Some standard content:
NY412-2000
This standard amends the phosphorus bacteria fertilizer part of the original standard NY/T227--1994 "Microbial Fertilizer". The main modifications are as follows:
- Added terms, sampling and judgment rules; - Added strains, including strain effectiveness, bacterial characteristics, and colony morphology; - Deleted the indicators of finished product harmlessness. Because phosphorus bacteria fertilizers are generally carried by peat, there is no harm from heavy metals. After high-temperature sterilization of peat, coliform bacteria and insect eggs no longer exist. From the date of implementation, this standard will replace the phosphorus bacteria fertilizer part in NY/T227-1994. Appendix A, Appendix B, Appendix C, and Appendix D of this standard are all appendices to the standard. This standard was proposed by the Ministry of Agriculture of the People's Republic of China. The drafting units of this standard: Microbial Fertilizer Quality Supervision, Inspection and Testing Center of the Ministry of Agriculture, Soil and Fertilizer Research Institute of the Chinese Academy of Agricultural Sciences. The main drafters of this standard: Liang Shaofen, Jiang Ruibo, Ge Cheng, Li Jun, Shen Delong 3
1 Scope
Agricultural Industry Standard of the People's Republic of China
Phosphate bacteria fertilizer
Phosphate bacteria fertilizerNY 412 2000
This standard specifies the classification, technical requirements, sampling, inspection methods, inspection rules, packaging, labeling, transportation and storage of phosphorus bacteria fertilizer products.
This standard applies to living microbial products containing beneficial phosphorus bacteria microorganisms that can decompose insoluble phosphides in the soil, improve the phosphorus nutrition status of crops, and secrete stimulants to stimulate the growth and development of crops. 2 Reference 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 the parties using this standard should explore the possibility of using the latest versions of the following standards. GB/T6543--1986 Corrugated paper box
NY411--2000 Nitrogen-fixing bacteria fertilizer
3 Definitions
This standard adopts the following definitions.
3.1 Phosphorus bacteria fertilizer
Living microbial products that can convert insoluble phosphorus in the soil into effective phosphorus nutrients that can be used by crops and can secrete hormones to stimulate crop growth.
4 Product classification
4.1 According to the dosage form, it can be divided into: liquid phosphorus bacteria fertilizer, solid powdered phosphorus bacteria fertilizer and granular phosphorus bacteria fertilizer. 4.2 According to the strain and the characteristics of the fertilizer, it can be divided into: organic phosphorus bacteria fertilizer and inorganic phosphorus bacteria fertilizer. 4.2.1 Organic phosphorus bacteria fertilizer: a microbial fertilizer made by beneficial microorganisms that can decompose organic phosphides (lecithin, nucleic acid and phytochemicals, etc.) in the soil. Bacteria that decompose organic phosphates include species in the genus Bacillus (Bcillus sp.) and species in the genus Paenibucillus sp.
