Some standard content:
NY413-2000
This standard amends the silicate bacterial fertilizer part of the original standard NY/T227-1994 "Microbial Fertilizer".
The main modifications are as follows:
--Add the terms, sampling and judgment rules. Add the tolerance in the inspection method;--Delete the finished product harmlessness index in the technical requirements. This standard will replace the silicate bacterial fertilizer part in NY/T227-1994 from the date of entry into force. Appendix A, Appendix B and Appendix C of this standard are all appendices to the standard. This standard is 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: Li Huiquan, Wu Guanyi, Li Yuanfang, Ge Cheng, Shen Delong 515
1 Scope
Agricultural Industry Standard of the People's Republic of China
Silicate bacteria fertilizer
Silicate bacteria fertilizerNY413—2000
This standard specifies the classification, technical requirements, test methods, inspection rules, packaging, labeling, transportation and storage of silicate bacteria fertilizer products. This standard applies to live microbial fertilizer products made by fermentation of beneficial microorganisms that can release potassium, phosphorus and ash elements and improve crop nutrition conditions.
2 Cited 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/T1250--1989 Methods for expressing and determining limit values GB/T6543--1996 Corrugated paperboard
NY411-2000 Nitrogen-fixing bacteria fertilizer
NY/T227-1994 Microbial fertilizer
3 Definitions
This standard adopts the following definitions.
3.1 Silicate bacterial fertilizer
Through its life activities in the soil, it increases the supply of plant nutrients, stimulates crop growth, inhibits the activities of harmful microorganisms, and has a certain yield-increasing effect.
4 Product classification
According to the dosage form, it is divided into: liquid bacterial agent, solid bacterial agent and granular bacterial agent. 5 Technical requirements
5.1 Bacteria
Non-pathogenic bacteria, can grow on nitrogen-free culture medium containing potassium feldspar powder, mica and other minerals, and potassium and hormone substances that stimulate plant growth exist in the bacteria and fermentation liquid.
5.1.1 The strains used shall be a variant strain of Bacillus mucilaginosus or Bacillus cirulans and other strains identified for silicate bacterial fertilizer production. The use of various genetically engineered microorganisms (GEM) shall be strictly controlled. Any strains other than those in this standard must be identified. 5.1.2 The size of the bacteria is 4-7um×1-1.2μm, long rod-shaped, with blunt ends, and often 1-2 large fat particles in the cell. Gram staining is negative, with capsule and oval spores. Approved by the Ministry of Agriculture of the People's Republic of China on December 22, 2000. 516
Implemented on April 1, 2001
NY 413--2000
5.1.3 The colonies grown on nitrogen-free culture medium are sticky, elastic, round, with neat, smooth and shiny edges, large protuberances, and colorless and transparent
5.1.4 It basically does not grow on beef extract protein culture medium. 5.2 Technical indicators of finished products (see Table 1)
Technical indicators of finished products
Table 1
Water content, %
Fineness Residue on sieve, %
Aperture 0. 18 mm
Diameter 5.0 mm~~2.5 mm
Valid during the validity period
Live bacteria count: 10%/ml. (g)
Miscellaneous bacteria rate,
Valid period 2",
Special odor
1) Including 10\\No mold on Martin culture medium plate. Solid
Dark brown or brown powder, moist, loose, no odor
20.0~~50.0
6. 5~ 8.5
2) This instrument is only tested when the supervisory department or the arbitration inspection parties deem it necessary. 6 Sampling
Black or brown particles
The products made from the bacterial liquid in each 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, sterile plastic bags (or plastic bottles), metal spoons, scissors, sealing machines, kraft paper sealing bags, labels, sampling seals and glue are prepared in advance.
6.2 Sampling quantity and sampling method
Sampling in the finished product warehouse can be carried out according to the "\ shape layered point setting, and the sampling is in pieces. Small packaged products are considered as one piece per packaging box. Large packaged (30-50kg) product bags (one barrel) are considered as pieces. The number of sampling pieces is determined by the size of the sample base. All samples from 1 to 10 pieces are sampled; 10 pieces are sampled from 11 to 200 pieces; 20 pieces are sampled from 201 to 400 pieces. If the sample base is greater than 400 pieces, the number of samples shall be increased by 2% of the excess. The total number of pieces shall not exceed 40 pieces.
