plant growth-promoting rhizobacteria (PGPR) that can promote nitrogen fixation and growth, which are mainly nitrogen-fixing bacteria. 2 Referenced standards
The provisions contained in the following standards constitute the provisions of this standard through reference in this standard. When this standard is published, the versions shown are valid. All standards will be revised, and parties using this standard should explore the possibility of using the latest versions of the following standards. GB/T625--1989
Chemical reagents Sulfuric acid
GB/T629—1997
Chemical reagents Sodium hydroxide
GB/T1250—1989
Expression method and judgment method of limit values Corrugated paperboard
GB/T65431986
GB/T6682—1992
3 Definitions
Specifications and test methods for water used in analytical laboratories This standard adopts the following definitions.
3.1 Self-generated nitrogen-fixing bacteria
Microorganisms that can fix nitrogen in the air in the soil, provide nitrogen nutrition for crops, and secrete hormones to stimulate crop growth. 3.2 Rhizosphere-associated nitrogen-fixing bacteria
Microorganisms that not only rely on the rhizosphere environment for growth, but also fix nitrogen in the air in the rhizosphere, and have a positive effect on the growth and development of crops. 3.3 Nitrogen fixation efficiency
The number of milligrams of nitrogen that nitrogen-fixing bacteria absorb from the air for every gram of carbohydrate (sugar) consumed by nitrogen-fixing bacteria. The nitrogen fixation efficiency is expressed in mg nitrogen/g sugar. 4 Product classification
4.1 According to the dosage form, it is divided into: liquid nitrogen-fixing bacteria fertilizer, solid nitrogen-fixing bacteria fertilizer and freeze-dried nitrogen-fixing bacteria fertilizer. 4.2 According to the strain and characteristics, it is divided into: self-generated nitrogen-fixing bacteria fertilizer, rhizosphere-associated nitrogen-fixing bacteria fertilizer, and compound nitrogen-fixing bacteria fertilizer. 5 Technical requirements
5.1 Bacterial species
Can fix molecular nitrogen in a nitrogen-free medium containing an organic carbon source and have a certain nitrogen-fixing efficiency. The bacteria are generally short rod-shaped (the bacteria of the genus Azospirillum are spiral-shaped) and Gram-negative. Approved by the Ministry of Agriculture of the People's Republic of China on December 22, 2000 and implemented on April 1, 2001
5.1.1 Self-generated nitrogen-fixing bacteria
NY411--2000
Use bacteria of the genera Azotobacter and Azomonas. The strains of Azorhizobium caulinodans and Paenibacillus azotofixans can also be used. 5.1.2 Rhizosphere-associated nitrogen-fixing bacteria
The following strains can be used: Azospirillum, Enterobacter cloacae, strains identified as non-pathogenic bacteria, Alcaligenes faecalis, strains identified as non-pathogenic bacteria, and Klebsiella pneumoniae.
When using strains other than those specified in this guideline to produce nitrogen-fixing fertilizers, the strains must be identified and meet the requirements of 5.1. For strains used in the production of nitrogen-fixing microbial fertilizers, it is necessary to perform strain staining identification (see Appendix B) and strain nitrogen fixation efficiency determination (see Appendix C).
5.2 Technical indicators of finished products (see Table 1)
Appearance, odor
Water content, %
Fineness, residue after passing through standard sieve with aperture of 0.18 mm
Sieve residue, %
Effective viable bacteria count, pieces/mL
(pieces/g, pieces/bottle)
Miscellaneous bacteria rate\, %
Validity period", months
Table 1 Technical indicators of finished products
Milky white or light brown liquid, turbid, slightly precipitated, no peculiar smell
1) Including 10~ Martin medium plate without mold solid
Dark brown or brown powder, moist, loose, no peculiar smell
25.0~35.0||tt| |2) This item is only tested when the supervisory department or the arbitration inspection parties deem it necessary. 6 Sampling
Milky white crystals, odorless
The products made from the bacterial liquid in each formaldehyde fermentation tank are considered as a batch, and sampling and inspection are carried out batch by batch. The sampling process must strictly avoid contamination by miscellaneous bacteria. 6.1 Sampling tools and supplies
Before sampling, prepare sterile plastic bags (or plastic bottles), metal spoons, scissors, sealers, kraft paper sealing bags, labels, sampling seals and glue.
