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GB/T 13093-2023 English PDF (GB/T 13093-2006, GB/T 13093-1991)

GB/T 13093-2023_English: PDF (GB/T13093-2023)
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GB/T 13093-2023English155 Add to Cart 0--9 seconds. Auto-delivery Determination of bacterial count in feeds Valid GB/T 13093-2023
GB/T 13093-2006English105 Add to Cart 0--9 seconds. Auto-delivery Examination of bacterial count in feeds Obsolete GB/T 13093-2006
GB/T 13093-1991English199 Add to Cart 2 days [Need to translate] Method for determination of aerobic bacterial count in feeds Obsolete GB/T 13093-1991


BASIC DATA
Standard ID GB/T 13093-2023 (GB/T13093-2023)
Description (Translated English) Determination of bacterial count in feeds
Sector / Industry National Standard (Recommended)
Classification of Chinese Standard B46
Classification of International Standard 65.120
Word Count Estimation 10,156
Date of Issue 2023-08-06
Date of Implementation 2024-03-01
Older Standard (superseded by this standard) GB/T 13093-2006
Drafting Organization Institute of Agricultural Quality Standards and Testing Technology, Chinese Academy of Agricultural Sciences, Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences
Administrative Organization National Feed Industry Standardization Technical Committee (SAC/TC 76)
Proposing organization National Feed Industry Standardization Technical Committee (SAC/TC 76)
Issuing agency(ies) State Administration for Market Regulation, National Standardization Administration

BASIC DATA
Standard ID GB/T 13093-2006 (GB/T13093-2006)
Description (Translated English) Examination of bacterial count in feeds
Sector / Industry National Standard (Recommended)
Classification of Chinese Standard B46
Classification of International Standard 65.120
Word Count Estimation 9,912
Date of Issue 2006-12-12
Date of Implementation 2007-03-01
Older Standard (superseded by this standard) GB/T 13093-1991
Quoted Standard GB/T 6682; GB/T 14699.1; GB/T 20195-2006
Drafting Organization National Feed Quality Supervision and Inspection Center (Beijing)
Administrative Organization National Standardization Technical Committee Feed Industry
Regulation (derived from) National Standard Approval Announcement 2006 No.13 (Total No.100)
Proposing organization National Feed Industry Standardization Technical Committee
Issuing agency(ies) Administration of Quality Supervision, Inspection and Quarantine of People's Republic of China; Standardization Administration of China
Summary This standard specifies the determination of feed bacteria. This standard applies to feed, concentrated feed, feed ingredients (fish meal), cattle feed concentrate supplement the total number of bacteria measured.

BASIC DATA
Standard ID GB/T 13093-1991 (GB/T13093-1991)
Description (Translated English) Method for determination of aerobic bacterial count in feeds
Sector / Industry National Standard (Recommended)
Classification of Chinese Standard B46
Classification of International Standard 65.12
Word Count Estimation 4,425
Date of Issue 1991/7/16
Date of Implementation 1992/4/1
Adopted Standard ISO 4833-1978, NEQ
Regulation (derived from) Announcement of Newly Approved National Standards No. 13, 2006 (No. 100 overall)
Proposing organization National Feed Industry Standardization Technical Committee
Issuing agency(ies) State Bureau of Technical Supervision


