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Standard ID | GB 4789.10-2016 (GB4789.10-2016) | Description (Translated English) | National Food Safety Standard -- Food Microbiological Examination -- Examination of Staphylococcus Aureus | Sector / Industry | National Standard | Classification of Chinese Standard | C53 | Classification of International Standard | 07.100.30 | Word Count Estimation | 17,189 | Date of Issue | 2016-12-23 | Date of Implementation | 2017-06-23 | Older Standard (superseded by this standard) | GB 4789.10-2010; SN/T 0172-2010; SN/T 2154-2008 | Regulation (derived from) | National Health and Family Planning Commission Notice No.17 of 2016 | Issuing agency(ies) | National Health and Family Planning Commission of the People Republic of China, State Administration of Food and Drug Administration | Summary | This standard specifies the method for the determination of Staphylococcus aureus in food. The first method of this standard applies to the qualitative test of Staphylococcus aureus in food; the second method is applicable to the counting of Staphylococcus aureus in food with high Staphylococcus aureus content; the third method is suitable for Staphylococcus aureus content Low count of Staphylococcus aureus in food. | Standard ID | GB 4789.10-2010 (GB4789.10-2010) | Description (Translated English) | National food safety standard. Food microbiological examination. Staphylococcus aureus | Sector / Industry | National Standard | Classification of Chinese Standard | C53 | Classification of International Standard | 07.100.30 | Word Count Estimation | 16,184 | Date of Issue | 2010-03-26 | Date of Implementation | 2010-06-01 | Older Standard (superseded by this standard) | GB/T 4789.10-2008; GB/T 4789.37-2008 | Regulation (derived from) | Circular of the Ministry of Health (2010)7 | Issuing agency(ies) | Ministry of Health of People's Republic of China | Summary | This Chinese standard specifies the food Staphylococcus aureus (Staphylococcus aureus) testing methods. This standard applies to the first law of Staphylococcus aureus in foods qualitative test, second law applies to higher levels of Staphylococcus aureus in food count, Third Law applies to Staphylococcus aureus content than low and high levels of bacteria Staphylococcus aureus in food count. | Standard ID | GB/T 4789.10-2008 (GB/T4789.10-2008) | Description (Translated English) | Microbiological examination of food hygiene. Detection of staphylococcus aureus | Sector / Industry | National Standard (Recommended) | Classification of Chinese Standard | C53 | Classification of International Standard | 07.100.30 | Word Count Estimation | 15,136 | Date of Issue | 2008-11-21 | Date of Implementation | 2009-03-01 | Older Standard (superseded by this standard) | GB/T 4789.10-2003 | Adopted Standard | AOAC 987.09, NEQ; ISO 6888-1-1999, NEQ | Drafting Organization | China Disease Prevention and Control Center of Nutrition and Food Safety | Administrative Organization | Ministry of Health | Regulation (derived from) | National Standard Approval Announcement 2008 No.22 (Total No.135); Health-Communication (2010) 7; National Standard Approval Announcement 2010 No.5 (Total No.160) | Proposing organization | People's Republic of China Ministry of Health | Issuing agency(ies) | Ministry of Health of People's Republic of China; Standardization Administration of China | Summary | This standard specifies test methods Staphylococcus aureus in food. |
GB 4789.10-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard -
Food microbiological examination -
Staphylococcus aureus
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the
People’s Republic of China;
China Food and Drug Administration.
3. No action is required - Full-copy of this standard will be automatically &
immediately delivered to your EMAIL address in 0~60 minutes.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Equipment and materials ... 4
3 Medium and reagents ... 5
4 Examination procedure ... 5
5 Operating procedure ... 6
6 Result and report ... 8
7 Examination procedure ... 8
8 Operating procedure ... 8
9 Result calculation ... 10
10 Report ... 11
11 Examination procedure ... 12
12 Operating procedure ... 12
13 Result and report ... 13
Annex A Medium and reagents ... 14
Annex B Staphylococcal enterotoxin detection ... 19
Annex C Retrieval table for most probable number (MPN) of staphylococcus
aureus ... 24
Foreword
This Standard replaces GB 4789.10-2010 “National food safety standard - Food
microbiological examination. Staphylococcus aureus”, SN/T 0172-2010
“Determination of Staphylococcus aureus in foods for import and export”, SN/T 2154-
2008 “Determination of coagulase-positive staphylococci in import and export food -
Technique using rabbit plasma fibrinogen agar medium”.
Compared with GB 4789.10-2010, the main changes of this standard are as follows.
- The enrichment fluid for test is modified to 7.5 % sodium chloride broth.
National food safety standard -
Food microbiological examination –
Staphylococcus aureus
1 Scope
This Standard specifies the examination method for Staphylococcus aureus in foods.
Method I of this Standard applies to the qualitative examination of Staphylococcus
aureus in foods; Method II applies to the counting of Staphylococcus aureus in foods
with high Staphylococcus aureus content; Method III applies to the counting of
Staphylococcus aureus in foods with low Staphylococcus aureus content.