6.2 Sampling quantity and sampling method
Sampling in the finished product warehouse can be carried out in a ""-shaped layered manner. The sampling is based on pieces, and small-packaged products are counted as one box. Large-packaged (30-50kg) products are counted as one bag (or one barrel). The number of sampling pieces is determined by the sample base. Size is determined. For 110 pieces, all will be sampled; for 11-200 pieces, 10 pieces will be sampled; for 201-400 pieces, 20 pieces will be sampled; for those with a sample base of more than 400 pieces, the number of samples will be increased by 2% of the excess, but the total number of pieces should not exceed 40 pieces.
Take a small bag from each packaged product and sample 200g from each bag under sterile conditions. Then mix all the samples, reduce them to 2000g by quartering and divide them into 4 bags, which are then sealed. Every 2 bags are put into a kraft paper sealed sample bag. Take 10mL of each liquid product and put them into a sterile conical flask together. After mixing, divide them into two portions, each of 250mL. For freeze-dried products, take one tube from each piece and put it into a sterile conical flask, add sterile liquid culture medium, dissolve and mix, and then divide them into two portions, each of 50mL. Label and seal (One copy is retained by the sampling unit, and one copy is submitted to the testing center for testing). 2
7 Inspection methods
7.1 Instruments and reagents
7.1.1 Instruments
Biological microscope (10×100);
Colony counter,
Constant temperature incubator;
Constant temperature drying oven;
Constant temperature shaker;
Autoclave;
Sterile room or clean bench;
Acidity meter:
NY411-2000
Test tube: q15mm×150mm, 18mm×180mmCulture blood: diameter 9cm;
Triangular flask: 500mL;
Sterile pipette: 0.5, 1, 5, 10mL,
Glass scraper;
Filter paper,
Alcohol lamp;
Standard sieve: aperture 0.18mm and 0.15mm; No. 1 wool brush;
Sterile water,
Deionized water.
7.1.2 Reagents
Selective culture medium (see Appendix A);
Congo red dye solution (0.5%).
7.2 Finished product inspection
7.2.1 Odor inspection
Take a sample of nitrogen-fixing bacteria fertilizer and open the package for sensory inspection. 7.2.2 Appearance inspection
Open the package of solid nitrogen-fixing bacteria fertilizer and put it in a white sugar porcelain dish. Shake the liquid nitrogen-fixing bacteria fertilizer and conduct a sensory inspection under bright light.
7.2.3 pH value determination
pH value determination of liquid nitrogen-fixing bacterial fertilizer: take the test bacterial solution to directly determine the pH value, and take the average of three determination results. pH value determination of solid nitrogen-fixing bacterial fertilizer: take a 50mL beaker, add 25g of bacterial fertilizer and 25mL of deionized water. After the solution is made uniform by a magnetic stirrer, the pH value is determined with an acidometer. Take the average of the three determination results. 7.2.4 Water content determination
Weigh three portions of solid nitrogen-fixing bacterial fertilizer, 20.00g each, accurate to 0.01g, and put them into an aluminum box that has been weighed to a constant weight. Bake in a 105℃ drying oven for 4h, move to a desiccator, and weigh after cooling. Calculate the water content according to formula (1) based on the mass difference before and after drying. Take the average of the three sample determinations. The absolute deviation of parallel samples should be less than 1%. Water content (%) = mo = m × 100
Where: mo—-mass of sample before drying, g;. (1)
-mass of sample after drying, g.