GB/T 13093-2023 GB NATIONAL STANDARD OF THE PEOPLE'S REPUBLIC OF CHINA ICS 65.120 CCS B 46 Replacing GB/T 13093-2006 Determination of bacterial count in feeds ISSUED ON: AUGUST 6, 2023 IMPLEMENTED ON: MARCH 1, 2024 Issued by: State Administration for Market Regulation; Standardization Administration of PRC. Table of Contents Foreword ... 3 1 Scope ... 5 2 Normative references ... 5 3 Terms and definitions ... 5 4 Principle... 5 5 Reagents or materials ... 6 6 Instruments and equipment ... 6 7 Samples ... 7 8 Test steps ... 7 9 Experimental data processing ... 9 Appendix A (Normative) Plate count agar medium preparation ... 11 Appendix B (Informative) Examples of bacterial count calculation ... 12 Determination of bacterial count in feeds 1 Scope This document describes the plate count method for the detection of the bacterial count in feed. This document is applicable to the determination of the bacterial count in compound feed, concentrated feed, concentrate supplements, and feed ingredients of animal and plant origin. 2 Normative references The provisions of the following documents constitute the essential clauses of this document through normative references in this text. Among them, for referenced documents with dates, only the versions corresponding to the dates are applicable to this document; for referenced documents without dates, the latest versions (including all amendments) are applicable to this document. GB/T 6682 Water for analytical laboratory use - Specification and test methods GB/T 20195 Animal feeding stuffs - Preparation of test samples 3 Terms and definitions The following terms and definitions apply to this document. 3.1 bacterial count The number of colonies obtained per gram or per milliliter of the specimen after the feed specimen is processed and cultured under certain conditions (such as using a specific culture medium, culture temperature, and time). NOTE: The number of colonies is expressed in colony-forming units (CFU). 4 Principle After the feed sample is properly treated and diluted, the microorganisms in it are fully dispersed into single cells. A certain amount of the diluted sample solution is inoculated on a plate. After cultivation, each single cell grows and multiplies to form colonies visible to the naked eye, that is, a single colony shall represent a single cell in the original sample; the number of colonies is counted, and the number of bacteria in the feed can be calculated based on the dilution multiple and the sampling inoculation amount. 5 Reagents or materials Unless otherwise specified, only analytical-grade reagents are used. 5.1 Water: The grade 3 specified in GB/T 6682. 5.2 Sodium hydroxide solution (1 mol/L): Weigh 20 g of sodium hydroxide, dissolve it in water, cool to room temperature, and make up to 500 mL with water. 5.3 Hydrochloric acid solution (1 mol/L): Measure 42 mL of concentrated hydrochloric acid, dilute to 500 mL with water, and shake well. 5.4 Phosphate buffer stock solution: Weigh 34.0 g potassium dihydrogen phosphate and dissolve it in 500 mL water. Adjust the pH to 7.2 with about 175 mL of 1 mol/L sodium hydroxide solution, dilute to 1000 mL with water, mix well, and store at 2 °C~8 °C. 5.5 Sterile phosphate buffer: Take 1.25 mL of phosphate buffer stock solution (5.4), dilute to 1000 mL with water, dispense into suitable containers or 9 mL per tube, and sterilize by autoclaving at 121 °C for 15 min. 5.6 Sterile physiological saline: Weigh 8.5 g of sodium chloride, dissolve it in water, and make up to 1000 mL. Dispense into suitable containers or 9 mL per tube and sterilize by autoclaving at 121 ℃ for 15 min. 5.7 Plate count agar medium: Prepare according to Appendix A. 5.8 Sterile liquid paraffin: Take liquid paraffin and sterilize it by autoclaving at 121 ℃ for 20 min. 5.9 Sterile Tween 80: Take Tween 80 and sterilize it by autoclaving at 121 ℃ for 20 min. 6 Instruments and equipment 6.1 Constant temperature incubator: The temperature control accuracy is ±1 °C. 6.2 Slap homogenizer. 6.3 Constant temperature device: The temperature control accuracy is ±2 ℃. 8.1.2.2 Follow the procedure in 8.1.2.1 to make 10-fold incremental dilutions. The sterile pipette or micropipette tip shall be replaced after each incremental dilution. 8.1.2.3 According to the estimation of the contamination status of the sample, select 2 ~3 sample homogenates of appropriate serial dilutions, and pipette 1 mL of sample homogenate of each dilution into a sterile plate; make two plates for each dilution. At the same time, pipette 1 mL of sterile phosphate buffer or sterile physiological saline into a sterile plate as a blank control. 