4.2.2 Inorganic phosphorus bacterial fertilizers: They can dissolve inorganic phosphates in the soil that are difficult to dissolve and cannot be directly absorbed and utilized by crops and convert them into effective phosphates that can be absorbed and utilized by crops. Bacteria that decompose inorganic phosphates include species in the genus Pseudomonus sp., species in the genus Aicatigenes sp., and species in the genus Thiobacillus sp. When using strains other than those specified in this standard to produce phosphorus bacterial fertilizers, the strains must be identified and must be non-pathogenic strains. 5 Technical requirements
5.1 Effectiveness of strains
The strains used to produce phosphorus bacterial fertilizers must be non-pathogenic strains introduced from national strain centers or national scientific research institutions and identified as non-pathogenic to animals and plants; these strains can be cultured on agar plates containing lecithin or tricalcium phosphate, and obvious phosphorus dissolution zones can be observed; the amount of phosphorus solubilized after fermentation culture is significantly different from that of the uninoculated control (p≤0.05). 5.1.1 Organophosphorus bacteria: Bacillus bacteria are Gram-positive, can produce heat-resistant spores, have oval or cylindrical peritrichal or lateral flagella, can move, and can produce catalase. 5.1.2 Inorganic phosphorus bacteria: Pseudomonas bacteria are Gram-negative rods with polar single flagella or clustered flagella, can move, and are catalase positive. Some strains of this genus are pathogenic bacteria and must be strictly identified before they can be used in production. Bacteria of the genus Alcaligenes. The cells are rod-shaped, with 1 to 4 peritrichous flagella, can move, are Gram-negative, and are catalase positive. Bacteria of the genus Thiobacillus are Gram-negative rods with a single polar flagellum, can move, and are strictly autotrophic. 5.2 Product Technical Specifications
5.2.1 Technical specifications of liquid phosphorus bacterial fertilizers are shown in Table 1. Table 1 Technical indicators of liquid phosphorus bacterial fertilizer
Appearance, odor
Effective live bacteria count
100 million/mL
Organic phosphorus bacterial fertilizer
Inorganic phosphorus bacterial fertilizer
Miscellaneous bacteria rate\, %
Validity period, month
Light yellow or grayish white turbid liquid, with slight precipitation, slight odor or no odor 2.0
1) The miscellaneous bacteria rate includes the number of miscellaneous bacteria on the selective culture medium and the number of molds on the Martin culture medium. The regulations on the number of molds are as follows: the number of molds for general phosphorus bacterial fertilizers is required to be less than 30.0×105 pieces/ml (g), and the number of molds for seed dressing phosphorus bacterial fertilizers is required to be less than 10.0×10+ pieces/ml (g) 5. 2.2
The technical indicators of solid (powdered) phosphorus bacterial fertilizers are shown in Table 2 Table 2 Technical indicators of solid (powdered) phosphorus bacterial fertilizers Item
Appearance, odor
Moisture, %
Effective number of live bacteria
100 million pieces/g
Fineness (particle size)
Organic phosphorus bacterial fertilizers
Inorganic phosphorus bacterial fertilizers
Miscellaneous bacteria rate, %
Validity period, month
5.2.3 The technical indicators of solid (granular) phosphorus bacterial fertilizers are shown in Table 3. Refers to
Powdered, loose, moist, no mold block, no moldy smell, slight odor2550
The residue passing through the standard sieve with an aperture of 0.20mm≤10%6.0~7.5
Technical indicators of solid (granular) phosphorus bacterial fertilizer3
Appearance, odor
Water content, %
Effective number of viable bacteria
100 million/g
Organic phosphorus bacterial fertilizer
Inorganic Phosphorus bacterial fertilizer
Fineness (particle size)
Loose, black or gray particles, slightly smelly
All pass through the standard sieve with an aperture of 2.5~4.5mm 50
6 Sampling
Miscellaneous bacteria rate, %
Validity period. Month
NY412--2000
Table 3 (end)
The product made from the bacterial liquid in each fermentation tank is considered as one 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, sealing machines, kraft paper sample bags, labels, sample seals and glue.
6.2 Sampling quantity and sampling method
Sampling in the finished product warehouse can be carried out by setting points in a “:” shape. The sampling is based on pieces. For small-packaged products, one package box is one piece. For large-packaged products (30-50kg), one bag (or one barrel) is one piece. The number of sampling pieces is determined by the size of the sample base. All samples within 10 pieces are sampled; 10 pieces are sampled for 11-200 pieces, and 20 pieces are sampled for 201-400 pieces. For sample bases greater than 400 pieces, the number of samples is increased by 2% of the excess. However, the total number of pieces should not exceed 40 pieces.