7.2.5 Determination of adsorbent particle fineness
NY411-2000
Weigh 10.00g of sample (accurate to 0.01g), place it in a standard sieve (diameter 0.18mm or 0.15mm respectively), rinse with water, and brush it lightly with a brush until the powder no longer passes through. Dry the sieve residue at 105-110℃ and weigh it. The percentage of sieve residue is calculated according to formula (2):
Sieve residue (%) = sieve residue amount + (1 = percentage of water content of sample crystal) × 100 sample mass
7.2.6 Determination of effective viable bacteria and content of foreign bacteria The effective viable bacteria and foreign bacteria rate are determined by plate count method. 7.2.6.1 Determination steps
· (2)
The sample should be no less than 500g. Weigh 10.00g from it and add it to 100mL of sterile water with glass beads (take 10mL of liquid bacterial agent and add it to 90mL of sterile water). After standing for 20min, fully shake it on a rotary shaker at 200r/min for 30min to form a bacterial suspension of the mother solution.
Use a sterile pipette to draw 5mL of the bacterial suspension of the above mother solution and add it to 45mL of sterile water. Mix it to form a 1110 diluted bacterial suspension. Dilute it in this way to obtain concentrations of 1:1×10°, 1:1×10°, 1:1×10°, 1:1×105, etc. (the sterile pipette must be changed for each dilution).
Use a 0.5mL sterile pipette to take 0.1mL of bacterial suspension of different dilutions, add it to the surface of the selective culture medium (see Appendix A) with a diameter of 9cm, and use a sterile glass scraper to evenly apply the bacterial suspension on the agar surface, or take 1mL of bacterial suspension of different dilutions and add it to the culture medium, mix it with the agar medium, take 3 consecutive appropriate dilutions for each sample, repeat each dilution 3 times, and add a blank control of sterile water at the same time. After culturing at 28℃ for 2-3 days, take 5-10 colonies of bacteria from each dilution and stain them with smears (see Appendix B). After microscopic observation and identification, select an appropriate dilution (a plate with 30-300 colonies), and calculate the average number of colonies on three plates of the same dilution. The number of miscellaneous bacteria refers to the sum of the number of colonies of other bacteria except effective live bacteria on the selective culture medium and the number of molds on the Martin culture medium. Martin culture medium 10-dilution bacterial suspension is added, and the operation steps are the same as the effective live bacteria detection operation of the sample. 7.2.6.2 Allowable error
Parallel sample error is calculated according to formula (3) and shall comply with the provisions of Table 2. )
Where: 8—single standard deviation;
A—the measurement value of any plate with the same dilution;
A—the average value of the measurement of the plate with the same dilution;
Table 2 Single standard deviation corresponding to the number of colonies on the plate Number of colonies on the plate, pieces/blood
Single standard deviation,
7.2.6.3 Calculation of measurement results
Calculate the effective viable count and foreign bacteria rate of the sample according to formula (4) and formula (5) Effective viable count (pieces/g) = average number of colonies × dilution factor × volume of mother solution bacterial suspension (mL) Number of bacteria in grams or milliliters
Number of foreign bacteria
Further bacteria rate (%) =
Effective count + number of foreign bacteria|| tt||100～300
Sample volume (mL)
（3）
....(4)
....(5)
7.2.7 Shelf life inspection 10 days before the expiration date indicated in the product manual, measure the various indicators of the finished product, the method is the same as 7.2.1～7.2.6.4
8 Inspection rules
8.1 Inspection classification
8.1.1 Product factory inspection
Inspection conducted when the product is delivered.