8.2 Cultivation After the dilution is transferred to the plate, promptly pour 15 mL~20 mL of the culture medium cooled to 46 ℃~50 ℃ (can be placed in a 48 ℃±2 ℃ constant temperature device) into the plate, and carefully rotate the plate to fully mix the sample and the culture medium. The time from diluting the sample to pouring the culture medium shall not exceed 30 min. If it is estimated that the sample may diffusely grow on the surface of the culture medium, after the culture medium is completely solidified, a thin layer of plate count agar medium (about 4 mL) cooled to 48 ℃±2 ℃ can be covered on the solidified agar surface. After the agar solidifies, invert the plate horizontally and culture it in a 36 ℃±1 ℃ constant temperature incubator for 48 h±2 h. 8.3 Bacterial colony counting 8.3.1 When counting bacterial colonies, observation can be made with the naked eye. If necessary, a magnifying glass or colony counter can be used to check to prevent omissions. Bacterial counts are expressed in colony-forming units (CFU). 8.3.2 Select plates with colony counts between 30 CFU ~ 300 CFU and no spreading colony growth for counting. For plates with less than 30 CFU, record the specific colony count; for plates with more than 300 CFU, record as TNTC (too numerous to count). 8.3.3 When one of the two plates has large flake-like colonies growing, the plate without large flake-like colonies shall be selected for colony counting; if the flake-like colonies are less than half of the plate, and the colonies on the other half are evenly distributed, the half of the plate with evenly distributed colonies shall be selected for colony counting, and the count result multiplied by 2 represents the colony number on this plate. 8.3.4 If there are chain-like growths without clear boundaries between colonies on the plate, each single chain shall be counted as one colony. 8.3.5 If there is colony growth on the blank control, the test result will be invalid. 9 Experimental data processing 9.1 Calculation of bacterial count 9.1.1 If the number of colonies on only one dilution plate is within the appropriate counting range, calculate the average value of the colony counts on the two plates and then multiply the average value by the corresponding dilution multiple to obtain the bacterial count per gram or per milliliter of the sample. See Example 1 in Appendix B. 9.1.2 If the colony counts on two serial dilution plates are within the appropriate counting range, calculate it according to formula (1): where: N -- The bacterial count in the sample; C -- The number of colonies on the plates (the plates on which the number of colonies is in the appropriate range); n1 -- The number of plates of the first dilution (low dilution multiple); n2 -- The number of plates of the second dilution (high dilution multiple); d -- Dilution factor (first dilution). The calculated result is the bacterial count per gram or per milliliter of the sample, see Example 2 in Appendix B. 9.1.3 If the colony counts on all dilution plates are greater than 300 CFU, count the plate with the highest dilution and record the other plates as TNTC. The result is calculated by multiplying the average colony count by the highest dilution multiple, see Example 3 in Appendix B. 9.1.4 If the colony counts on the plates of all dilutions are less than 30 CFU, the calculation shall be based on the average colony count of the lowest dilution multiplied by the dilution multiple, see Example 4 in Appendix B. 9.1.5 If no bacteria grow on any dilution plate, calculate by multiplying the lowest dilution multiple by less than 1, see Example 5 in Appendix B. 9.1.6 If the colony counts on the plates of all dilutions are not between 30 CFU ~ 300 CFU, and some of them are less than 30 CFU or greater than 300 CFU, the average colony count closest to 30 CFU or 300 CFU shall be multiplied by the dilution multiple ......


