Some standard content:
NY 410—-2000
This standard is based on the original standard NY/T227-1994 "Microbial Fertilizer", and modifies the rhizobium fertilizer part. The main modifications are as follows:
Add the definition, sampling and judgment rules; add the strain part, including the strain validity, bacterial characteristics, and colony morphology; delete the finished product harmlessness index. Because rhizobium fertilizer generally uses peat as a carrier, and only 750-7500g of rhizobium fertilizer is used per hectare per year for seed dressing, there is no harm from heavy metals. After high-temperature sterilization of peat, coliform bacteria and roundworm eggs will no longer survive. From the date of implementation, this standard will also replace the rhizobium fertilizer part in NY/T227-1994. Appendix A of this standard is the appendix 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: 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: Ning Guozan, Liu Huiqin, Ma Xiaotong, Ge Cheng, Li Jun481
1Scope
Agricultural Industry Standard of the People's Republic of China
Rhizobium fertilizer
Rhizobium fertilizer
NY 410—2000
This standard specifies the classification, technical requirements, inspection methods, inspection rules, marking, packaging, transportation and storage of leguminous rhizobium fertilizers. This standard applies to rhizobium liquid fertilizers produced by liquid fermentation using rhizobium strains with excellent symbiotic nitrogen fixation performance as production bacteria, and rhizobium solid fertilizers made by using carriers with good water holding performance as adsorbents; it also applies to composite rhizobium fertilizers made of rhizobia mainly containing Bacillus, Pseudomonas or other beneficial growth-promoting bacteria that can promote nodulation and nitrogen fixation. No. 2 Standards
The clauses contained in the following standards constitute the clauses 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 version 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 Rhizobium fertilizer
An inoculant used for inoculation of leguminous crops to make leguminous crops nodulate and fix nitrogen. 3.2 Composite rhizobium fertilizer
Rhizobium fertilizer with rhizobium as the main component and a small amount of bacillus, pseudomonas or other beneficial growth-promoting microorganisms that can promote nodulation and nitrogen fixation is called composite rhizobium fertilizer. The growth-promoting microorganisms added must be strains that are harmless to humans, animals and plants. 4 Product classification
4.1 According to different forms, it is divided into liquid rhizobium fertilizer and solid rhizobium fertilizer. 4.2 According to different host species, it is divided into bean rhizobium fertilizer, soybean rhizobium fertilizer, peanut rhizobium fertilizer, clover rhizobium fertilizer, pea rhizobium fertilizer, blue rhizobium fertilizer, lotus root rhizobium fertilizer, astragalus rhizobium fertilizer and sand rhizobium fertilizer, etc. 5 Technical requirements
5.1 Bacterial species
5.1.1 Validity of bacterial species
The bacterial species used to produce rhizobium fertilizers belong to different rhizobia species in the genera such as Rizobium, Brutyrhizobium, Azorhizobium and Mesorhizobium. These bacterial species must be identified strains or strains that have significantly increased yields in two-year multi-point field trials. The bacterial species must be identified in a nitrogen-free nutrient solution pot inoculation test within one year before the production of bacterial fertilizers, with excellent nodulation and nitrogen fixation performance, and the weight of inoculated plants significantly increased compared with the control. 5.1.2 Bacterial characteristics
Approved by the Ministry of Agriculture of the People's Republic of China on December 22, 2000 482
Implemented on April 1, 2001
Short rod, no spores, Gram-negative. 5.1.3 Colony morphology
NY 410-2000
Round, with neat edges and slightly raised, milky white or colorless and translucent on the mannitol-yeast medium plate containing Congo red 5.2 Product technical indicators
Liquid rhizobium fertilizer (see Table 1)
Table 1 Technical indicators of liquid rhizobium fertilizer
Appearance, odor
Number of live rhizobia, 10%/mL
Rate of foreign bacteria, %
Minimum dilution of host nodulation|| tt||Validity period, month
5.2.2 Solid rhizobium fertilizer (see Table 2) Item
Appearance, odor
Moisture content, %
Number of live rhizobia, 10°/g
Rate of foreign bacteria, %
Adsorbent particle fineness
Minimum dilution of host nodulation
Validity period, month