Take a small bag for each small-packaged product, and sample 200g from each bag under sterile conditions. Then mix all the samples, reduce them to 2000g by quartering, and pack them into 4 bags, and then seal them. Each 2 bags are packed into a cowhide sample bag. Finally, affix the sampling label and sampling seal (the same method is also used for small packages of liquid fertilizer). One copy of the sample is retained by the sampled unit, and one copy is submitted to the testing center for testing. 7 Inspection methods
7.1 Instruments and reagents
7.1.1 Instruments
Constant temperature incubator;
Constant temperature drying oven;
Constant temperature shaking table;
High-speed centrifuge;
Biological microscope (10X100);
Autoclave;
Acidity meter;
Torsion balance (division value 0.01g)
Colony counter;
Sterile room or clean bench;
Petri dish: 9cm in diameter;
Erlenmeyer flask: 500mL, 100mL;
Beaker: 500ml;
Measuring cylinder: 100ml;
Sterile pipette: 0.5, 1.0, 5.0, 10.0mL; Glass scraper;
Filter paper;
Alcohol lamp;
Standard sieve: pore size 0.18mm, 2.5mm, 5.0mm; s0r
Aluminum box;
No. 1 wool brush.
7.1.2 Reagents and culture media
7.1.2.1 Reagents
Distilled water;
Deionized water;
Sterile water.
7.1.2.2 Culture media (see Appendix D)
7.1.2.2.1 Broth culture media (see Appendix D1). NY 412--2000
7.1.2.2.2 Organic phosphorus bacterial culture media (see Appendix D2). 7.1.2.2.3 Inorganic phosphorus bacterial culture media (see Appendix D3). 7.1.2.2.4 Martin culture media (see Appendix D4). 7.2 Finished product inspection
7.2.1 Odor inspection
Open the package of liquid or solid phosphorus bacterial fertilizer and smell its odor by manually using the air at the packaging opening. 7.2.2 Appearance inspection
Open the package of the national phosphorus bacterial fertilizer, place it in a white porcelain plate, shake the liquid fertilizer, and observe it under bright light. 7.2.3 pH value determination
Determination of pH value of liquid phosphorus bacterial fertilizer: Take the test bacterial solution in a 50mL beaker and directly measure the pH value with an acidometer, and take the average of three measurement results.
Determination of pH value of solid phosphorus bacterial fertilizer: Weigh 15.00g of the test sample on a torsion balance with a graduation value of 0.01g, place it in a 500mL beaker, measure 30.0mL of distilled water with a measuring cylinder, stir it with a magnetic force or shake it by hand for 1min, then let it stand for 30min, measure its pH value with an acidometer, repeat 3 times, and take the arithmetic mean. 7.2.4 Determination of moisture content
Weigh 3 portions of solid phosphorus bacterial fertilizer, 20.00g each, accurate to 0.01g, 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 dryer, and weigh them after cooling. Take the average of the three sample measurements. The absolute deviation of parallel samples should be less than 1%. The moisture content is calculated according to formula (1):
Moisture content (%) = m = ㎡ × 100
Wu Zhong: ma-sample mass before drying, g
m.sample mass after drying·g.
7.2.5 Determination of adsorbent particle fineness
· (1)
Accurately weigh 10.0g of sample, place it in a standard sieve with an aperture of 0.20mm, rinse with water, and brush it lightly with a brush until the sample 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):
Weight of sieve residue (1-percentage of sample moisture content) × 100Sieve residue (%) =
Sample mass
Particle size of granular fertilizer can be directly passed through 2.5mm and 4.5mm standard sieves after weighing. 7.2.6 Determination of effective viable bacteria count and foreign bacteria rate of phosphorus bacterial fertilizer The effective viable bacteria count and foreign bacteria rate are determined by plate count method. 7.2.6.1 Preparation of broth culture plate and Martin culture plate (formula see Appendix D) 7.2.6.2 Sample dilution culture and counting
Weigh 10)g of sample for inspection (accurate to 0.01g) and put it into a conical flask with glass beads and 100ml sterile water (if the sample for inspection is liquid 507
NY 412—2000
, take 10ml and add it to a triangular flask containing 90mL sterile water), place it on a shaker with a speed of 200r/min for 30min, then let it stand for 20min and dilute it to 101 (liquid sample is diluted to 108) by 10-fold dilution method. Take 0.1mL from each of the last three concentrations of suspension and add it to a plate with a diameter of 9cm containing culture medium, and spread the bacterial solution evenly with a glass scraper. Repeat each dilution 3 times. Culture at 28~30C for 2~5d. Add 10-3 dilution suspension to Martin culture medium to determine the number of molds. 7.2.6.3 Colony identification and counting