GB/T 13093-2006 GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA ICS 65.120 B 46 Replacing GB/T 13093-1991 Examination of bacterial count in feeds ISSUED ON: DECEMBER 12, 2006 IMPLEMENTED ON: MARCH 01, 2007 Issued by: General Administration of Quality Supervision, Inspection and Quarantine of PRC; Standardization Administration of PRC. Table of Contents Foreword ... 3 1 Scope ... 4 2 Normative references ... 4 3 Terms and definitions ... 4 4 Principles ... 5 5 Equipment and materials ... 5 6 Media and reagents ... 5 7 Preparation of specimens ... 6 8 Measurement procedure ... 6 9 Measurement steps ... 7 Appendix A (Normative) Media and reagents ... 10 References ... 12 Examination of bacterial count in feeds 1 Scope This standard specifies the method for the determination of the bacterial count in feed. This standard is applicable to the determination of the bacterial count in compound feed, concentrated feed, feed raw materials (fishmeal, etc.), supplementary cattle concentrate. 2 Normative references The provisions in following documents become the provisions of this Standard through reference in this Standard. For the dated references, the subsequent amendments (excluding corrections) or revisions do not apply to this Standard; however, parties who reach an agreement based on this Standard are encouraged to study if the latest versions of these documents are applicable. For undated references, the latest edition of the referenced document applies. GB/T 6682 Water for analytical laboratory use - Specification and test methods GB/T 14699.1 Feeding stuffs - Sampling GB/T 20195-2006 Animal feeding stuffs - Preparation of test samples 3 Terms and definitions The following terms and definitions apply to this standard. 3.1 Bacterial count After the feed specimen is processed and cultured under certain conditions (such as culture ingredients, culture temperature and time, etc.), the bacterial count in 1 g (mL) of the specimen obtained. Note: It is mainly used as a sign to determine the degree of contamination of feeds. This method can also be used to observe the dynamics of bacterial reproduction in feed, so as to provide a basis for hygienic evaluation of the tested samples. 4 Principles After the specimen is processed, it is diluted to an appropriate concentration, cultured under certain conditions (such as using a specific medium, culturing at a temperature of 30 °C ± 1 °C for 72 h ± 3 h, etc.), the bacterial count in the resulting 1 g (mL) of specimen is obtained. 5 Equipment and materials 5.1 Analytical balance: Sensitivity is 0.1 g. 5.2 Oscillator: Reciprocating. 5.3 Pulverizer: It is not a whirlwind mill, with good airtightness. 5.4 Autoclave: Sterilization pressure 0 ~ 3 kg/cm2. 5.5 Refrigerator: Ordinary refrigerator. 5.6 Constant temperature water bath: 46 °C ± 1 °C. 5.7 Constant temperature incubator: 30 °C ± 1 °C. 5.8 Micromixer. 5.9 Sterile conical flasks of 100 mL, 250 mL, 500 mL. 5.10 Sterile pipette: 1 mL, 10 mL. 5.11 Sterile test tube: 16 mm x 160 mm. 5.12 Sterile glass beads: 5 mm in diameter. 5.13 Sterile petri dish: 90 mm in diameter. 5.14 Sterilize metal spoons, knives, etc. 6 Media and reagents 6.1 Nutrient agar medium: See Appendix A. 6.2 Phosphate buffer solution (diluent): See Appendix A. 6.3 0.85% normal saline: See Appendix A. 6.4 Water agar medium: See Appendix A. 9 Measurement steps 9.1 Specimen dilution and incubation 9.1.1 Weigh 25 g (or 10 g) of the specimen (Chapter 7) by aseptic operation. Put it in a sterilized conical flask, which contains 225 mL (or 90 mL) of diluent or normal saline (the bottle is pre-filled with appropriate number of glass beads). Place it on an oscillator, to oscillate it for 30 minutes. After fully shaking, prepare a uniform dilution of 1:10. It is best to process in a homogenizer, at a speed of 8000 r/min ~ 10000 r/min for 1 min. 9.1.2 Use a 1 mL sterilized pipette, to absorb 1 mL of the 1:10 diluent. Slowly pour it into a test tube, which contains 9 mL of the sterilized diluent or normal saline, along the tube wall (note that the tip of the pipette shall not touch the diluent in the tube). Shake the test tube. OR put it on a micromixer, to mix for 30 s. Mix well. Make a 1:100 diluent. 9.1.3 Take another 1 mL sterilized straw. Make a 10-fold incremental dilution, according to the above operation method. Every time the incremental dilution is performed, replace a sterilized straw. 9.1.4 According to the requirements of the feed hygiene standards or the estimation of the contamination degree of the specimen, select 2 ~ 3 appropriate dilutions, respectively. While making 10-fold incremental dilutions, pipette 1 mL of the dilution into the sterilized petri dish, to make two petri dishes for each dilution. 