6 Sampling
Milky white or grayish white uniform turbid liquid, or with slight precipitation. No sour smell
Table 2 Technical indicators of solid rhizobium fertilizer
Powder, loose, moist, no mold, no sour smell, no mold
6. 0～7.2
The pH value of the bacterial solution produced by acid-resistant strains can be greater than 7.2
This item is only tested when the supervisory department or the arbitration inspection parties deem it necessary
This item is only tested when the supervisory department or the arbitration inspection parties deem it necessary
The bacterial fertilizer used for large seeds (soybeans, peanuts, peas, etc.) can pass through a hole with a diameter of 0.18 The residue on the standard sieve of mm is ≤10%
The residue on the standard sieve of 0.15mm for the bacterial fertilizer used for small seeds (clover, clover, astragalus) is ≤10%
This item is tested only when the supervisory department or the arbitration inspection parties deem it necessary
This item is tested only when the supervisory department or the arbitration inspection parties deem it necessary
The products made from the bacterial liquid in each fermentation tank are considered as a batch, and the batch sampling inspection is carried out. The sampling process must strictly avoid contamination by foreign bacteria. 6.1 Sampling tools and supplies
Before sampling, prepare sterile plastic bags (or plastic bottles), metal spoons, scissors, sealers (or sealing tapes), kraft paper sample bags, labels, sampling seals and glue in advance
6.2 Sampling quantity and sampling method
NY 410—2000
Sampling in the finished product warehouse, according to the "" shape layered point sampling, sampling in pieces, small package products are one package box as one piece. Large package (30-50kg) products are one bag (or one barrel) as one piece. The number of sampling pieces is determined by the size of the sample base. 1-10 pieces, all samples. 11-200 pieces, sample 10 pieces; 201-400 pieces, sample 20 pieces. If the sample base is greater than 400 pieces, increase the number of sampling pieces by 2% of the excess. The total number of pieces shall not exceed 40 pieces.
Take a small bag from each small package product, and sample 200g from each bag under sterile conditions. Mix all the samples, reduce them to 2000g by quartering, and pack them into 4 bags, then seal them. Put 2 bags into a kraft paper sealed bag. Finally, affix the sampling label and sampling seal (refer to this method for sampling small package products of liquid rhizobium 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
Biological microscope (10×100);
Colony counter;
Constant temperature incubator;
Constant temperature drying oven;
Constant temperature shaker;
Autoclave;
Aseptic room or clean bench;
Acidity meter;
Greenhouse or plant light incubator: the light intensity of the plant leaf surface is greater than 80001x, and the temperature control range is 20~30℃; Test tube: 915 mmX150mm, $18 mmX180mm; culture IIIl: 9cm in diameter;
Triangular flask: 500mL;
sterile pipettes: 1, 2, 5, 10mL;
glass scraper;
filter paper;
alcohol lamp;
standard sieve: 0.18mm and 0.15mm in aperture; No. 1 wool brush;
sterile water;
deionized water.
7.1.2 Reagents
7.1.2.1 Gram staining reagents
crystal violet;
safranine;
iodine solution;
95% alcohol.
7.1.2.2 Rhizobium agar medium
Mannitol (CH,O.)
Sucrose (C/2H2On)
Yeast powder
Dipotassium hydrogen phosphate (K,HPO4·3H2O)
Magnesium sulfate (MgSO4·7H,O）
Sodium chloride (NaCI)
Calcium sulfate (CaSO)
Sodium molybdate (NazMo0)4·2H,O) solution
Manganese sulfate (MnSO4·4H,O) solution
Ferric citrate (FeCH.O,) 1% solution
Acid (H.BO,) 1% solution
Congo red C32H2gNgO,S,N a) 1% solution
7.1.2.3 Martin's medium (for testing mold count) Potassium dihydrogen phosphate (KH,PO.·7H2O)
Glucose (C,H12O·H,O)
Magnesium sulfate (MgSO4*7H.0)
Protein Chen
1% Bengal red alcohol (anhydrous) solution
Distilled water
Fluomycin
NY 410---2000
1 000 ml.
1000 mL
7.1.2.4 Agar slant medium for nodulation test of legumes Potassium dihydrogen phosphate (KH,PO.)
Potassium chloride (KCI)
Magnesium sulfate (MgSO,7H,0)
Zinc sulfate (ZnSO7H.0)
Copper sulfate (CuSO.·5H,O)
Boric acid (H,BO)
Sodium hydroxide (Na2MoO2H,0)
Ferric citrate (FeC.H,O,*rH,0)
Sodium nitrate (NaNO,)
7.2 Finished product inspection
7.2.1 Odor inspection
5~10 g
1000 mL
Take a sample of rhizobium fertilizer and open the package for sensory inspection. 7.2.2 Appearance inspection
Open the package of solid rhizobium fertilizer, put it in a white porcelain plate, and shake the liquid rhizobium fertilizer. Perform sensory inspection under bright light.