2 Equipment and materials
In addition to routine sterilization and culture equipment for microbiological laboratories,
other equipment and materials are as follows.
2.1 Constant temperature incubator. 36 °C ± 1 °C.
2.2 Refrigerator. 2 °C ~ 5 °C.
2.3 Constant temperature water bath. 36 °C ~ 56 °C.
2.4 Balance. with division of 0.1 g.
2.5 Homogenizer.
2.6 Oscillator.
2.7 Sterile pipette. 1 mL (with 0.01 mL scale), 10 mL (with 0.1 mL scale) or micro pipette
and tip.
2.8 Sterile Erlenmeyer flask. with capacity of 100 mL and 500 mL.
2.9 Sterile Petri dish. with diameter of 90 mm.
2.10 Spreader.
2.11 pH meter or pH colorimetric tube or precision pH test paper.
5.4 Preliminary identification
Staphylococcus aureus on the Baird-Parker plate is round, smooth-surface, raised,
moist, with colony diameter of 2 mm ~ 3 mm, color of gray black to black color, shiny,
and usually light (non-white) edge, around the opaque circle (precipitation) and a clear
band outside. When the inoculating needle touches the colony, there is a butter-like
sticky feeling. Sometimes strains of fats can be decomposed can be seen, except
opaque circle and clear band, the appearance is basically the same. Colonies isolated
from long-term stored frozen or dehydrated foods are often with lighter black than
typical colonies, and more rough appearance and more dry texture. On the blood plate,
the colony is larger, round, smooth raised, moist, golden (sometimes white), with
transparent hemolytic circle seen around the colony. Pick the above-mentioned
suspicious colonies for Gram’s stain microscopic examination and plasma coagulase
test.
5.5 Confirmation identification
5.5.1 Stain microscopic examination. Staphylococcus aureus is Gram’s positive cocci,
arranged in grape-like, no spores, no capsule, with the diameter of about 0.5 μm ~ 1
μm.
5.5.2 Plasma coagulase test. PICK at least 5 suspicious colonies (select all when there
are less than 5) on Baird-Parker plate or blood plate; INOCULATE them to 5 mL of BHI
and nutrient agar slants respectively; CULTURE at 36 °C ± 1 °C for 18 h ~ 24 h.
TAKE 0.5 mL of newly prepared rabbit plasma into a small test tube, ADD 0.2 mL ~ 0.3
mL of BHI culture, SHAKE well; PLACE it in temperature box or water bath box at
36 °C ± 1 °C; OBSERVE every half hour for a total of 6 h; if there is solidification (that
is, when the test tube is tilted or inverted, there are clots) or if the solidification volume
is greater than half the original volume, it is determined to be positive results. While
the broth culture of the positive and negative Staphylococcus aureus strains of plasma
coagulase test is used as a control. It can also use commercial reagents, operate
according to the instructions, carry out the plasma coagulase test.
If the result is suspicious, pick the colonies on the nutrient agar slant into 5 mL of BHI
and culture at 36 °C ± 1 °C for 18 h ~ 48 h.
5.6 Staphylococcal enterotoxin examination (optional)
For identification of suspicious food poisoning samples or Staphylococcus aureus
strains producing staphylococcal enterotoxin, Staphylococcal enterotoxin shall be
tested according to Annex B.
T - the number of Staphylococcus aureus colonies in the sample;
A - the total number of typical colonies of a certain dilution;
B - the number of colonies identified as positive of a certain dilution;
C - the number of colonies used for identification test of a certain dilution;
d - the dilution factor.
Equation (2).
where.
T - the number of Staphylococcus aureus colonies in the sample;
A1 - the total number of typical colonies of the first dilution (low dilution factor);
B1 - the number of colonies identified as positive of the first dilution (low dilution
factor);
C1 - the number of colonies used for identification test of the first dilution (low dilution
factor);
A2 - the total number of typical colonies of the second dilution (high dilution factor);
B2 - the number of colonies identified as positive of the second dilution (high dilution
factor);
C2 - the number of colonies used for identification test of the second dilution (high
dilution factor);
1.1 - the calculation coefficient;
d - the dilution factor (first dilution).
10 Report
According to the calculation results of the equations in Clause 9, report the number of
Staphylococcus aureus in per g (mL) of sample, expressed as CFU/g (mL); if the T
value is 0, report the lowest dilution factor multiplies by less than 1.
95 % ethanol 20.0 mL
1 % aqueous ammonium oxalate solution 80.0 mL
A.8.1.2 Preparation method
Completely DISSOLVE the crystal violet in ethanol, and then MIX with ammonium
oxalate solution.
A.8.2 Gram’s liquid iodine
A.8.2.1 Ingredients
Iodine 1.0 g
Potassium iodide 2.0 g
Distilled water 300 mL
A.8.2.2 Preparation method
MIX iodine and potassium iodide first; ADD a small amount of distilled water and
SHAKE well; after completely dissolved, ADD distilled water to 300 mL.