Distinguish the strains of the tested product from miscellaneous bacteria according to their colony characteristics. If necessary, perform Gram staining (see Appendix A) or spore staining (see Appendix B), observe under a microscope, and take 20 to 300 plates for colony counting. Calculate the average number of effective phosphorus bacterial colonies for three replicates. The bacterial colonies other than the effective phosphorus bacterial colonies are the bacterial and miscellaneous bacteria colonies. The mold colonies are counted on Martin medium. The number of live effective phosphorus bacteria is calculated according to formula (3):
The number of live effective phosphorus bacteria (pieces/g) = the average number of effective colonies / sample volume (mL) × dilution factor / mother liquor volume (mL)
The number of foreign bacteria is the sum of the number of molds on Martin medium and the number of foreign bacteria on the selection medium. The number of foreign bacteria is calculated according to formula (4): The number of foreign bacteria (pieces/g) = the average number of foreign bacteria colonies × dilution factor × sample volume (g) × sample volume (mL) / mother liquor volume (mL)
The foreign bacteria rate is calculated according to formula (5):
The average number of foreign bacteria colonies
The foreign bacteria rate (%) = the number of phosphorus bacteria colonies / the average number of foreign bacteria colonies × 1007.2.6.4 Determination of phosphorus bacteria phosphate solubilization efficiency
7.2.6.4.1 Qualitative determination of blood
.( 5 )
After melting the culture medium of organic phosphorus bacteria or inorganic phosphorus bacteria, cool it to about 45℃ and pour it into a sterile plate, about 15mL per plate. After solidification, when there is no condensed water on the surface of the culture medium, set it aside for use. After the test bacteria are activated, add an appropriate amount of sterile water, scrape off the bacterial moss, and make a uniform bacterial suspension. Use a sterile dropper to drop it onto a spare plate, and four points can be dropped evenly on each plate. After the dropped bacterial suspension is dry, it can be inverted and placed in a constant temperature box at 28-30℃ for culture. The results can be observed after 2-3 days. The colonies with transparent rings around them are capable of dissolving or solubilizing phosphorus, while those without transparent rings are insoluble phosphorus, less soluble phosphorus, or insoluble phosphorus or less solubilized phosphorus. 7.2.6.4.2 Quantitative determination by molybdenum antimony colorimetric method Inoculate phosphorus bacteria into the corresponding triangular flask of phosphorus bacteria liquid culture medium, place it on a shaker (200r/min) and incubate at 30°C for 72h. After the shaker is removed, add phosphorus-free activated carbon for decolorization and centrifuge at high speed, and pour out the supernatant for testing. Determine the effective phosphorus content by molybdenum antimony colorimetric method (see Appendix B). ||t t||7.2.7 Inspection of validity period
Measure the number of live bacteria 10 days before the expiration date indicated in the product manual. If the number of live bacteria reaches the standard requirements, the validity period is qualified. 8 Inspection rules (determination rules)
8.1 Inspection classification
8.1.1 Product factory inspection
Inspection conducted when the product is delivered.
8.1.2 Product type inspection
New product identification or quality supervision inspection by national quality inspection agencies. 8.2 Inspection items
The inspection items of type inspection shall be carried out in accordance with the requirements of 5.2.1 or 5.2.2. The expiration date is not checked in factory inspection. 8.3 Determination rules
The number of effective live bacteria in the fertilizer is the key indicator of microbial fertilizer quality and is an indicator that all microbial fertilizer products must meet. The specified appearance, moisture, fineness, organic matter and pH value are used as reference indicators for microbial fertilizer quality inspection. The following regulations are made for whether the product is qualified:
NY 412---2000
a) If the effective live bacteria count is unqualified, the product is judged as unqualified; b) If the effective live bacteria count is qualified, the miscellaneous bacteria rate is qualified, and more than four (including four) reference indicators are unqualified, the product is judged as unqualified; c) If the effective live bacteria count is qualified, but the miscellaneous bacteria rate is not qualified, but less than 2 times the reference indicator specified in 5.2.3, if one of the reference indicators is unqualified, the product is judged as qualified; if more than two (including two) reference indicators are unqualified, the product is judged as unqualified; d) If the effective live bacteria count is qualified and the miscellaneous bacteria rate is greater than 2 times the reference indicator specified in 5.2.3; or the mold count of microbial fertilizer is greater than or equal to 6.0×10°/g (ml.) and the mold count of seed dressing agent is greater than or equal to 3.0×105/g (ml), the product is judged as unqualified. If the other items meet the requirements, it can be judged as a qualified product.