Take a small bag from each small packaged product and sample 200g from each bag under sterile conditions. Then mix all the samples, reduce them to 2000g by quartering, divide them into 4 bags, and seal them. Every 2 bags are put into a kraft paper sealed sample bag. Finally, affix the sampling label and sampling seal. Take 10ml of each liquid product, put it in a sterile triangular bottle, mix it, and divide it into two portions, each 250mL. One portion is retained by the sampled unit, and the other is submitted to the testing center for testing.
7 Test 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:
NY 413--2000
Test tube: 915mm×150mm, q18mm×180mm; culture blood: diameter 9cm;
Triangular flask: 500mL, 150mL;
Sterile pipette: 1, 2, 5, 10mL;
Glass scraper;
Filter paper;
Alcohol lamp;
Balance (one thousandth);
Standard sieve: aperture 0.18mm, 2.5mm, 50.0mm; No. 1 wool brush;
Sterile water;
Deionized water.
7.1.2 Reagents (see Appendix A)
7.2 Finished product inspection
7.2.1 Odor inspection
Take the silicate bacterial fertilizer sample, open the package, and conduct sensory inspection. 7.2.2 Appearance inspection
Open the package of solid silicate bacterial fertilizer and put it in a white porcelain plate. Shake the liquid silicate bacterial fertilizer and conduct a sensory inspection under bright light.
7.2.3 pH value determination
Liquid sample: Use a 50ml measuring cylinder to take 40.0ml of sample and put it into a 50mL beaker. Measure it directly on the acidometer and record the pH value of each sample. Repeat three times for each sample. Powder and granular samples: Weigh 15.0g of sample and put it into a 50mL beaker. Use a 50ml measuring cylinder to measure 30ml of deionized water at a ratio of 1:2 (sample and deionized water) and add it to the beaker with the sample (if the water content of the sample is less than 1%, deionized water can be added at a ratio of 1:35). Stir each sample for 1min, let it stand for 30min, and then measure the pH of the sample. Repeat three times for each sample. Take the average value and be accurate to one tenth place. Granular samples should be ground and passed through a 1mm sieve first, and the steps are the same as for powdered samples. 7.2.4 Determination of moisture content
Weigh three portions of solid bacterial fertilizer, 20.0 g each, accurate to 0.01g, and place them in an aluminum box that has been weighed to a constant weight. Bake in a 105℃ drying oven for 4 hours, move to a dryer, and weigh after cooling. Calculate the moisture content based on the mass difference before and after drying. 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):
ml×100
Moisture content (%)= m.
Where: m.…-the mass of the sample before drying, g; mr
-the mass of the sample after drying, g.
7.2.5 Determination of fineness
Accurately weigh 50.0g of the powdered sample and place it in a 300mL beaker.Add 250ml of tap water and soak for 5 minutes. Pour the soaked sample onto a 0.18mm sieve and rinse with tap water (be careful not to splash the sample during rinsing). During the rinsing process, brush gently with a brush until clear water flows down. Put the sieve together with the sample on the sieve into an oven and bake at 105°C for 41. After cooling, weigh the sample on the sieve. The percentage of the sample on the sieve is calculated according to formula (2), and the percentage of the sample passing through the 0.18mm aperture sieve is calculated according to formula (3): Sample on the sieve (%) Residue on the sieve weighs the main sample mass Water content of the sample × 10 Sample mass
Note: The percentage of the sample on the sieve shall not be 20%, the percentage of the sample passing through the 0.18mm aperture sieve minus 1 minus the percentage of the sample on the sieve ++***(2)
(3)
When measuring the particle sample through the sieve, it is not necessary to soak it in water. Rub the two sieves with a pore size of 5.0mm and 2.5mm together, with the larger pore size on top, and pass them through at one time. The amount of the sieve on the sieve must be 10%.
7.2.6 Determination of the number of effective live bacteria and foreign bacteria Plate counting method, using selective culture medium (see Appendix B), according to 5.2 of NY/T227--1994. 7.2.6.1 Determination steps
Accurately weigh 10.0 g of the sample to be tested and add it to 100 mL of sterile water with glass beads (500 mL Erlenmeyer flask). If it is a liquid bacterial agent, take 10-20 mL and add it to 90-180 mL of sterile water. After standing for 20 minutes, fully shake it on a rotary shaker at 200 r/min for 30 minutes at room temperature to prepare the stock solution.