A.8.3 Safranin counter stain
A.8.3.1 Ingredients
Safranin 0.25 g
95 % ethanol 10.0 mL
Distilled water 90.0 mL
A.8.3.2 Preparation method
DISSOLVE safranin in ethanol, and then DILUTE with distilled water.
A.8.4 Staining method
a) FIX the smear on the flame, DROP crystal violet stain, STAIN for 1 min, RINSE.
b) DROP Gram’s liquid iodine, REACT for 1 min, RINSE.
c) DROP 95 % ethanol to decolorize for about 15 s ~ 30 s, until the stain is rinsed
away, do not over-decolorize, RINSE.
d) DROP counter stain to stain for 1 min, RINSE and DRY for microscopic
Annex B
Staphylococcal enterotoxin detection
B.1 Reagents and materials
Unless otherwise specified, the reagents used are analytical regents, and the test
water shall comply with the provisions of Grade I water in GB/T 6682.
B.1.1 A, B, C, D, E-type Staphylococcal enterotoxin ELISA kit.
B.1.2 pH test paper, with the range of 3.5 ~ 8.0, and the accuracy of 0.1.
B.1.3 0.25 mol/L Tris buffer with pH 8.0. DISSOLVE 121.1 g of Tris in 800 mL of
deionized water; after cooling to room temperature, ADD 42 mL of concentrated HCL
to pH 8.0.
B.1.4 Phosphate buffer with pH 7.4. WEIGH 0.55 g of NaH2PO4...
......
GB 4789.10-2010
National food safety standard.Food microbiological examination.Staphylococcus aureus
National Standards of People's Republic of China
National Food Safety Standard
Food Microbiology testing of Staphylococcus
National food safety standard
Food microbiological examination. Staphylococcus aureus
People's Republic of China Ministry of Health issued
Issued on. 2010-03-26
2010-06-01 implementation
Foreword
This standard replaces GB/T 4789.10-2008 "Microbiological examination of food hygiene inspection Staphylococcus aureus" and
GB/T 4789.37-2008 "Microbiological examination of food hygiene Staphylococcus aureus count."
This standard and GB/T 4789.10-2008 and GB/T 4789.37-2008 compared to major changes are as follows.
- Modify the standard English name;
- Modify the range;
- Specification of the sample preparation process;
- An increase of 1.1 coefficient calculation formula explained;
- Amend Appendix A in trypticase soy broth name, specification of 10% sodium chloride trypticase soy broth;
- Added a second law of Staphylococcus aureus Baird-Parker plate count and the third law of Staphylococcus aureus MPN count.
The Standard Appendix A, Appendix B, Appendix C is a normative appendix.
This standard replaces the standards previously issued as follows.
--GB 4789.10-84, GB 4789.10-1994, GB/T 4789.10-2003, GB/T 4789.10-2008.
--GB/T 4789.37-2008.
National Food Safety Standard
Food Microbiology testing of Staphylococcus
1 Scope
This standard specifies the food Staphylococcus aureus (Staphylococcus aureus) test method.
The first method is suitable for standard qualitative test foods Staphylococcus aureus; second method is suitable for a high content of Staphylococcus aureus
Food counting Staphylococcus aureus; third law applies to Staphylococcus aureus bacteria content is low and the higher gold content in foods
Staphylococcus count.
2 Equipment and Materials
In addition to the microbiological laboratory conventional sterilization and cultivation equipment, other equipment and materials as follows.
2.1 incubator. 36 ℃ ± 1 ℃.
2.2 Refrigerator. 2 ℃ ~ 5 ℃.
2.3 constant temperature water bath. 37 ℃ ~ 65 ℃.
2.4 Balance. a sense of the amount of 0.1 g.
2.5 homogenizer.
2.6 oscillator.
2.7 sterile pipette. 1 mL (0.01 mL with scale), 10 mL (0.1 mL with scale) or micro pipettes and tips.
2.8 sterile conical flask. capacity 100 mL, 500 mL.
2.9 sterile Petri dish. a diameter of 90 mm.
2.10 Syringe. 0.5 mL.
2.11 pH meter or pH colorimetric tubes or precision pH test paper.
3 media and reagents
3.1 10% sodium chloride trypticase soy broth. See Appendix A, A.1.
3.2 7.5% sodium chloride broth. See Appendix A A.2.
3.3 blood agar plates. A A.3 in the Appendix.
3.4 Baird-Parker agar plates. See Appendix A A.4.
3.5 brain heart infusion broth (BHI). See Appendix A A.5.
3.6 Rabbit plasma. A A.6 in the appendix.
3.7 Diluent. Phosphate buffer. See Appendix A A.7.
3.8 nutrient agar small slant. See Appendix A A.8.
3.9 Gram Staining Solution. See Appendix A, A.9.
Sterile saline 3.10. See Appendix A A.10.
36 ± 1 ℃ ℃ 18 h ~ 24 h
36 ± 1 ℃ ℃ 18 h ~ 24 h
The first law of Staphylococcus aureus qualitative test
4 inspection procedures
Staphylococcus aureus qualitative test program shown in Figure 1.