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. Granular phosphorus bacterial fertilizers can also be packaged in woven bags. 9.1.2 Outer packaging
Outer packaging is in 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 the product is accompanied by a product certificate and instructions for use, which indicate the method of use, dosage and precautions. 9.2 Labeling, transportation and storage
The labeling, transportation and storage of phosphorus bacterial fertilizers shall comply with 9.2, 9.3 and 9.4 of NY411--2000. 509
A1 Stain
A1.1 Crystal violet solution (Hucker's formula) Solution A: Crystal violet
Ethanol (95%)
Solution B: Ammonium oxalate
Distilled water
NY412-2000
Appendix A
(Standard Appendix)
Gram staining
Mix solutions A and B and let them stand for 48 hours before use. This stain is relatively stable and can be stored for several months in an airtight dark bottle. A1.2 Lugol's iodine solution
Iodine (12) 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 water to 300mL. Store in a brown bottle for later use.
A1.3 Decolorizing solution
95% ethanol solution.
A1.4 Counterstaining solution
0.5% safranin aqueous solution
20mL safranin 2.5% alcohol solution
Distilled water
Store this solution in a brown bottle and dilute it before use. A2 Smear
A2.1 Write the date and slide number on a clean slide without oil stains. A2.2 Add a small drop of sterile water to the slide, pick up a small amount of bacterial moss with an inoculation loop, and lightly smear it on the edge of the water drop. A2.3 Air dry naturally or heat it slightly to speed it up. After drying, pass it over a flame 2~3 times to fix the smear. A3 Staining steps
A3.1 Add crystal violet solution and cover it for about 1 minute. A3.2 Rinse the crystal violet solution with water.
A3.3 First rinse the residual water with iodine solution, then add iodine solution to cover it for about 1 minute. A3.4 Rinse the iodine solution with water, shake off the water on the slide or absorb it with filter paper. A3.5 Tilt the slide and set it against a white background. Drip 95% alcohol solution for about 20~30 seconds, and immediately rinse the alcohol with water. A3.6 Stain with safranin solution for 1~2 (or more) minutes. A3.7 Wash off the safranin with water, absorb the surface water with filter paper or air dry. Prepare for microscopic examination. A4 Observation results
Use a mouse microscope oil lens to observe directly on the slide. Red is Gram-negative, and blue-purple is Gram-positive. Observe the individual shape, arrangement, size, presence of spores and shape, size and location of the bacteria NY 412-2000
. A5 Precautions
A5.1 There are many recipes for Gram's staining, but the success of staining depends largely on experience. When you are not sure, it is best to use Escherichia coli and Staphylococcus aureus as Gram-negative and Gram-positive control bacteria on the same slide. A5.2 In the operation of Jungfrau staining, the smear and decolorization steps are the most important. The smear must not be too thick. For bacteria that are easy to emulsify. Gently apply the inoculation loop stained with bacterial moss on the edge of the water droplet once or twice. During microscopic examination, the Gram staining reaction of dispersed bacteria shall prevail. Too dense bacteria often show false positives. For pure bacterial smears, 95% ethanol is easy to handle. The more water in ethanol, the stronger the decolorization power, which is easy to form false negatives. Therefore, try to reduce the residual water on the slide. Shake off or rinse with ethanol to remove the residual water, and decolorize for 20 seconds; use filter paper to absorb the residual water, and use ethanol to decolorize for nearly 30 seconds. A5.3 Do not overheat when fixing with a flame, so that the slide is not hot. Overheating will cause incorrect staining reaction. In fact, for pure bacterial smears on the slant, they can be stained after air drying without flame fixation. A5.4 The recommended counterstain is 0.5% safranin solution, which is twice as concentrated as the concentration in general literature, so it is better to use. A5.5 Gentian violet is not a single-component dye. Unlike crystal violet, it is often difficult to decolorize after staining, resulting in false positives. In this case, dilute the stain before use. The trial gentian violet can be used after diluting it to one-tenth of the concentration, but the color is still not as good as crystal violet. A5.6 For heterotrophic bacteria that are easy to grow, it is advisable to check bacteria that have been cultured for 18 to 24 hours. The staining reaction of Gram-negative bacteria is stable and not easily affected by the age of the bacteria. The staining reaction of Gram-positive bacteria is sometimes affected by the age of the bacteria: younger cells cultured for 18 to 24 hours or less will show a positive reaction; older cells cultured for 24 hours or more than 48 hours will partially or completely turn into a negative reaction. Be careful when distinguishing Gram staining reactions. Appendix B