Serial dilution: Use a 10mL sterile pipette to draw 5mL of the mother solution that has been shaken evenly, add it to 45mL of sterile water, mix it to a 1:10 bacterial suspension, use a 5mL sterile pipette to draw 5L of the 1:10 bacterial solution into another triangular flask containing 45mL of sterile water, mix it to a 1*1×10-2 bacterial suspension, dilute it step by step, and get a series of dilution bacterial suspensions such as 1:1×10-3.1:1×10-41:1×10~5. When diluting, the sterile pipette must be changed for each dilution. Sample addition: Use a 1mL or 0.5mL sterile pipette to draw 0.1mL of bacterial suspensions of different dilutions, add it to the surface of a 9cm diameter silicate bacterial culture medium, and use a sterile scraper to evenly spread the bacterial suspension on the entire agar surface. Take three consecutive appropriate dilutions for each sample. The sterile pipette must be replaced when adding samples for each dilution. Repeat each dilution three times, and use a blank with sterile water as a control in between. Culture: After adding samples, cover the plate with sterile paper and place it in a 28~30℃ incubator for 48 hours. Colony count: According to the relevant technical data of the sample to be tested, after confirming the colony morphology, staining reaction and individual morphology of the effective bacteria through staining, microscopy and other techniques, count the number of effective bacteria and the number of miscellaneous bacteria respectively. When the number of colonies in each plate III of the blank control is greater than 4, the test result is invalid and must be repeated.
Take 5 to 10 colonies of bacteria for each dilution multiple, smear stain (see Appendix A, Appendix B), observe and identify under a microscope, and count the plates with 30 to 300 colonies. Count the average number of colonies on three plates with the same dilution. The effective viable bacteria count is calculated according to formula (4):
Number of bacteria in the microbial agent (pieces/) - average number of colonies × dilution factor × volume of mother solution bacterial suspension (mL)
Number of bacteria in the microbial agent in grams or milliliters
× amount of sample added (mL)
.++9#**(4)
Miscellaneous bacteria refers to the general term for other types of microorganisms other than the effective viable bacteria count contained in the sample. In the bacterial microbial fertilizer currently used, the number of miscellaneous bacteria is the sum of the number of molds appearing on Martin medium and the number of miscellaneous bacteria appearing on selective medium (except for dancing fungi). The number of miscellaneous bacteria appearing on selective medium can be obtained by counting the colonies of the effective viable bacteria count of the test sample. 7.2.6.2 Determination of mold count
Add 0.1mL of 10-2 or 10~3 diluted bacterial suspension to the Martin medium plate (the operating steps are the same as 7.2.6.1) and culture at 28℃ for 48h. The rate of foreign bacteria is calculated according to formula (5):
Far bacteria rate (%) = effective bacteria count + foreign bacteria count × 100 Foreign bacteria count
** (5)
Upper limit of bacteria count: The number of molds in the microbial agent used for seed dressing shall not exceed 10×104 per gram or per milliliter; the number of molds in the microbial agent not used for seed dressing shall not exceed 30×10° per gram or per milliliter. The number of foreign bacteria refers to the sum of the number of colonies other than effective live bacteria on the selective culture medium and the number of molds on the Martin medium. When using Martin medium to measure molds, take a 10-* dilution of bacterial suspension for determination. Culture at 28°C. 7.2.7 Inspection of validity period
The method for determining the various indicators of the finished product 10 days before the expiration date indicated in the product manual is in accordance with 7.2.17.2.6. The standard requirements are met to meet the validity period requirements.
8 Inspection rules
8.1 Inspection categories
8.1.1 Product factory inspection
Inspection conducted when the product is delivered.