1 Staphylococcus aureus inspection procedures
5 steps
5.1 sample handling
Weigh 25 g containing sample to within 225 mL 7.5% sodium chloride broth or 10% sodium chloride trypticase soy broth sterile homogeneous cup,
8000 r/min ~ 10000 r/min homogenized 1 min ~ 2 min, or placed containing 225 mL 7.5% sodium chloride and 10% sodium chloride broth or trypticase
Soy broth sterile homogenization bags with slap-type homogenizer beat 1 min ~ 2 min. If the sample is a liquid sample to draw 25 mL containing
225 mL 7.5% sodium chloride and 10% sodium chloride broth or trypticase soy broth in a sterile Erlenmeyer flask (flask preset appropriate number of sterile glass
Beads), the Shakers.
5.2 Enrichment and Isolation
36 ± 1 ℃ ℃ blood agar 18 h ~ 24 h
Baird-Parker plates 18 h ~ 24 h or 45 h ~ 48 h
Baird-Parker flat, blood agar
Test sample
25 g (mL) sample 225 mL 7.5% sodium chloride and 10% sodium chloride broth or trypticase soy broth, homogeneous
Coagulase test
BHI broth and nutrient agar small slant
Observation of hemolysis smear
report
5.2.1 The sample was homogenized culture 18 h ~ 24 h at 36 ℃ ± 1 ℃. Staphylococcus aureus was born in 7.5% sodium chloride cloudy broth
Long and serious pollution in 10% sodium chloride trypticase soy broth was cloudy growth.
5.2.2 The above culture were streaked onto Baird-Parker and blood agar plates, blood agar 36 ℃ ± 1 ℃ cultured 18 h ~ 24 h.
Baird-Parker flat 36 ℃ ± 1 ℃ cultured 18 h ~ 24 h or 45 h ~ 48 h.
5.2.3 Staphylococcus aureus on Baird-Parker plates, colonies having a diameter of 2 mm ~ 3 mm, color gray to black edges
Pale, surrounded by a cloudy band is in its outer layers have a transparent circle. Inoculating needle in contact with the colony like cream to the gum-like hardness, accidental encounter
Similar colonies of non-fat soluble; but no opacity and transparency with a circle. Frozen or dried foods in the long-term preservation of isolated colonies than typical bacteria
Black drop produced some lighter, it may look rough and dry. On blood agar, the formation of large colonies, round, smooth, convex, moist,
Golden yellow (sometimes white), completely transparent to visible hemolytic ring around the colony. Colonies were picked above Gram staining and plasma coagulation
Enzyme assay.
5.3 Identification
5.3.1 staining. Staphylococcus aureus Gram-positive cocci, arranged in a spherical grapes, no spores, no capsule diameter of about
0.5 μm ~ 1 μm.
5.3.2 coagulase test. picked, tablet or suspected colonies on blood agar Baird-Parker 1 or more, were inoculated into 5 mL BHI
And nutrient agar small slant, 36 ± 1 ℃ ℃ cultured 18 h ~ 24 h.
Fresh configuration rabbit plasma 0.5 mL, into a small test tube, then add BHI culture was 0.2 mL ~ 0.3 mL, shaking shake, set 36
± 1 ℃ ℃ incubator or water bath tank, every half-hour observation time, observation 6 h, as presented solidification (coming sideways or upside down when the tube, showing condensate
Block) or coagulation volumes greater than half of the original volume, was judged to be positive results. While test-positive and coagulase-negative staphylococci bacteria
Broth culture of strain as a control. Also using commercially available reagents, by manual operation, carried coagulase test.
The results are suspicious colonies were picked nutrient agar small slant to 5 mL BHI, 36 ± 1 ℃ ℃ cultured 18 h ~ 48 h, repeat the test.
5.4 staphylococcal enterotoxin test
Identification of suspected food poisoning samples or produce staphylococcal enterotoxin of Staphylococcus aureus strains, Appendix B shall detect staphylococci
Enterotoxin.
6 Results and reporting
6.1 Result judgment. in line with 5.2.3,5.3 may be assessed as Staphylococcus aureus.
6.2 Results Report. detection or Staphylococcus aureus in 25 g (mL) sample.
36 ℃ ± 1 ℃
The second law of Staphylococcus aureus Baird-Parker plate count
7 Inspection Program
Staphylococcus aureus plate count procedure shown in Figure 2.
Figure 2 aureus Baird-Parker plate method test program
8 steps
8.1 sample dilution
8.1.1 solid and semi-solid samples. Weigh 25 g sample set containing 225 mL of phosphate buffered saline solution or sterile within a homogeneous Cup 8000
r/min ~ 10000 r/min homogenized 1 min ~ 2 min, or set containing 225 mL of sterile diluent bag homogenized by homogenizer slap-style beat 1
min ~ 2 min, a sample was evenly 1.10.
8.1.2 Liquid samples. 25 mL sterile pipette sample set containing 225 mL of phosphate buffered saline solution or sterile conical flask (bottles
Within a preset appropriate number of sterile glass beads), and mix well to form uniform sample 1.10.