(Standard Appendix)
Spore staining
B1 Staining agent
B1.17.6% Malachite green
Malachite green
Distilled water
100 mL
B1.20.5% Safranin solution
Safranine.2.5% alcohol solution
Distilled water
B2 Smear (Method tube A2)
B3 Staining
a) Dye the slide with malachite green stain for about 10 minutes. The effect will be better if heated to make steam appear; b) Rinse with tap water;
c) Restain with 0.5% safranin solution for 30 seconds;
d) Rinse with water, blot dry and examine under a microscope.
B4 Result observation
Observed under the oil microscope, the spores are green, and the bacteria and spores are slightly red. However, when there are metachromatic particles in the bacteria, they can also appear green. 511
C1 Reagent preparation
NY 412—2000
Appendix C
(Standard Appendix)
Determination of effective phosphorus by molybdenum antimony colorimetric method
C1.12,6-dinitrophenol [CsH, OH (NO,) 2] indicator: 0.25g 2,6-dinitrophenol is dissolved in 100mL water (saturated solution). C1.2 Phosphorus-free activated carbon
C1.2.1 Activated carbon with low phosphorus content is directly soaked in 0.5mol sodium bicarbonate (NaHCO:) for 24h. Filter by vacuum on a Buchner funnel, rinse with a small amount of distilled water several times each time until the filtrate is checked to be phosphorus-free, then dry and bottle for later use. C1.2.2 Activated carbon with high phosphorus content should first be soaked in 2mol/L hydrochloric acid (HCi) overnight, rinsed with distilled water several times, and then soaked in 0.5mol sodium bicarbonate (NaHCO).
C1.3 Molybdenum antimony antimony solution
153mL of concentrated sulfuric acid (H2SO4) (analytical grade) is slowly poured into about 400mL of distilled water, stirred and cooled. 10.0g of ammonium molybdate (analytical grade) is dissolved in 300mL of water at about 60°C and cooled. Then the sulfuric acid solution is slowly dripped into the ammonium molybdate solution, and then 100ml.0.5% potassium antimony tartrate solution [K(SbO)CH.O. ·H2O is analytically pure, and finally diluted with water to 1L and stored in a brown bottle. C1.4 Molybdenum antimony colorimetric agent: weigh 1.5g ascorbic acid (CH.O left-handed specific rotation +21~22℃ analytical grade). Dissolve in 100mL saw antimony storage solution. This solution is used immediately after preparation and is valid for 1d. C1.5 5mg/L phosphorus standard solution: weigh 0.439g potassium dihydrogen phosphate (KH,PO4 analytical grade) that has been dried in an oven at 105℃ for 2h and then cooled, dissolve in 200mL water, add 5mL concentrated sulfuric acid (HzSO.), transfer to a 1L volumetric flask, and dilute to the mark with distilled water to obtain a 100mg/I phosphorus standard solution, which can be stored for a long time. Take this solution and dilute it 20 times to obtain a 5mg/L phosphorus standard solution. This solution should not be stored for a long time. C1.6 Prepare 10% sulfuric acid (H,SO4) and 10% sodium hydroxide (NaOH). C2 Operation steps
C2.1 Add appropriate amount of activated carbon to decolorize the fermentation liquid, centrifuge, draw 2~10mL of supernatant (containing 5~25μg phosphorus) into a 50ml volumetric flask, dilute to 20mL with water, add 2 drops of 2,6-dinitrophenol indicator, adjust pH with 10% sodium hydroxide or dilute sulfuric acid solution until the solution is just slightly yellow (add slowly and carefully, shake while adding to prevent the generated carbon dioxide from spraying the solution out of the bottle mouth), then add 5mL of molybdenum antimony anti-color developer, shake well, and adjust to the scale. After placing at room temperature 20℃~25C for 30min, use a wavelength of 680~~700nm (red filter) on a spectrophotometer to compare colors, and use the blank test solution as the reference solution to adjust the zero point. Read the value and find the reading of phosphorus in the color developing solution on the working curve. The color can remain stable within 8h.