7.2.3 pH value determination
Determination of pH value of liquid rhizobium fertilizer: Take the test bacterial solution and measure the pH value, and take the average of three measurement results. Determination of pH value of solid rhizobium fertilizer: Take a 50mL beaker, add 25g of bacterial fertilizer and 25mL of deionized water. Stir the solution magnetically to make it homogenous, and then use an acidometer to measure the pH value. Take the average of three measurement results. 7.2.4 Determination of moisture content
NY410---2000
Weigh three portions of solid rhizobium fertilizer, 20.00g 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 4h, move to a desiccator, and weigh after cooling. Calculate the moisture content according to formula (1) 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%. Moisture content (%) =㎡two\×100
Where: mo---sample mass before drying·g; sample mass after drying, g.
7.2.5 Determination of adsorbent particle fineness
Weigh 10g of sample (accurate to 0.01g), place it in a standard sieve (diameters are 0.18mm and 0.15mm respectively), rinse with water, and brush lightly with a brush until no powder passes through. Dry the residue at 105-110℃ and weigh it. The percentage of the residue is calculated according to formula (2): Residue (%) = Residue mass ± (maximum water content of sample) × 100 (2)
Sample mass
7.2.6 Determination of live rhizobia and foreign bacteria rate The live rhizobia and foreign bacteria rate are determined by plate counting method. 7.2.6.1 Prepare rhizobia culture medium plates (7.1, 2.2) and Martin culture medium plates (7.1.2.3) 7.2.6.2 Sample dilution culture
Weigh 10g of the sample to be tested (accurate to 0.01g), put it into a triangular flask with glass beads and 100mL of sterile water (if the sample to be tested is liquid, take 10mL and add it to a triangular flask containing 90mL of sterile water), and place it on a shaker for 20min at a speed of 200r/min. After the shaken sample is left to stand for 20 minutes, it is diluted to 10-? (liquid sample is diluted to 10-8) by 10-fold dilution method. Take 0.1mL of each of the last three concentration suspensions and add them to a culture medium plate with a diameter of 9cm. Use a glass scraper to spread the bacterial solution evenly. Repeat each dilution three times. Incubate at 25-28℃ for 3-7 days. Use the same method to add 0.1mL of 10-3 dilution bacterial suspension to Martin culture medium plate. Incubate at 28℃ for 48h. 7.2.6.3 Colony identification and counting
According to the colony characteristics described in the standard of the product to be tested, identify rhizobium colonies and miscellaneous bacteria colonies. If necessary, perform Gram staining (method see Appendix A) and microscopic examination to distinguish rhizobium colonies from other colonies. Take plates with 30-300 colonies and count them. Calculate the average number of rhizobium and miscellaneous bacteria colonies in three replicates. Miscellaneous bacteria refers to the general term for other types of microorganisms other than the effective live bacteria (including rhizobia and growth-promoting bacteria) contained in the sample. The total number of miscellaneous bacteria is the sum of the number of molds appearing on the Martin medium and the number of miscellaneous bacteria appearing on the rhizobia medium (excluding molds). The number of rhizobia is calculated according to formula (3), and the miscellaneous bacteria rate is calculated according to formula (4): Number of rhizobia [100 million/g (mL)) = Average number of colonies × dilution factor × amount of Jia release plus amount of base liquid volume
plus +++* (3)
7.2.7 Dilution nodulation test
Miscellaneous bacteria rate (%) = Number of grain nodules + Total number of miscellaneous bacteria Total number of tea fungi
The host plant seeds are surface disinfected with 0.1% mercuric chloride solution and then germinated. When the seed buds are 1-2 double m long, they are inoculated with a 10-510-dilution suspension of the test sample and planted on the agar slope of the test tube. Set up a negative control without inoculation and a positive control inoculated with known bacteria. Repeat 6 times. The plant culture temperature is 20~25℃, the light intensity on the plant leaves is greater than 8000Ix, and the light is on for 12 hours every day. Observe the nodulation condition after 10~20 days. If the blank control nodulates, the test needs to be repeated. If the blank control does not nodulate, but the positive control nodulates, it can be judged. In the 10 dilution, 70% of the plants nodulate, which is qualified for dilution nodulation.