8.1.3 with 1 mL sterile pipette or micro-pipette evenly 1.10 sample solution 1 mL, slowly along the wall in Note 9 mL diluent containing no
Bacteria tube (note pipette tip or not to tip touched diluted liquid), shake the tube or replaced with a 1 mL sterile pipette pipetting repeatedly make
Mixed uniformly to make a 1. 100 sample liquid uniform.
8.1.4 press 8.1.3 operating procedures to prepare 10-fold serial dilution of the sample was homogenized. Each increment diluted once, for once with 1 mL sterile pipette or
Tips.
8.2 inoculation samples
According to the estimates of sample contamination situation, select the sample uniform was 2 to 3 appropriate dilutions (liquid sample may include a liquid), in
Diluted 10-fold increments, each dilution Pipette 1 mL sample was homogenized in 0.3 mL, 0.3 mL, 0.4 mL inoculum were added
10 fold serial dilutions
Select 2 ~ 3 consecutive samples appropriate dilutions were uniformly inoculated plates Baird-Parker
Count and plasma coagulase test
report
Test sample
25 g (mL) 225 mL Sample diluent, homogeneous
45 h ~ 48 h
Baird-Parker into three plates, and then with sterile L bar coating the entire plate, be careful not to touch the plate edges. Before use, such as Baird-Parker
Flat surface water droplets can be placed in the culture box 25 ℃ ~ 50 ℃ dry, flat surface until the water droplets disappear.
8.3 Training
8.3.1.1 Under normal circumstances, after coating, the plate was allowed to stand for 10 min, the sample solution is not easily absorbed e.g., the plates were placed in the incubator can be 36 ± 1 ℃
℃ for 1 h; after absorption of fluid sample such as homogenized flip plate upside down in an incubator, 36 ℃ ± 1 ℃ culture, 45 h ~ 48 h.
8.4 typical colony counting and verification
8.4.1 Staphylococcus aureus on Baird-Parker plates, colonies having a diameter of 2 mm ~ 3 mm, color gray to black edges
Pale, surrounded by a cloudy band is in its outer layers have a transparent circle. Inoculating needle in contact with the colony like cream to the gum-like hardness, accidental encounter
Similar colonies of non-fat soluble; but no opacity and transparency with a circle. Frozen or dried foods in the long-term preservation of isolated colonies than typical bacteria
Black drop produced some lighter, it may look rough and dry.
8.4.2 Select the typical S. aureus colonies flat, and the same dilution plates all three colonies in total 20 CFU ~
Tablet 200 CFU between typical count the number of colonies. in case.
a) the number of colonies only a dilution plate between 20 CFU ~ 200 CFU and typical colonies counted on the dilution plate
Typical colonies;
Colonies b) the minimum dilution plate number is less than 20 CFU and have typical colonies, the dilution counting typical colonies on the plate;
c) the number of colonies a dilution plate and has more than 200 CFU typical colonies, but there is no typical colonies on the next dilution plate,
Accruals typical colonies on the plate dilution;
Colonies d) a dilution greater than 200 CFU flat and typical colonies, typical colonies and the next dilution plate, but
Its colonies on the plate is not between 20 CFU ~ 200 CFU, should count the dilution typical colonies on the plate;
According to the above formula (1) calculations.
e) plate count two successive dilutions were between 20 CFU ~ 200 CFU, according to the formula (2) calculation.
8.4.3 optionally five colonies from typical colonies (less than 5 Select All), respectively, according to 5.3.2 do coagulase test.
9 result of the calculation.
Formula 1).
Cd
ABT = (1)
Where.
T-- samples of Staphylococcus aureus colonies;
Total A-- a dilution typical colonies;
B-- a dilution of coagulase-positive colonies;
C-- a dilution for the number of colonies coagulase test;
d-- dilution factor.
Equation (2).
(2)
Where.
T - samples of Staphylococcus aureus colonies;
A1-- first dilution (lower dilution) Total number of typical colonies;
A2-- second dilution (high dilution) Total number of typical colonies;
B1-- first dilution (lower dilution) coagulase-positive colonies;
CBACBAT
1.1
222 111 =
B2-- second dilution (high dilution) coagulase-positive colonies;
C1-- first dilution (lower dilution) for the number of colonies coagulase test;
C2-- second dilution (high dilution) for the number of colonies coagulase test;
1.1 - Calculation coefficients;
d - dilution factor (first dilution).
10 Results and Reports
According to the typical number of colonies on the plate Baird-Parker Staphylococcus aureus, according to the formula 9, report every g (mL) sample gold
Aureus count in CFU/g (mL) represented; T as a value of 0, 1 places less than the minimum dilution factor multiplied by the report.
The third law of Staphylococcus aureus MPN count
11 inspection procedures
Staphylococcus aureus MPN counting procedure shown in Figure 3.
Figure 3 Staphylococcus aureus MPN method testing procedures
12 Procedure
12.1 diluted sample
Press 8.1.