8.1.2 Product type inspection
NY411—2000
New product identification or quality supervision inspection by national quality inspection agencies. 8.2 Inspection items
Type inspection items shall be carried out in accordance with the requirements of 5.2. Factory inspection does not check the shelf life. 8.3 Judgment rules
8.3.1 Qualified products:
a) Products whose test results meet the technical indicators specified in 5.2 are qualified products; b) When the effective viable bacteria count of the product meets the indicators, and the foreign bacteria rate does not meet the indicators, but is less than twice the requirements of this standard (10% for liquids, 30% for solids, and 4% for freeze-dried bottles), and the mold count on the Martin culture medium plate is less than 30×105, and one of the other indicators does not meet the requirements, it can also be judged as a qualified product;
c) When the effective viable bacteria count and foreign bacteria rate of the product meet the indicators, and three of the secondary test items such as moisture, appearance, pH value, and fineness do not meet the technical indicators, it can also be judged as a qualified product. 8.3.2 Unqualified products:
a) If the number of effective live bacteria of the product does not meet the technical indicators or the input bacteria are not completely detected, it is judged as an unqualified product; b) If the rate of foreign bacteria in the product exceeds twice the standard (10% for liquid, 30% for solid, and 4% for freeze-dried bottles), it is judged as an unqualified product; c) If the number of effective live bacteria of the product meets the indicator, the rate of foreign bacteria does not meet the indicator, but is less than twice the standard (10% for liquid, 30% for solid, and 4% for freeze-dried bottles), and two of the other indicators do not meet the indicator, it is judged as an unqualified product; d) If the number of molds on the Martin medium plate is ≥30×105, the product is judged as an unqualified product; e) If 4 of the secondary test items such as moisture, pH value, fineness, and appearance of the product do not meet the technical indicators, it is judged as an unqualified product. 8.3.3 When testing nitrogen-fixing fertilizer products containing plant growth-promoting rhizobacteria (PGPR), only the number of nitrogen-fixing bacteria is measured, and the number of plant growth-promoting rhizobacteria is not measured, and it is not considered as foreign bacteria. 8.3.4 The quality indicators in this standard are judged to be qualified by comparison with the rounded values in GB/T1250. 9 Packaging, labeling, transportation and storage
9.1 Packaging
9.1.1 Inner packaging
Liquid fertilizers are packaged in plastic bottles or glass bottles for small packages and plastic barrels for large packages. Solid fertilizers are packaged in opaque polyethylene plastic bags. Freeze-dried bacterial agents are vacuum dried in glass finger tubes.
9.1.2 Outer packaging
The outer packaging is made of cartons, and the quality of the cartons should meet the requirements of GB/T6543. The outside of the box is reinforced with nylon strapping tape. 9.1.3 Each box (bag) of products is accompanied by a product certificate and instructions for use, and the instructions for use indicate the method of use, dosage and precautions. 9.2 Labeling
The packaging box (bag) is printed with the product name, trademark, standard number, fertilizer registration certificate number, production unit, factory address, production date, batch number and net weight, and printed with sun protection, moisture-proof, anti-freeze and other marks. If necessary, fragile and anti-inversion marks should also be printed. The inner packaging has the product name, trademark, standard number, fertilizer registration certificate number, effective bacteria content, production date, validity period, product performance, instruction manual and production unit, factory address, etc.
9.3 Transportation
It is suitable for common transportation tools. There should be covers during transportation to prevent rain, sun exposure and high temperatures above 35°C. When the temperature is below 0°C, take 5
NY411-2000
insulation measures to prevent the bacterial fertilizer from freezing. Load and unload gently during transportation to avoid damage to the packaging. 9.4 Storage
The product should be stored in a cool, dry and ventilated warehouse. The optimum temperature is 10℃～25℃. It should not be piled in the open air to prevent rain and sun, freezing and prolonged high temperature above 35℃. NY411—2000
Appendix A
(Standard Appendix)
Selective culture medium for determination of effective viable count and foreign bacteria (molds) A1 Ashby medium for nitrogen-fixing bacteria Potassium dihydrogen phosphate (KHPO.)