8.1.2 Product type inspection
NY 413--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 5.2. The validity period shall not be checked during factory inspection. 8.3 Judgment rules
8.3.1 Qualified products:
) Products whose test results meet the technical indicators specified in 5.2 are qualified products: 6) When the rate of foreign bacteria does not meet the indicators, but is less than 2 times the requirements of this standard, no mold appears on the 103 Martin culture medium plate, and other indicators meet the requirements, it can also be judged as a qualified product; C) Among the secondary test items such as pH value, moisture, adsorbent particle fineness, appearance and odor, two of them do not meet the technical indicators, but the number of viable bacteria and the rate of foreign bacteria meet the requirements of the indicators, it can also be judged as a qualified product. 8.3.2 Unqualified products:
a) The number of viable silicate bacteria does not meet the technical indicators, it is judged as an unqualified product; b) The rate of foreign bacteria exceeds 2 times the requirements of this standard, it is judged as an unqualified product; () Mold appears on the 10-6 Martin culture medium plate, it is judged as an unqualified product; d) Among the secondary test items such as pH value, moisture, fineness, appearance, etc., three of them do not meet the technical indicators, it is judged as an unqualified product. 8.3.3 For silicate bacterial fertilizer products containing plant growth promoting rhizobacteria (PCPR), only the number of silicate bacteria shall be measured during testing, and the number of plant growth promoting rhizobacteria shall not be measured, nor shall they be considered as miscellaneous bacteria. 8.3.4 The qualified judgment of the quality indicators in this standard shall be compared 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. Granular silicate bacterial fertilizers can also be packaged in woven bags. Freeze-dried bacterial agents are vacuum dried in glass finger tubes. 9.1.2 Outer packaging
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 product is accompanied by a product certificate and instructions for use, which shall indicate the method of use, dosage and precautions. 9.2 Labeling, transportation and storage
The labeling, transportation and storage of silicate bacterial fertilizers shall comply with 9.2.9.3 and 9.4 of NY411-2000. 520
A1 Preparation of dye solution
A1.1 Carbolic acid fuchsin dye solution
Liquid A: alkaline fuchsin
95% alcohol
Liquid B: carbolic acid
NY 413--2000
Appendix A
(Standard Appendix)
Dye and dyeing method
Distilled water
Mix liquid A and liquid B and dilute 5 to 10 times before use. A1.2 Methylene blue staining solution (methylene blue)
Solution A: Methylene blue
95% alcohol
Solution B: Potassium hydroxide
Distilled water
Mix solution A and solution B.
A1.3 Malachite green staining solution
Malachite green
Distilled water
A1.4 Safranin staining solution
95% alcohol
Take 10 mL of the above safranin alcohol solution and mix it with 90 mL of distilled water to make a dilute safranin dilution solution. A1.5 Alkaline fuchsin [0.5% (m/V)]
Alkaline fuchsin
95% alcohol
A2 Bacteria staining method
Put a small drop of sterile water on a clean slide, dip a small amount of bacterial moss or liquid with an inoculation loop, mix with water and apply to form a uniform thin layer, and dry naturally in the air. Hold one end of the slide and quickly pass it through the flame of an alcohol lamp 2 to 3 times to fix it. Add a drop of carbolic fuchsin or methylene blue staining solution, stain for about 1 minute, gently rinse the dye with tap water, absorb excess water from the slide with absorbent paper, dry it, and examine it under a microscope. A3 Membrane staining method
Prepare the slide the same as A2. After staining with carbolic fuchsin for 1 minute, wash it with water, absorb the water with absorbent paper, dry it naturally, add a drop of ink on one end of the slide, push it to the other end with absorbent paper, apply it into a thin layer, and examine it under a microscope after drying. The staining results show that the background is black, the bacteria are red, and the bacteria are surrounded by a transparent circle, which is the membrane. You can also add carbolic acid fuchsin to stain for 1 minute, wash with water, and examine under a microscope after drying. The staining results show that the bacteria are red, and the part surrounded by a layer of non-colored is the membrane.
A4 spore staining method
The preparation is the same as A2. Add 3~~5 drops of malachite green staining solution to the smear, heat with the flame of an alcohol lamp until steam is emitted, but not boiling, and add staining solution at any time to keep the smear from drying. Stain for about 5~10 minutes, and rinse with tap water for 30 seconds. Use 0.05% alkaline fuchsin or 0.5% safranin to stain for 1 minute, wash with water, and examine under a microscope. The staining results show that the spores are green and the bacteria are red. 521
A5 Gram staining method
A5.1 Staining agent
A5.1.1 Crystal violet staining solution
Liquid A: Crystal violet
95% alcohol
Liquid B: Ammonium oxalate
Distilled water
Mix liquid A and liquid B and let stand for 48 hours before use. A5.1.2 Lugol's iodine solution
Iodine (12) tablets
Potassium iodide (KI)
Distilled water
NY 413—2000
First take a small amount (35mL) 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 2 times the amount of water to dilute it into Lugol's iodine solution.
