Standard category: GB-national standard, Key words: Feed mould inspection method, Standard number: GB13092-91, Standard name: Mold test method in feed, Standard classification: Agricultural feed standard.
1 Topic content and scope of application This standard specifies the test methods for moulds in feed.
This standard applies to the inspection of mold in feed.
2 Principle According to the physiological characteristics of the mold, select the medium that is suitable for mold growth and not suitable for bacterial growth. Use the plate counting method to determine the number of molds.
3 Equipment and Materials
3.1 Balance; Sensitivity 1g, maximum weighing 1000g.
3.2 Microscope: 1500.
3.3 Thermostat: 25-281°C.
3.4 refrigerator: ordinary refrigerator.
3.5 Autoclave: 2.5kg.
3.6 Drying oven: 50-2501°C.
3.7 Water bath: 45-771°C.
3.8 Oscillator: Reciprocating.
3.9 Micromixer; 2900r/min.
3.10 Electric stove.
3.11 alcohol lamp.
3.12 Inoculation rod: Nichrome wire.
3.13 Thermometer: 100+1°C.
3.14 Slides.
3.15 coverslip.
3.16 breast milk.
3.17 Test tube racks.
3.18 glass flask: 250,500 mL.
3.19 Test tube: 15150mm.
3.20 Petri dishes: 9 cm in diameter.
3.21 Pipette: 1,10 mL.
3.22 bead: diameter 5mm.
3.23 jar: 100,500 mL.
3.24 metal spoons, knives, etc.
3.25 nipples.
4 medium and diluent
4.1 High Salt Catch's medium.
4.2 Diluent.
4.3 Commonly used sterile drugs in the laboratory.
5 Inspection Procedures The mold inspection procedures are as follows:
Sample
|
--------------------------
↓ ↓ ↓
Grinding ↠- block feed grain feed powder feed
Raw materials |
| ↓ |
--> Add sterile diluent 10 times diluted shaking 30min â†--
↓
Make several dilutions ↓
Select 3 appropriate dilutions, add 1ml each to the sterile plate.
Add appropriate amount of high-salt medium per dish
25-281°C culture for 1 week
Colony count
report
6 Operation steps
6.1 Sampling When sampling, special attention must be paid to the representation of the sample and to avoid contamination during sampling. Prepare sterilizing containers and sampling tools first, such as sterilized kraft paper bags or jars, metal spoons and knives. Take representative samples on the basis of hygiene investigations. Samples should be inspected as soon as possible after sample collection; otherwise samples should be Put it in a cool, dry place.
According to the size and type of feed storage and feedstuff, the sampling at different levels is usually divided into three layers or five points or randomly sampled at different levels. After the samples at different points are fully mixed, about 500g is sent for inspection, and a small amount of feed is stored. A metal spoon can be used to sample the upper, middle, and lower parts of the sample.
Seaborne import feed sampling: Each shiphouse takes four samples of the surface, upper, middle and lower layers. Each layer is sampled and mixed from five points. If the shiphouse holds more than 10,000 tons of feed, a sample shall be taken. If necessary, take samples of questionable inspections.
6.2 Weigh 25 g (or 25 mL) of the sample in a sterile manner and place it into a glass stopper triangle containing 225 mL of sterile diluent. Place it on a shaker and shake it for 30 minutes. This is a 1:10 dilution.
6.3
Use a sterile pipette to draw 10 mL of a 1:10 dilution and inject it into a test tube with glass beads. Mix on the micromixer for 3 minutes, or inject it into a test tube. Use a sterile 1 mL pipette with a rubber nipple to inhale and breathe 50 times. Mold spores spread out.
6.4 Take 1mL1 10 dilution, inject a tube containing 9mL sterile diluent, and then change to another pipette and pipette 5 times. This solution is 1100 dilution.
6.5 Use 10 times increasing dilution according to the above operation sequence. Use one for each dilution.
1mL sterilized pipette, according to the estimate of the sample contamination, select three suitable dilutions, respectively, while making a 10-fold dilution, while drawing
1mL. Dilutions were sterilized in petri dishes. Two plates were used for each dilution. Then, the high-salt Cach medium, which was cool to about 45° C., was poured into the petri dish and mixed well. After the agar solidified, the arm was 25-28 liters.
In the incubator, observations were started 3 days after culture and should be observed for a week.
6.6 Computational methods Usually, the number of colonies with a colony count of 30-100 is selected to count, and the average number of colonies on the 2 plates of the dilution is multiplied by the dilution factor, which is the mold contained in each sample (or per milliliter). number.
6.7 Report the number of molds per gram (or per milliliter) of feed contained in units/g (pieces/mL).
Appendix A
Culture medium and dilution preparation (supplements)
Except for special regulations, the chemical reagents used in this standard are analytically pure or chemically pure; biological agents are used for bacterial culture; and water is distilled water.
A1 High Salt Catch Medium
A1.1 Ingredients Sodium Nitrate (GB636)
Potassium Dihydrogen Phosphate (GB1274) 1g
Magnesium sulfate (MgSO47H20, GB671) 0.5g
Potassium chloride (GB646) 0.5g
Ferrous sulfate (GB664) 0.01g
Sodium chloride (GB1266) 60g
Sucrose (HG3-1001) 30g
Agar 20g
Distilled water 1000mL
A1.2 method of heating dissolved, after dispensing, 115 autoclave 30min. If necessary, increase the amount of agar.
A2 diluent
A2.1 Sodium Chloride (GB1266) 8.5g
Distilled water 1000mL
A2.2 heating method to dissolve, after packing, 121 autoclave 30min.