Magnesium sulfate (MgSO,·7HO)
Sodium chloride (NaCI)
Calcium carbonate (CaCO,)
Mannitol (C.HuO)
Calcium sulfate (CaSO·2H,0)
A2 Ammonia-fixing (stem nodule) rhizobium culture medium
Sodium lactate (C,H,O,Na)
Dipotassium hydrogen phosphate (K ,HPO）
Potassium dihydrogen phosphate (KHPO)
Sodium chloride (NaCI)
Calcium chloride (CaCl2)
Ferric chloride (FeCl,)
Magnesium sulfate (MgSO·7HO)
Yeast extract
(or yeast powder
A3 combined nitrogen-fixing bacteria culture medium
Sodium D-gluconate (CH,O,Na)
Potassium dihydrogen phosphate (KH,PO,)
Potassium dihydrogen phosphate (K,HPO,)
Magnesium sulfate (MgSO.·7H.O)
Alcohol master paste
(or alcohol master powder
Sodium chloride (NaCI)
Calcium chloride (CaClz)
Iron chloride (FeCl3)
Sodium molybdate (NazMoO,)
A4 Martin medium (for testing mold count)
Dipotassium hydrogen phosphate (K,HPO)
Magnesium sulfate (MgSO·7H,O)
Glucose (C.Hi2O.H,O)
Protein Chen
NY411—2000
1% Bengal red aqueous solution [tetrafluoroethylene tetraiodofluorescein (C2H,CI,IO,) Add 0.1g chloranil violet to each liter of culture medium and sterilize together. Note: The above culture media need to be distilled 1000mL water, 18~20g agar. Appendix B
(Standard Appendix)
Staining agent and staining method
B1 Capsule staining method
B1.1 Staining agent
Carbofuchsin staining solution
Solution A: Basic fuchsin (Basicfuchsin) (CzoHzoCIN,) 95% alcohol (CHCHOH)
Solution B: Carbofuchsin (CH,OH)
Distilled water
a) Grind basic fuchsin in a mortar, gradually add 95% alcohol, continue grinding to dissolve it, and prepare solution A. b) Dissolve carbofuchsin in distilled water to prepare solution B. c) Mix solution A and solution B and shake to make carbofuchsin. The mixed solution can usually be diluted 5 to 10 times for use. The diluted solution is easy to deteriorate and become ineffective, so it is not advisable to prepare more at one time. B1.2 Staining method
B1.2.1 Take a small amount of cultured bacteria and apply it to the water droplets on the slide to form a thin layer. B1.2.2 Dry naturally or heat slightly.
B1.2.3 After staining with carbolic acid fuchsin for 1 minute, wash with water and examine under a microscope after drying. B1.3 Staining results
The bacteria are red, and there is a transparent circle around the bacteria, which is the membrane. B2
Spore staining method
B2.1 Staining agentwww.bzxz.net
B2.1.15% Malachite green
Malachite green (C23H2sCIN,)
Distilled water
B2.1.20.05% basic fuchsin
Basic fuchsin (CH2CIN,)
95% alcohol
B2.2 Staining method
B2.2.1 Preparation of slides.
B2.2.2 Add 3-5 drops of Malachite green dye solution to the smear, heat with an alcohol lamp until steam is emitted but not boiling, and add dye solution at any time to keep the smear from drying out. Stain for about 5-10 minutes.
B2.2.3 Rinse with tap water for 30 seconds.
NY411--2000
B2.2.4 Stain with 0.05% alkaline fuchsin for 1 min, wash with water, and examine under a microscope (if the concentration of 0.05% alkaline fuchsin is too high, it can be diluted appropriately according to the specific situation).
B2.3 Staining results
Spores are green, and bacteria are red.
B3 Gram staining method
B3.1 Staining agent
B3.1.1 Crystal violet staining solution
Solution A: Crystal violet (C2H3CIN,·9H,O) ethanol (95%) (CH,CH2OH)
Solution B: Ammonium oxalate [(NH),C,·H,O)
Distilled water
Solution A and B are mixed and allowed to stand for 48 hours before use. B3.1.2 Lugol's iodine solution
Iodine (I2) tablets
Potassium iodide (KI)
Distilled water
First dissolve potassium iodide with a small amount (3~5mL) of distilled water, then add the iodine tablets. After the iodine is completely dissolved, add 100mL of water to make a mother solution. When using, add twice the amount of water to dilute it to Lugol's iodine solution. B3.1.3 Decolorizing solution: 95% ethanol.
B3.1.4 Counterstaining solution: 0.5% safranin aqueous solution 2.5% safranin alcohol solution
Distilled water
B3.2 Staining method
B3.2.1 Preparation of slides.
Add crystal violet solution dropwise and cover for 1min.
Rinse the crystal violet solution with water.