A5.1.3 Decolorizing agent: 95% alcohol.
A5.1.4 Counterstaining solution: 0.5% safranin aqueous solution. 2.5% safranin alcohol solution
Distilled water
A5.2 Staining method
A5.2.1 Preparation.
A5.2.2 Add crystal violet solution and cover for 1 minute. 20mL
A5.2.3 Rinse the crystal violet with water and air dry (or blow dry). A5.2.4 Add iodine solution and cover for 1 minute.
A5.2.5 Rinse the iodine solution with water, absorb the water with absorbent paper, and air dry (or blow dry). A5.2.6 Add a few drops of 95% alcohol and shake gently to decolorize. After 30 seconds, wash with water, absorb the water, and air dry (or blow dry). A5.2.7 After staining with safranin solution for 1~～2 (or longer) minutes, rinse with tap water. Dry and examine under a microscope. A5.3 Staining results
Gram-positive bacteria are purple, and Gram-negative bacteria are red. Appendix B
(Standard Appendix)
Medium for determination of effective viable count and content of foreign bacteria B1 Medium formula for determination of effective viable count of silicate bacteria Sucrose
Sodium dihydrogen phosphate (Na,HPO)
Magnesium sulfate (MgSO·7H,O)
Calcium carbonate (CaCO;)
Ferric chloride (FeCl,)
Glass powder
Distilled water
1 000 mL
18~20g
B2 Martin medium (for testing mold count)
Potassium dihydrogen phosphate (KHPO)
Magnesium sulfate (MgSO,*7H,O)
Glucose (C,H12O·H,O)
Protein Chen
1% Bengal red alcohol (anhydrous) solution
Distilled water
NY 413--2000
18~20g
1000mL
When using, add 0.3mL (30mg/kg) of 1% streptomycin solution to every 100mL of culture medium, or add 0.1g of chloramphenicol to every 1000rnL of culture medium for sterilization.
Appendix ℃
(Standard Appendix)
Determination Method of Total Potassium and Gold in Fermentation Broth of Silicate Bacteria—Flame Photometry C1 Principle
A flame photometer is an instrument that measures the intensity of spectral energy emitted by a flame when a certain element to be tested is excited. The sample is prepared into a simple solution, which is sprayed into mist with compressed air, and then burned after being mixed with acetylene or other combustible gases. The potassium, sodium and other ions in the solution emit their special emission bright line spectra. After being separated and selected by a filter plate, the light energy emitted by the flame is converted into photocurrent by a photocell, and then the current size is measured by an ammeter. The size of the photocurrent is positively correlated with the content of the element in the solution, and then the corresponding concentration is found from the curve of the standard solution measured under the same conditions for quantitative measurement.
Drawing of C2 potassium oxide standard curve
Accurately weigh 1.5830g of analytical pure potassium chloride dried at 105C for 4-6h, dissolve it in steamed stuffing water, dilute to 1000mL and shake well, which is 1000ng/kg potassium oxide standard solution. Dilute this solution to 100mg/kg, use it to prepare 2, 4, 6, 8.10, 15, 20mg/kg potassium oxide standard solution series, 50mL (or 250mL) each, and measure the current reading with a flame photometer. Draw a standard curve on graph paper with the current meter reading as the ordinate and the potassium oxide concentration as the abscissa. C3 Determination Steps
The culture solution is composed of: glucose (C.H2O·H0) 5.0g, magnesium sulfate (MgSO, 7H.O) 0.5g, calcium carbonate (CaCO3) 0.1g + disodium hydrogen phosphate (NazHPO.) 42.0g, ferric chloride (FeC1s) 0.005g, feldspar powder (glass powder, potassium-containing ore powder) 1.0g, distilled water 1000mL, pH 7.0. Put 100mL of the above tower culture solution into a 250mL triangular flask and sterilize it at 121℃ for 30min. After cooling, inoculate the silicate bacterial suspension. After culturing on a 28C200r/min shaker for 4d, transfer all the culture to an evaporator, concentrate it to about 10ml on a water bath, add a little 6% (V/V) HO, continue to cook, and stir continuously. Repeat this process until the silicate bacterial mucus disappears. Add water and filter into a 100mL volumetric flask. After constant volume, use a flame photometer to determine potassium. According to the current reading, the number of potassium (KO) dissolved by silicate bacteria can be found on the standard curve. The result shows that the amount of potassium oxide (K20) contained in each 100mL fermentation liquid is expressed in mg/kg. The value is rounded to two decimal places. 3232.3 Rinse the crystal violet with water and air dry (or blow dry). A5.2.4 Add drops of iodine solution and cover for 1 minute.