7.2.8 Inspection of validity period
Measure the number of viable bacteria 10 days before the expiration date indicated in the product manual, or inoculate the host according to the usage indicated in the product manual (7.2.7). The validity period is qualified when the number of viable bacteria reaches the standard or 70% of the plants nodulate. 8 Inspection rules
8.1 Inspection classification
8.1.1 Product factory inspection
Inspection performed when the product is delivered.
8.1.2 Product type inspection
NY 410—-2000
New product identification or quality supervision inspection by national quality inspection institutions. 8.2 Inspection items
Type inspection items shall be carried out in accordance with the requirements of 5.2.1 or 5.2.2. The validity period shall not be inspected for factory inspection. 8.3 Judgment rules
8.3.1 Qualified products:
a) Products whose inspection results meet the technical indicators specified in 5.2.1 (or 5.2.2) are qualified products; b) Products whose foreign bacteria rate does not meet the indicators, but the foreign bacteria rate of liquid samples does not exceed 10%, the foreign bacteria rate of solid samples does not exceed 20%, no mold appears on the 10-6 rhizobium plate, and other indicators meet the requirements, can also be judged as qualified products; c) Among the minor inspection items such as pH value, moisture, adsorbent particle fineness, appearance and odor, if two of them do not meet the technical indicators, but the number of viable bacteria and foreign bacteria rate meet the indicator requirements, can also be judged as qualified products. 8.3.2 Unqualified products:
a) If the number of live rhizobia does not meet the technical indicators, it shall be judged as unqualified product; b) If the mixed bacteria rate of liquid samples exceeds 10%, and the mixed bacteria rate of solid samples exceeds 20%, it shall be judged as unqualified product; c) If mold appears on the 10-6 rhizobium culture medium plate, it shall be judged as unqualified product; d) If the residue of adsorbent passing through the standard sieve with a pore size of 0.15mm for rhizobium fertilizer for small seeds is ≥30%, it shall be judged as unqualified product. 8.3.3 When testing rhizobium fertilizer products containing growth-promoting bacteria such as Bacillus or Pseudomonas, the judgment of Bacillus and Pseudomonas shall be carried out according to the basis provided by the product enterprise standard. 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. 9.1.2 Outer packaging
Outer packaging shall be made of cartons, the quality of which shall comply with the requirements of GB/T6543. The outside of the box shall be reinforced with nylon strapping tape. 9.1.3 Each box (bag) of product shall be accompanied by a product certificate and instruction manual, which shall specify the method of use, dosage and precautions. 9.2 Labels
The packaging box (bag) shall be 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 protection, anti-freeze and other marks. If necessary, fragile and anti-inversion marks shall also be printed. The inner packaging shall be printed with the product name, trademark, standard number, fertilizer registration certificate number, effective bacteria content, production date, validity period, product performance, instruction manual, production unit, factory address, etc.
9.2.1 Product Name
The product name of rhizobium fertilizer shall be consistent with the name of rhizobium in the product. For example: the product name of fertilizer produced with soybean rhizobium is soybean rhizobium fertilizer.
9.2.2 Product Performance
The name of the leguminous host that the product can be inoculated shall be indicated in the product performance. 9.3 Transportation and Storage
The transportation and storage of rhizobium fertilizers shall be carried out in accordance with 9.3 and 9.4 of NY411-2000. 187
NY 410-2000
Appendix A
(Standard Appendix)
Gram staining
The cell walls of Gram-positive bacteria and Gram-negative bacteria have different permeabilities to crystal violet-iodine complexes, resulting in positive or negative staining reactions.
A1 Stain
A1.1 Crystal violet solution (Hucker's formula) Solution A: Crystal violet
Ethanol (95%)
Solution B: Ammonium oxalate
Distilled water
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 (1) tablets
Potassium iodide (KI)
Distilled water
First dissolve potassium iodide in a small amount (35 mL) of distilled water, then add the iodine tablets. After the iodine is completely dissolved, dilute with water. This stain is stable and can be stored for several months in an airtight dark bottle.
A1.3 Decolorizing solution (95% ethanol)
A1.4 Restaining solution (0.5% safranin aqueous solution) Take 2.5 g of safranin and dissolve it in 100 mL of anhydrous alcohol. Take 20 mL of safranin alcohol solution and add 80 mL of distilled water to make a 0.5% safranin aqueous solution.