B3.2.4 Add iodine solution dropwise to rinse off the residual water and cover for 1min. B3.2.5
Rinse the iodine solution with water and absorb the water with absorbent paper. 10ml
Add a few drops of 95% alcohol and shake gently to decolorize. After 30s, wash with water and absorb the water. After dyeing with safranin stain solution for 10s, rinse with tap water. Dry and examine under a microscope. B3.3 Staining results
Gram's positive reaction bacteria are purple, and negative reaction bacteria are red. Flagella staining
B4.1 Staining agents
Liquid A: Tannic acid (C6Hs2046)
Ferric fluoride (FeCL)
Formaldehyde (15%) (HCHO)
Sodium hydroxide (NaOH) (1%)
Distilled water
Liquid B: Silver nitrate (AgNO,)
Distilled water1 Staining agent
Carbofuchsin staining solution
Liquid A: Basic fuchsin (Basicfuchsin) (CzoHzoCIN,) 95% alcohol (CHCHOH)
Liquid B: Carbofuchsin (CH,OH)
Distilled water
a) Grind basic fuchsin in a mortar, gradually add 95% alcohol, continue grinding to dissolve it, and prepare liquid A. b) Dissolve carbofuchsin in distilled water to prepare liquid B. c) Mix liquid A and liquid B and shake to make carbofuchsin. The mixture can usually be diluted 5 to 10 times for use. The diluted solution is easy to deteriorate and become ineffective, so it is not advisable to prepare more at one time. B1.2 Staining method
B1.2.1 Take a small amount of cultured bacteria and apply it to the water droplets on the slide to form a thin layer. B1.2.2 Dry naturally or heat slightly.
B1.2.3 After dyeing with carbolic acid fuchsin for 1 minute, wash with water, dry and examine under a microscope. B1.3 Staining results
The bacteria are red, and there is a transparent circle around the bacteria, which is the membrane. B2
Spore staining method
B2.1 Staining agent
B2.1.15% malachite green
Malachite green (C23H2sCIN,)
Distilled water
B2.1.20.05% basic fuchsin
Basic fuchsin (CH2CIN,)
95% alcohol
B2.2 Staining method
B2.2.1 Preparation of slides.
B2.2.2 Add 3-5 drops of malachite green dye to the smear, heat with an alcohol lamp until steam is emitted but not boiling, and add dye at any time to keep the smear from drying out. Stain for about 5-10 minutes.
B2.2.3 Rinse with tap water for 30 seconds.
NY411--2000
B2.2.4 Stain with 0.05% alkaline fuchsin for 1 minute, wash with water, and examine under a microscope (if the concentration of 0.05% alkaline fuchsin is too high, it can be appropriately diluted according to the specific situation).
B2.3 Staining results
Spores are green, and bacteria are red.
B3 Gram staining method
B3.1 Staining agent
B3.1.1 Crystal violet staining solution
Solution A: Crystal violet (C2H3CIN, ·9H,O) ethanol (95%) (CH,CH2OH)
Solution B: Ammonium oxalate [(NH),C,·H,O)
Distilled water
Solution A and B are mixed and allowed to stand for 48 hours before use. B3.1.2 Lugol's iodine solution
Iodine (I2) tablets
Potassium iodide (KI)
Distilled water
First, use a small amount (3~5mL) of distilled water to dissolve potassium iodide, then add iodine tablets. After the iodine is completely dissolved, add 100mL of water to make a mother solution. When using, add twice as much water to dilute it into Lugol's iodine solution. B3.1.3 Decolorizing solution: 95% ethanol.
B3.1.4 Counterstaining solution: 0.5% safranin aqueous solution 2.5% safranin alcohol solution
Distilled water
B3.2 Staining method
B3.2.1 Preparation.
Add crystal violet solution and cover for 1 minute.
Rinse the crystal violet solution with water.