9.1.5 After the dilution liquid is transferred into the petri dish, about 15 mL of the medium, which is cooled to 46 °C ± 1 °C (it can be kept in a water bath at 46 °C ± 1 °C), shall be poured into the petri dish in time. Rotate the culture dish carefully, to make the specimen mix well with medium. The time -- between diluting the specimen and pouring the culture medium -- shall not exceed 30 min. If it is estimated that the microorganisms, which are contained in the specimen, may grow on the surface of the medium plate, after the medium is completely set, pour 4 mL of water agar medium, which is cooled to 46 °C ± 1 °C, on the surface of the medium. 9.1.6 After the agar has solidified, invert the plate and incubate it in a constant temperature incubator at 30 °C ± 1 °C, for 72 h ± 3 h. Take it out. Calculate the bacterial count in the plate. Multiply the bacterial count by the dilution factor, to obtain the bacterial count per gram of specimen. 9.2 Calculation method of bacterial count When doing plate colony counting, it can be observed with the naked eye. If the colony shape is small, it can be checked with the help of a magnifying glass, to prevent omissions. After calculating the bacterial count on each plate, calculate the average value of the two plate colonies, which have the same dilution. 9.3 Reporting of bacterial counts 9.3.1 Selection of the bacterial count on the plate Select a plate, which has a bacterial count between 30 and 300, as the standard for the determination of the bacterial count. For each dilution, use the average number of colonies on two plates. If one of the two plates has larger flake colonies, it should not be used. Instead, the average number of plate colonies without flake colonies shall be used as the bacterial count of this dilution. If the flake colony is less than half of the plate, whilst the other half of the colony is evenly distributed, THEN, the half plate can be calculated and multiplied by 2, to represent the bacterial count on the plate. 9.3.2 Selection of dilution 9.3.2.1 The dilution, which has an average bacterial count between 30 and 300, shall be selected, multiplied by the dilution factor and reported (see case 1 in Table 1). 9.3.2.2 If there are two dilutions and the bacterial count grown is within 30 ~ 300, it shall be decided based on the ratio of the two. If the ratio is less than or equal to 2, the average value shall be reported; if it is greater than 2, the smaller number shall be reported (see case 2 and case 3 in Table 1). 9.3.2.3 If the average bacterial count in all dilutions is greater than 300, it shall be reported as the average bacterial count in the highest dilution multiplied by the dilution factor (see case 4 in Table 1). 9.3.2.4 If the average bacterial count in all dilutions is less than 30, it shall be reported as the average bacterial count in the lowest dilution multiplied by the dilution factor (see case 5 in Table 1). 9.3.2.5 If there is no colony growth at all dilutions, report it as less than 1 multiplied by the lowest dilution (see case 6 in Table 1). 9.3.2.6 If the average bacterial count in all dilutions is not between 30 and 300, meanwhile some of them are greater than 300 or less than 30, then it is reported as the average bacterial count closest to 30 or 300 multiplied by the dilution factor (see case 7 of Table 1). 9.3.3 Report of total bacterial count When the bacterial count is less than 100, it is reported according to the actual number; when it is greater than 100, it takes two significant figures, meanwhile the value after the two significant figures is rounded off. In order to shorten the number of zeros after the number, it can also be represented by an exponent of 10 (see Table 1). Appendix A (Normative) Media and reagents Unless otherwise specified, all reagents used are of analytical grade; water complies with the requirements of grade-3 water in GB/T 6682. A.1 Nutrient agar medium A.1.1 Composition Peptone: 10 g Beef extract: 3 g Sodium chloride: 5 g Agar: 15 g ~ 20 g Distilled water: 1000 mL A.1.2 Preparation method Dissolve all components, except agar, in distilled water. Add about 2 mL of 15% sodium hydroxide solution, to correct the pH to 7.2 ~ 7.4. Add agar and heat to boil, to dissolve the agar. Divide into conical flasks and autoclave at 121 °C for 20 min. Note: This medium is used for general bacterial culture. It can be poured into a flat plate or made into a slope. For colony counting, the agar shall be 1.5%, and if it is made into a plate or slope, it shall be 2%. A.2 Phosphate buffer (diluent) A.2.1 Storage solution Potassium dihydrogen phosphate: 34 g 1 mol/L sodium hydroxide solution: 175 mL Distilled water: 1000 mL A.2.2 Preparation method Dissolve phosphate in 500 mL of distilled water. Use 1 mol/L sodium hydroxide solution, to correct the pH to 7.0 ~ 7.2. Then use distilled water to dilute it to 1000 mL. ......

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