A5.2.5 Rinse the iodine solution with water, absorb the water with absorbent paper, and air dry (blow dry). A5.2.6 Add a few drops of 95% alcohol and shake gently to decolorize. After 30 seconds, wash with water, absorb the water, and air dry (or blow dry). A5.2.7 After staining with safranin solution for 1~~2 (or longer) minutes, rinse with tap water. Dry and examine under a microscope. A5.3 Staining results
Gram-positive bacteria are purple, and Gram-negative bacteria are red. Appendix B
(Standard Appendix)
Medium for determination of effective viable count and foreign bacteria content B1 Medium formula for determination of effective viable count of silicate bacteria Sucrose
Sodium hydrogen phosphate (Na,HPO)
Magnesium sulfate (MgSO·7H,O)
Calcium carbonate (CaCO;)
Ferric chloride (FeCl,)
Glass powder
Distilled waterwww.bzxz.net
1 000 mL
18~20g
B2 Martin medium (for testing mold count)
Potassium dihydrogen phosphate (KHPO)
Magnesium sulfate (MgSO,*7H,O)
Glucose (C,H12O·H,O)
Protein Chen
1% Bengal red alcohol (anhydrous) solution
Distilled water
NY 413--2000
18~20g
1000mL
When using, add 0.3mL (30mg/kg) of 1% streptomycin solution to every 100mL of culture medium, or add 0.1g of chloramphenicol to every 1000rnL of culture medium for sterilization.
Appendix ℃
(Standard Appendix)
Determination Method of Total Potassium and Gold in Fermentation Broth of Silicate Bacteria—Flame Photometry C1 Principle
A flame photometer is an instrument that measures the intensity of spectral energy emitted by a flame when a certain element to be tested is excited. The sample is prepared into a simple solution, which is sprayed into mist with compressed air, and then burned after being mixed with acetylene or other combustible gases. The potassium, sodium and other ions in the solution emit their special emission bright line spectra. After being separated and selected by a filter plate, the light energy emitted by the flame is converted into photocurrent by a photocell, and then the current size is measured by an ammeter. The size of the photocurrent is positively correlated with the content of the element in the solution, and then the corresponding concentration is found from the curve of the standard solution measured under the same conditions for quantitative measurement.
Drawing of C2 potassium oxide standard curve
Accurately weigh 1.5830g of analytical pure potassium chloride dried at 105C for 4-6h, dissolve it in steamed stuffing water, dilute to 1000mL and shake well, which is 1000ng/kg potassium oxide standard solution. Dilute this solution to 100mg/kg, use it to prepare 2, 4, 6, 8.10, 15, 20mg/kg potassium oxide standard solution series, 50mL (or 250mL) each, and measure the current reading with a flame photometer. Draw a standard curve on graph paper with the current meter reading as the ordinate and the potassium oxide concentration as the abscissa. C3 Determination Steps
The culture solution is composed of: glucose (C.H2O·H0) 5.0g, magnesium sulfate (MgSO, 7H.O) 0.5g, calcium carbonate (CaCO3) 0.1g + disodium hydrogen phosphate (NazHPO.) 42.0g, ferric chloride (FeC1s) 0.005g, feldspar powder (glass powder, potassium-containing ore powder) 1.0g, distilled water 1000mL, pH 7.0. Put 100mL of the above tower culture solution into a 250mL triangular flask and sterilize it at 121℃ for 30min. After cooling, inoculate the silicate bacterial suspension. After culturing on a 28C200r/min shaker for 4d, transfer all the culture to an evaporator, concentrate it to about 10ml on a water bath, add a little 6% (V/V) HO, continue to cook, and stir continuously. Repeat this process until the silicate bacterial mucus disappears. Add water and filter into a 100mL volumetric flask. After constant volume, use a flame photometer to determine potassium. According to the current reading, the number of potassium (KO) dissolved by silicate bacteria can be found on the standard curve. The result shows that the amount of potassium oxide (K20) contained in each 100mL fermentation liquid is expressed in mg/kg. The value is rounded to two decimal places. 3232.3 Rinse the crystal violet with water and air dry (or blow dry). A5.2.4 Add drops of iodine solution and cover for 1 minute.