12.2 seeded and grown
12.2.1 According to the estimate of sample contamination situation, select the sample was homogenized three suitable dilutions (liquid sample may include a liquid), the intake
10-fold dilution when the line is incremented each dilution Pipette 1 mL sample was evenly inoculated into 10% sodium chloride trypticase soy broth tube, dilute each
Interpretation of inoculation 3, the above inoculum train 45 h ~ 48 h at 36 ± 1 ℃ ℃.
10 fold serial dilutions
Select three suitable dilutions liquid sample uniform, each draw 1 mL,
3 were inoculated in 10% sodium chloride trypticase soy broth.
Inoculated plate Baird-Parker
Coagulase test
Test sample
25 g (mL) 225 mL Sample diluent, homogeneous
Charles MPN table
Report Results
45 h ~ 48 h 36 ℃ ± 1 ℃
36 ℃ ± 1 ℃ 45 h ~ 48 h
12.2.2 inoculation loop from the growth of bacteria in each tube, pipette 1 ring, were inoculated plate Baird-Parker, 36 ± 1 ℃ ℃ cultured 45 h ~
48 h.
12.3 typical colonies confirmed
12.3.1 see 8.4.1.
12.3.2 typical colonies were picked from at least one colony was inoculated into BHI broth and nutrient agar slant, 36 ± 1 ℃ ℃ cultured 18 h ~ 24 h.
Were coagulase test, see 5.3.2.
13 Results and Reports
Calculate the number of tube coagulase positive colonies corresponding enzyme test, check MPN search table (see Appendix C), the report per g (mL) sample gold
The most probable number aureus in MPN/g (mL) FIG.
Appendix A
(Normative)
Media and reagents
A.1 10% sodium chloride trypticase soy broth
A.1.1 ingredients
Trypticase (or tryptone) 17.0 g
Plant peptone (or soy peptone) 3.0 g
100.0 g of sodium chloride
Dipotassium hydrogen phosphate 2.5 g
10.0 g of sodium pyruvate
Glucose 2.5 g
1 000 mL of distilled water
pH 7.3 ± 0.2
A.1.2 Method
The above ingredients were mixed, heated gently with stirring and dissolved, adjusting the pH, dispensing bottle 225 mL, 121 ℃ autoclave 15 min.
A.2 7.5% sodium chloride broth
A.2.1 ingredients
Peptone 10.0 g
Beef extract 5.0 g
75 g of sodium chloride
1 000 mL of distilled water
pH 7.4
A.2.2 Method
The above ingredients were heated and dissolved, adjust the pH, dispensing bottle 225 mL, 121 ℃ autoclaving 15 min.
A.3 blood agar plate
A.3.1 ingredients
Flour agar (pH7.4 ~ 7.6) 100 mL
Defibrinated sheep blood (or rabbit blood) 5 mL ~ 10 mL
A.3.2 Method
Heated to melt the agar, cooling to 50 ℃, added aseptically defibrinated sheep blood, shake, pour plates.
A.4 Baird-Parker agar plates
A.4.1 ingredients
10.0 g tryptone
Beef extract 5.0 g
Yeast extract 1.0 g
10.0 g of sodium pyruvate
Glycine 12.0 g
Lithium (LiCl · 6H2O) chloride, 5.0 g
Agar 20.0 g
950 mL of distilled water
pH 7.0 ± 0.2
A.4.2 increased with agents of law
30% egg yolk and 50 mL brine through a 1% potassium tellurite solution of 10 mL sterile filtered mixed, stored in a refrigerator.
A.4.3 Method
The ingredients are added to distilled water and heated to boiling until complete dissolution, adjusting the pH. Dispensing bottle 95 mL, 121 ℃ autoclaving 15 min.
When you use heat to melt the agar, cooling to 50 ℃, each added 95 mL preheated to 50 ℃ yolk potassium tellurite by agents after pouring 5 mL shake
flat. Dense medium should be opaque. Before using stored in the refrigerator should not exceed 48 h.
A.5 brain heart infusion broth (BHI)
A.5.1 ingredients
Pancreatic protein peptone 10.0 g
Sodium chloride 5.0 g
Disodium hydrogen phosphate (Na2HPO4 · 12H2O) 2.5 g
Glucose 2.0 g
Bovine heart infusion 500 mL
pH 7.4 ± 0.2
A.5.2 Method
Dissolved by heating, adjusting pH, dispensing tube 16 mm × 160 mm, 5 mL per tube set 121 ℃, 15 min sterilization.
A.6 rabbit plasma
Take sodium citrate 3.8 g, add distilled water to 100 mL, dissolved filtration, bottling, 121 ℃ autoclaving 15 min.
Rabbit plasma preparation. Take a 3.8% sodium citrate solution, plus four rabbit whole blood, mixed with good standing (or 3000 r/min centrifugation 30 min),
Decreased blood cells, plasma can be obtained.
A.7 phosphate buffer
A.7.1 Ingredients.
Potassium dihydrogen phosphate (KH2PO4) 34.0 g
500 mL of distilled water
pH 7.2
A.7.2 Method.