C2.2 Drawing of working curve
Pipette 0, 2, 3, 4.5, 6 mL of 5 mg/L phosphorus standard solution into a 50 mL volumetric flask, dilute with water to about 20 mL, add 5 mL of antimony colorimetric agent, shake well to make up to volume, and obtain 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6mg/L phosphorus standard series solution, colorimetric with the solution to be tested, read the value, and draw a working curve on the grid paper with the measured value as the ordinate and the phosphorus mg/L as the abscissa. C3 result calculation
Effective phosphorus is calculated according to formula (C1):
Effective %）Colorimetric solution phosphorus reading×development volume×fractionation multiple 10.(1)W 10
Wherein: Colorimetric solution phosphorus (P) reading——colorimetric solution phosphorus reading obtained from the working curve; Colorimetric solution volume—50mL;
Fermentation solution volume (Triangular flask) (mL)/absorbed fermentation solution (mL);512
NY412—2000
W——mass of organic or inorganic phosphide added to the fermentation solution (referring to each triangular flask), g;106—convert μg (microgram) into g;The allowable error of two parallel determination results is <5%. Appendix D
(Standard Appendix)
Culture medium for determining the number of viable phosphate bacteria and the content of miscellaneous bacteria Broth culture medium (determining the number of phosphate bacteria)
Protein
Beef extract
Sodium chloride (NaCI)
Distilled water
D2 organic phosphorus bacteria culture medium (determining the phosphate solubilization efficiency of phosphate bacteria) Glucose
Ammonium sulfate [[(NH),SO
Yeast powder
Sodium chloride (NaCI)
Potassium chloride (KCI)www.bzxz.net
Magnesium sulfate (MgSO4·7H,0)
Ferrous sulfate (FeSO·7H,O)
|Manganese sulfate (MnSO4·4H,O)
Lecithin
Calcium carbonate (CaCO:)
Distilled water
Inorganic phosphorus bacterial culture medium (to measure the phosphorus-solubilizing efficiency of phosphorus bacteria)Glucose
Ammonium sulfate [(NH),SO]
Yeast powder
Sodium chloride (NaCI)
Potassium chloride (KCI)
Magnesium sulfate (MgSO4·7H20)
Iron sulfate (FeSO4·7H,O)
Manganese sulfate (MnSO4·4H,O)
Calcium phosphate (Cas(PO4)2)
18-20 g/L
1 000. 0 ml
0.2(alcohol solution)g/L
18~20 g/L
7. 0~~7. 5
0. 03 g/L
18~20g/L
distilled water
Martin's medium (for measuring the number of cysts)
potassium dihydrogen phosphate (KH,PO)
magnesium sulfate (MgSO.·7H.O)
1% Bengal red aqueous solution
glucose
protein
distilled water
NY 412---2000
18~20g/1
1 000.0ml.
Add 0.1g chloramphenicol to each liter of culture medium to sterilize. 511
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.