A5.2.5 Rinse the iodine solution with water, absorb the water with absorbent paper, and air dry (blow dry). A5.2.6 Add a few drops of 95% alcohol and shake gently to decolorize. After 30 seconds, wash with water, absorb the water, and air dry (or blow dry). A5.2.7 After staining with safranin solution for 1~~2 (or longer) minutes, rinse with tap water. Dry and examine under a microscope. A5.3 Staining results
Gram-positive bacteria are purple, and Gram-negative bacteria are red. Appendix B
(Standard Appendix)
Medium for determination of effective viable count and foreign bacteria content B1 Medium formula for determination of effective viable count of silicate bacteria Sucrose
Sodium hydrogen phosphate (Na,HPO)
Magnesium sulfate (MgSO·7H,O)
Calcium carbonate (CaCO;)
Ferric chloride (FeCl,)
Glass powder
Distilled water
1 000 mL
18~20g
B2 Martin medium (for testing mold count)
Potassium dihydrogen phosphate (KHPO)
Magnesium sulfate (MgSO,*7H,O)
Glucose (C,H12O·H,O)
Protein Chen
1% Bengal red alcohol (anhydrous) solution
Distilled water
NY 413--2000
18~20g
1000mL
When using, add 0.3mL (30mg/kg) of 1% streptomycin solution to every 100mL of culture medium, or add 0.1g of chloramphenicol to every 1000rnL of culture medium for sterilization.
Appendix ℃
(Standard Appendix)
Determination Method of Total Potassium and Gold in Fermentation Broth of Silicate Bacteria—Flame Photometry C1 Principle
A flame photometer is an instrument that measures the intensity of spectral energy emitted by a flame when a certain element to be tested is excited. The sample is prepared into a simple solution, which is sprayed into mist with compressed air, and then burned after being mixed with acetylene or other combustible gases. The potassium, sodium and other ions in the solution emit their special emission bright line spectra. After being separated and selected by a filter plate, the light energy emitted by the flame is converted into photocurrent by a photocell, and then the current size is measured by an ammeter. The size of the photocurrent is positively correlated with the content of the element in the solution, and then the corresponding concentration is found from the curve of the standard solution measured under the same conditions for quantitative measurement.
Drawing of C2 potassium oxide standard curve
Accurately weigh 1.5830g of analytical pure potassium chloride dried at 105C for 4-6h, dissolve it in steamed stuffing water, dilute to 1000mL and shake well, which is 1000ng/kg potassium oxide standard solution. Dilute this solution to 100mg/kg, use it to prepare 2, 4, 6, 8.10, 15, 20mg/kg potassium oxide standard solution series, 50mL (or 250mL) each, and measure the current reading with a flame photometer. Draw a standard curve on graph paper with the current meter reading as the ordinate and the potassium oxide concentration as the abscissa. C3 Determination Steps
The culture solution is composed of: glucose (C.H2O·H0) 5.0g, magnesium sulfate (MgSO, 7H.O) 0.5g, calcium carbonate (CaCO3) 0.1g + disodium hydrogen phosphate (NazHPO.) 42.0g, ferric chloride (FeC1s) 0.005g, feldspar powder (glass powder, potassium-containing ore powder) 1.0g, distilled water 1000mL, pH 7.0. Put 100mL of the above tower culture solution into a 250mL triangular flask and sterilize it at 121℃ for 30min. After cooling, inoculate the silicate bacterial suspension. After culturing on a 28C200r/min shaker for 4d, transfer all the culture to an evaporator, concentrate it to about 10ml on a water bath, add a little 6% (V/V) HO, continue to cook, and stir continuously. Repeat this process until the silicate bacterial mucus disappears. Add water and filter into a 100mL volumetric flask. After constant volume, use a flame photometer to determine potassium. According to the current reading, the number of potassium (KO) dissolved by silicate bacteria can be found on the standard curve. The result shows that the amount of potassium oxide (K20) contained in each 100mL fermentation liquid is expressed in mg/kg. The value is rounded to two decimal places. 323
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