Storage solution. Weigh 34.0 g of potassium dihydrogen phosphate was dissolved in 500 mL of distilled water, with about 175 mL of 1 mol L sodium hydroxide solution to adjust /
pH 7.2, diluted with distilled water to 1 000 mL after storage in a refrigerator.
Diluent. Take stock solution 1.25 mL, diluted with distilled water to 1 000 mL, packed in suitable containers, 121 ℃ autoclaving 15 min.
A.8 nutrient agar small slant
A.8.1 ingredients
Peptone 10.0 g
Beef extract 3.0 g
Sodium chloride 5.0 g
Agar 15.0 g ~ 20.0 g
1 000 mL of distilled water
pH 7.2 ~ 7.4
A.8.2 Method
The ingredients except the agar were dissolved in distilled water, 15% sodium hydroxide solution was added 2 mL pH adjusted to about 7.2 to 7.4. Jia Ruqiong
Fat, heated to boiling, melting agar, dispensing 13 mm × 130 mm tube, 121 ℃ autoclaving 15 min.
A.9 Gram stain solution
A.9.1 crystal violet staining solution
A.9.1.1 ingredient
1.0 g crystal violet
20.0 mL of 95% ethanol
1% aqueous solution of ammonium oxalate 80.0 mL
A.9.1.2 Method
The crystal violet was completely dissolved in ethanol and then mixed with ammonium oxalate solution.
A.9.2 Gram iodine solution
A.9.2.1 ingredient
Iodine 1.0 g
Potassium iodide 2.0 g
300 mL of distilled water
A.9.2.2 Method
Iodine and potassium iodide will first mixed, add a little distilled water and shake well, until completely dissolved, together with distilled water to 300 mL.
A.9.3 sand yellow counterstain
A.9.3.1 ingredient
0.25 g yellow sand
95% Ethanol 10.0 mL
90.0 mL of distilled water
A.9.3.2 Method
The yellow sand was dissolved in ethanol and then diluted with distilled water.
A.9.4 staining
a) smear fixed on the flame, a solution of crystal violet dye, dye 1 min, washed with water.
b) dropping Gram iodine solution, the role of 1 min, washed with water.
c) 95% ethanol solution of decolorized about 15 s ~ 30 s, until the staining solution is washed off, do not over-bleaching, washing with water.
d) dropping counterstain, stained 1 min, washed with water, until dry, microscopic examination.
A.10 sterile saline
A.10.1 ingredient
Sodium chloride 8.5 g
1 000 mL of distilled water
A.10.2 Method
Weigh 8.5g of sodium chloride dissolved in 1 000 mL of distilled water, 121 ℃ autoclaving 15 min.
Appendix B
(Normative)
Staphylococcal enterotoxin test
B.1 Reagents and materials
Unless otherwise specified, the reagents were of analytical grade, test water should be consistent with GB/T 6682 provides for a water.
B.1.1 A, B, C, D, E S. aureus enterotoxin type ELISA kit.
B.1.2 pH test strips, range of 3.5 to 8.0, precision 0.1.
B.1.3 0.25 mol/L, pH8.0 Tris buffer. The 121.1g of Tris was dissolved in 800mL of deionized water until the temperature cooled to
Room temperature, add 42 mL of concentrated HCl, pH was adjusted to 8.0.
B.1.4 pH 7.4 phosphate buffer. the weighing NaH2PO4 · H2O 0.55g (or NaH2PO4 · 2H2O 0.62g), Na2HPO4 · 2H2O
2.85g (or Na2HPO4 · 12H2O 5.73g), NaCl 8.7g dissolved in 1 000 mL of distilled water, mix well can be.
B.1.5 heptane.
B.1.6 10% sodium hypochlorite solution.
B.1.7 enterotoxin toxin-producing medium
B.1.7.1 ingredient
Peptone 20.0 g
Casein trypsin digest 200 mg (amino acid)
Sodium chloride 5.0 g
Dipotassium hydrogen phosphate 1.0 g
1.0 g of potassium dihydrogen phosphate
Calcium chloride 0.1 g
0.2 g of magnesium sulfate
Niacin 0.01 g
1 000 mL of distilled water
pH7.2 ~ 7.4
B.1.7.2 Method
All ingredients were mixed in water, the pH adjusted after dissolving, 121 ℃ autoclave 30min.
B.1.8 nutrient agar
B.1.8.1 ingredient
Peptone 10.0 g
Beef extract 3.0 g
Sodium chloride 5.0 g
Agar 15.0 g ~ 20.0 g
1 000 mL of distilled water
B.1.8.2 Method
The ingredients except the agar were dissolved in distilled water, was added 15% aqueous sodium hydroxide solution of about 2mL, correcting the pH to 7.2 to 7.4. plus
Into the agar, heated to boiling, agar dissolves. Dispensing flask, 121 ℃ autoclave 15min.
B.2 instruments and equipment
B.2.1 electronic balance. a sense of the amount of 0.01g.
B.2.2 homogenizer.
B.2.3 centrifuge. speed of 3000 g ~ 5000 g.