A2 Smear
A2.1 Use a crayon to draw a grid on a clean slide without oil stains, and write the date and slide number on the end of the slide. A2.2 Drop a small drop of sterile water or distilled water in each grid on the slide. Use an inoculation loop to pick up a small amount of bacterial moss and gently apply it to the edge of the water drop.
A2.3 Air dry naturally or heat it slightly to promote quick drying. After drying, pass it over a flame 1 to 2 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. Www.bzxZ.net
A3.3 Add iodine solution to rinse off the residual water. Cover it for about 1 minute. A3.4 Rinse off 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~~30s. Immediately rinse off the alcohol with water. A3.6 Stain with safranin solution for 1~~2 (or longer) minutes. A3.7 Wash off the safranin with water, absorb the surface water with filter paper or air dry it, and prepare for microscopic examination. A4 Observation results
Use the oil lens of the microscope to examine directly on the slide. Red is Gram-negative, and blue-purple is Gram-positive. 188
A5 Precautions
NY410—2000
A5.1 There are many recipes for Gram 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 Gram staining operation, the smear and decolorization are the most important steps. 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 drop. When examining under the microscope, the Gram staining reaction of dispersed bacteria shall prevail. Too dense bacteria often show false positives. For smears of pure bacteria, it is easy to control by decolorizing with 95% ethanol. The more water there is in ethanol, the stronger the decolorization power is, which is easy to form a false negative. Try to reduce the residual water on the slide. Just 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 ethanol can be used 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 It is recommended to use 0.5% red solution as the counterstain. Other counterstains, such as diluted carbolic acid red solution, can be selected according to specific circumstances. A5.5 Because gentian violet is not a single-component dye, unlike crystal violet, it is often difficult to decolorize after staining according to the above formula, resulting in false positives. In this case, the stain can be diluted before use. The trial gentian violet can be diluted 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 the 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, while 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. 4892 In the Gram staining operation, the smearing and decolorization steps are the most important. The smear must not be too thick. For bacteria that are easy to emulsify, gently smear the inoculation loop stained with bacterial moss on the edge of the water drop. During microscopic examination, the Gram staining reaction of dispersed bacteria shall prevail. Too dense bacteria often show false positives. For pure bacterial smears, it is easy to control the decolorization with 95% ethanol. The more water contained in ethanol, the stronger the decolorization power, which is easy to form a false negative. Try to reduce the residual water on the slide. Just shake off or rinse with ethanol to decolorize for 20s; use filter paper to absorb the residual water, and ethanol can be used for decolorization for nearly 30. 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 It is recommended to use 0.5% red solution as the counterstain. Other counterstains, such as diluted carbolic acid fuchsin solution, can be selected according to specific circumstances. A5.5 Since gentian violet is not a single-component dye, unlike crystal violet, it is often difficult to decolorize after staining according to the above formula, resulting in false positives. In this case, the dye can be diluted before use. The trial gentian violet can be diluted to one-tenth of the concentration, but the color is still not as good as crystal violet.
A5.6 For heterotrophic bacteria that are generally easy to grow, it is appropriate 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 that have been cultured for 18 to 24 hours or less will show a positive reaction, while older cells that have been 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. 4892 In the Gram staining operation, the smearing and decolorization steps are the most important. The smear must not be too thick. For bacteria that are easy to emulsify, gently smear the inoculation loop stained with bacterial moss on the edge of the water drop. During microscopic examination, the Gram staining reaction of dispersed bacteria shall prevail. Too dense bacteria often show false positives. For pure bacterial smears, it is easy to control the decolorization with 95% ethanol. The more water contained in ethanol, the stronger the decolorization power, which is easy to form a false negative. Try to reduce the residual water on the slide. Just shake off or rinse with ethanol to decolorize for 20s; use filter paper to absorb the residual water, and ethanol can be used for decolorization for nearly 30. 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 It is recommended to use 0.5% red solution as the counterstain. Other counterstains, such as diluted carbolic acid fuchsin solution, can be selected according to specific circumstances. A5.5 Since gentian violet is not a single-component dye, unlike crystal violet, it is often difficult to decolorize after staining according to the above formula, resulting in false positives. In this case, the dye can be diluted before use. The trial gentian violet can be diluted to one-tenth of the concentration, but the color is still not as good as crystal violet.
A5.6 For heterotrophic bacteria that are generally easy to grow, it is appropriate 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 that have been cultured for 18 to 24 hours or less will show a positive reaction, while older cells that have been 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. 489
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