GB 4789.2—2010 Aerobic plate count.doc

GB 4789.22010National food safety standardFood microbiological examination：Aerobic plate countAerobic plate count Food test sample is treated under certain conditions after (e.g. medium, culture temperature and culture time) culture, resulting per g (mL)The total number of microbial colonies formed seized samples1 Equipment and MaterialsIn addition to the microbiology laboratory and training routine sterilization equipment, other equipment and materials as follows:1.1 incubator: 36 1 , 30 1 .1.2 Refrigerator: 2 5 .1.3 thermostatic water bath: 46 1 1.4 Balance: a sense of the amount of 0.1 g.1.5 homogenizer.1.6 oscillators.1.7 Sterile pipette: 1 mL (0.01 mL with scale), 10 mL (0.1 mL with scale) or pipette and tip.1.8 sterile conical flask: capacity 250 mL, 500 mL.1.9 sterile Petri dish: diameter 90 mm.1.10 pH meter or pH colorimetric tubes or precision pH test paper.1.11 magnifier and / or colony counter.2 media and reagents2.1 plate count agar medium: See Appendix A, A.1.2.2 phosphate buffer: See Appendix A A.2.2.3 Sterile saline: See Appendix A A.33 Inspection ProgramThe total number of colonies inspection program in Figure 1Test sample25 g (mL) sample +225 mL diluent, homogeneousReportCalculate the total number of colonies10-fold serial dilutionsChoose two or three suitable dilution liquid sample uniform,From each 1 mL of sterile Petri dishes were added withinEach dish was added 15 mL 20 mLPlate count agar medium, mixCulture Count the number of colonies on each plateFigure1. The total number of colonies inspection procedures4 procedure4.1 dilution of samples4.1.1 solid and semi-solid samples: Weigh 25 g sample set containing 225 mL of phosphate buffered saline solution or sterile homogeneous within the cup, 8000 r / min 10000 r / min homogenized 1 min 2 min or placed filled with 225 mL of sterile diluent bag homogeneous, with slap-type homogenizer beat 1 min 2 min, a sample was evenly 1:10.4.1.2 Liquid samples: 25 mL sterile pipette sample set containing 225 mL of phosphate buffered saline solution or sterile Erlenmeyer flask (flask preset appropriate number of sterile glass beads), the full mix the sample was made uniform 1:10.4.1.3 with 1 mL sterile pipette or pipette to draw samples 1:10 uniform liquid 1 mL, along the wall slowly inject in a sterile tube containing 9 mL dilution (note pipette tip or not to tip touch diluted liquid), shake the tube or replaced with a sterile pipette repeated pipetting to mix evenly into 1: 100 of the liquid sample uniform.4.1.4 4.1.3 Procedure according to prepare 10-fold serial dilution of the sample was absorbed. Each incremental dilution once and replace it with a second 1 mL sterile pipette or tip.4.1.5 Based on the condition of sample contamination estimates, select the sample absorbed liquid 2 3 suitable dilution (liquid sample may include stock solution), during the 10-fold dilution increments, absorb 1 mL sample was homogenized in a sterile petri dish inside, do the two plates each dilution. Meanwhile, Pipette 1 mL dilution was added as blank blank in two sterile petri dish.4.1.6 promptly flat 15 mL 20 mL cooled to 46 count agar (can be placed at 46 1 water bath tank insulation) pour plate and turn the dish to mix evenly.4.2 Training5.2.1 until the agar solidified, the plates were inverted, 36 1 cultured 48 h 2 h. Aquatic 30 1 cultured 72 h 3 h.5.2.2 If the sample may contain the agar surface diffuse colonies grown, covering the agar surface in a thin layer of solidified agar medium (about 4 mL), after solidification flip tablet, according to 6.2.1 conditions bring up.4.3 colony countsNaked eye observation, if necessary, with a magnifying glass or a colony counter, recording the corresponding dilution factor and the number of colonies. Colony count in colony-forming units (colony-forming units, CFU), said.5 Results and reports5.1 Calculation of the total number of colonies5.1.1 If only a dilution of the number of colonies on the plate count in the appropriate range, calculate the average number of colonies of the two plates, and then flatMean multiplied by the corresponding dilution factor, as per g (mL) sample of the total number of colonies results.5.1.2 If two successive dilution plate count when the count in the appropriate range, according to the formula (1):N- colonies in the sample;C- tablet (tablet containing the appropriate range of the number of colonies) and the number of colonies;n1- first dilution (lower dilution) plate number;n2- second dilution (higher dilution factor) the number plate;d- dilution factor (first dilution).For example:Dilution1:100(first Dilution)1:1000(second Dilution)CFU232，24433，35Refer to 5.2.2, above data by rounding figures, expressed as 25,000 or 2.5 1045.1.3 If the number of colonies on the plate all dilutions were greater than 300 CFU, the highest degree of dilution plates were counted, other tablet canRecorded as uncountable, the result is multiplied by the average number of colonies highest dilution calculations.5.1.4 If the plate count all dilutions were less than 30 CFU, the dilution should be the lowest average number of colonies counted multiplied by the dilution factorCount.5.1.5 If all dilutions (including liquid sample liquid) tablet no colony growth, places less than a dilution factor is calculated by multiplying the minimum.6.1.6 If the plate count not at all dilutions between 30 CFU 300 CFU, some of which less than 30 CFU or greater than 300CFU, the places closest to 30 CFU or 300 CFU average dilution factor is calculated by multiplying the number of colonies.5.2 The report of total number colonies5.2.1 the number of colonies less than 100 CFU, press the rounding the principle of rounding to integer report.5.2.2 colony number greater than or equal to 100 CFU, the first three digits after the use of rounding the principle of rounding off, take the first two numbers, afterFace with a 0 instead of the median; 10 can also be used to represent the exponential form, press the rounding off after rounding principle, the use of two significant figures.5.2.3 If it is unable to counter the spread of colonies on all flat, the report colonies spread.5.2.4 If the colony growth on the control, then the test results invalid.5.2.5 Weighing sampled to CFU / g reported in units of volume sampled to CFU / mL reported in units ofAppendix A（Standard Appendix）Media and reagentsA.1 plate count agar，PCAA.1.1 CompositionTryptone 5.0 gYeast Extract 2.5 gGlucose 1.0 gAgar 15.0 gDistilled water 1000 mLPH 7.00.2A.1.2 MethodThe above ingredients were added to distilled