B.2.4 tube. 50 mL.
B.2.5 filter. Pore Size 0.2 μm.
B.2.6 micro pipette. 20 μL ~ 200 μL, 200 μL ~ 1000 μL.
B.2.7 micro multi-channel pipettes. 50 μL ~ 300 μL.
B.2.8 automatic plate washer (optional use).
B.2.9 microplate reader. a wavelength of 450 nm.
B.3 Principle
This method can be A, B, C, D, E S. aureus enterotoxin type ELISA kit complete. The method for the determination
It is based on the ELISA reaction (ELISA). 96 A ~ E hole every pore of the well microtiter plates were coated with A, B, C, D,
E-type staphylococcal enterotoxin antibody, H is the positive control hole, been coated with staphylococcal enterotoxin hybrid antibodies, F and G as negative control hole,
Coated with non-immune antibodies. If there is a sample of staphylococcal enterotoxin, staphylococcal enterotoxin is free with th......
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GB/T 4789.10-2008
Microbiological examination of food hygiene. Detection of staphylococcus aureus
ICS 07.100.30
C53
National Standards of People's Republic of China
Replacing GB/T 4789.10-2003
Microbiological examination of food hygiene
Staphylococcus aureus test
Posted 2008-11-21
2009-03-01 implementation
People's Republic of China Ministry of Health
Standardization Administration of China released
Foreword
This standard corresponds to international analysts Society (AOACINTERNATIONAL) AOAC987.09 "food in Staphylococcus aureus
mostprobablenumbermethodforisolationandenumeration) and the International Organization for Standardization ISO 6888-1.1999 "food
Products and animal feed coagulase positive Staphylococcus aureus and other staphylococci counting method "(Microbiology
um). The Standard and AOACOfficialMethod987.09, ISO 6888-1.1999 is not equivalent degree of consistency.
This standard replaces GB/T 4789.10-2003 "Microbiological examination of food hygiene inspection of Staphylococcus aureus."
This standard compared with GB/T 4789.10-2003 The main changes are as follows.
--- Standardized sample preparation process;
--- The original standard coagulase section has been modified.
This standard Annex A, Annex B normative appendix.
This standard is proposed and administered by the People's Republic of China Ministry of Health.
This standard by the People's Republic of China Ministry of Health is responsible for interpretation.
This standard is drafted by. China Center for Disease Control Nutrition and Food Safety.
Involved in the drafting of this standard. Shanghai Disease Prevention and Control Center, People's Republic of China Liaoning Exit Inspection and Quarantine, the Chinese people
Inner Mongolia, People's Republic of Exit Inspection and Quarantine, Jiangsu Province Center for Disease Control and Prevention.
The main drafters of this standard. Xiumei, Chen Min, Liu Hong, Liu high school, Lu Xingan, Yuan Baojun, Tian Jing.
This standard replaces the standards previously issued as follows.
--- GB 4789.10-1984, GB/T 4789.10-1994, GB/T 4789.10-2003.
Microbiological examination of food hygiene
Staphylococcus aureus test
1 Scope
This standard specifies the test methods in food Staphylococcus aureus.
This standard applies to all types of food and food poisoning Staphylococcus aureus samples tested.
2 Equipment and Materials
In addition to the microbiological laboratory conventional sterilization and cultivation equipment, other equipment and materials as follows.
2.1 incubator. 36 ℃ ± 1 ℃.
2.2 Refrigerator. 2 ℃ ~ 5 ℃.
2.3 constant temperature water bath. 37 ℃ ± 1 ℃.
2.4 Balance. a sense of the amount of 0.1g.
2.5 homogenizer.
2.6 oscillator.
2.7 sterile pipette. 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micro pipettes and tips.
2.8 sterile conical flask. capacity 100mL, 500mL.
2.9 sterile Petri dish. diameter of 90mm.
2.10 syringe. 0.5mL.
2.11 pH meter or pH colorimetric tubes or precision pH test paper.
3 media and reagents
3.1 10% sodium chloride trypticase soy broth. see Section A. Chapter 1.
3.2 7.5% sodium chloride broth. see Section A. 2.
3.3 blood agar plates. see Section A. 3.
3.4 Baird-Parker agar plates. see Section A. 4.
3.5 brain heart infusion (BHI) broth. See Section A. 5.
3.6 Rabbit plasma. see Section A. 6.
3.7 phosphate buffer. see Section A. 7.
3.8 nutrient agar slant. see Section A. 8.
3.9 Gram stain solution. See Section A. 9.
Sterile saline 3.10. Weigh 8.5g of sodium chloride dissolved in 1000mL of distilled water, 121 ℃ autoclave 15min.
3.11 1mol/L sodium hydroxide (NaOH). Weigh 40g of sodium hydroxide (NaOH) was dissolved in 1000mL of distilled water.
3.12 1mol/L hydrochloric acid (HCl). 37% concentrated hydrochloric acid 90mL, add distilled water to 1000mL.
4 inspection procedures
Test procedure shown in Figure 1.
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