B3.2.4 Add iodine solution to rinse off the residual water and cover for 1 minute. B3.2.5
Rinse off the iodine solution with water and absorb the water with absorbent paper. 10ml
Add a few drops of 95% alcohol and shake gently to decolorize. After 30 seconds, wash with water and absorb the water. After staining with safranin staining solution for 10 seconds, rinse with tap water. Dry and examine under a microscope. B3.3 Staining results
Gram-positive bacteria are purple, and negative bacteria are red. Flagella staining
B4.1 Staining agent
Liquid A: Tannic acid (C6Hs2046)
Ferrous fluoride (FeCL)
Formaldehyde (15%) (HCHO)
Sodium hydroxide (NaOH) (1%)
Distilled water
Liquid B: Silver nitrate (AgNO,)
Distilled water1 Staining agent
Carbofuchsin staining solution
Liquid A: Basic fuchsin (Basicfuchsin) (CzoHzoCIN,) 95% alcohol (CHCHOH)
Liquid B: Carbofuchsin (CH,OH)
Distilled water
a) Grind basic fuchsin in a mortar, gradually add 95% alcohol, continue grinding to dissolve it, and prepare liquid A. b) Dissolve carbofuchsin in distilled water to prepare liquid B. c) Mix liquid A and liquid B and shake to make carbofuchsin. The mixture can usually be diluted 5 to 10 times for use. The diluted solution is easy to deteriorate and become ineffective, so it is not advisable to prepare more at one time. B1.2 Staining method
B1.2.1 Take a small amount of cultured bacteria and apply it to the water droplets on the slide to form a thin layer. B1.2.2 Dry naturally or heat slightly.
B1.2.3 After dyeing with carbolic acid fuchsin for 1 minute, wash with water, dry and examine under a microscope. B1.3 Staining results
The bacteria are red, and there is a transparent circle around the bacteria, which is the membrane. B2
Spore staining method
B2.1 Staining agent
B2.1.15% malachite green
Malachite green (C23H2sCIN,)
Distilled water
B2.1.20.05% basic fuchsin
Basic fuchsin (CH2CIN,)
95% alcohol
B2.2 Staining method
B2.2.1 Preparation of slides.
B2.2.2 Add 3-5 drops of malachite green dye to the smear, heat with an alcohol lamp until steam is emitted but not boiling, and add dye at any time to keep the smear from drying out. Stain for about 5-10 minutes.
B2.2.3 Rinse with tap water for 30 seconds.
NY411--2000
B2.2.4 Stain with 0.05% alkaline fuchsin for 1 minute, wash with water, and examine under a microscope (if the concentration of 0.05% alkaline fuchsin is too high, it can be appropriately diluted according to the specific situation).
B2.3 Staining results
Spores are green, and bacteria are red.
B3 Gram staining method
B3.1 Staining agent
B3.1.1 Crystal violet staining solution
Solution A: Crystal violet (C2H3CIN, ·9H,O) ethanol (95%) (CH,CH2OH)
Solution B: Ammonium oxalate [(NH),C,·H,O)
Distilled water
Solution A and B are mixed and allowed to stand for 48 hours before use. B3.1.2 Lugol's iodine solution
Iodine (I2) tablets
Potassium iodide (KI)
Distilled water
First, use a small amount (3~5mL) of distilled water to dissolve potassium iodide, then add iodine tablets. After the iodine is completely dissolved, add 100mL of water to make a mother solution. When using, add twice as much water to dilute it into Lugol's iodine solution. B3.1.3 Decolorizing solution: 95% ethanol.
B3.1.4 Counterstaining solution: 0.5% safranin aqueous solution 2.5% safranin alcohol solution
Distilled water
B3.2 Staining method
B3.2.1 Preparation.
Add crystal violet solution and cover for 1 minute.
Rinse the crystal violet solution with water.
B3.2.4 Add iodine solution to rinse off the residual water and cover for 1 minute. B3.2.5
Rinse off the iodine solution with water and absorb the water with absorbent paper. 10ml
Add a few drops of 95% alcohol and shake gently to decolorize. After 30 seconds, wash with water and absorb the water. After staining with safranin staining solution for 10 seconds, rinse with tap water. Dry and examine under a microscope. B3.3 Staining results
Gram-positive bacteria are purple, and negative bacteria are red. Flagella staining
B4.1 Staining agent
Liquid A: Tannic acid (C6Hs2046)
Ferrous fluoride (FeCL)
Formaldehyde (15%) (HCHO)
Sodium hydroxide (NaOH) (1%)
Distilled water
Liquid B: Silver nitrate (AgNO,)
Distilled water
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