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Standard ID | GB 4789.30-2016 (GB4789.30-2016) | Description (Translated English) | National Food Safety Standard -- Food Microbiological Examination -- Examination of Listeria Monocytogenes | Sector / Industry | National Standard | Classification of Chinese Standard | X09 | Word Count Estimation | 16,187 | Date of Issue | 2016-12-23 | Date of Implementation | 2017-06-23 | Older Standard (superseded by this standard) | GB 4789.30-2010 | Regulation (derived from) | National Health and Family Planning Commission Notice No.17 of 2016 | Standard ID | GB 4789.30-2010 (GB4789.30-2010) | Description (Translated English) | National food safety standard - Food microbiological examination: Listeria monocytogenes | Sector / Industry | National Standard | Classification of Chinese Standard | C53 | Classification of International Standard | 07.100.30 | Word Count Estimation | 10,153 | Date of Issue | 2010-03-26 | Date of Implementation | 2010-06-01 | Older Standard (superseded by this standard) | GB/T 4789.30-2008 | Regulation (derived from) | Circular of the Ministry of Health (2010)7 | Issuing agency(ies) | National Health and Family Planning Commission of People's Republic of China | Summary | This Chinese standard specifies the food Listeria monocytogenes bacteria (Listeria monocytogenes) test. This standard applies to foods Listeria monocytogenes bacteria test. | Standard ID | GB/T 4789.30-2008 (GB/T4789.30-2008) | Description (Translated English) | Microbiological examination of food hygiene. Examination of listeria monocytogenes | Sector / Industry | National Standard (Recommended) | Classification of Chinese Standard | C53 | Classification of International Standard | 07.100.30 | Word Count Estimation | 17,178 | Date of Issue | 2008-11-21 | Date of Implementation | 2009-03-01 | Older Standard (superseded by this standard) | GB/T 4789.30-2003 | Drafting Organization | China Disease Prevention and Control Center of Nutrition and Food Safety | Administrative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 the foods monocytogenes Listeria (Listeria monocytogenes) test methods. This standard applies to food and foodborne disease samples monocytogenes Listeria test. |
GB 4789.30-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Food microbiological
examination - Examination of Listeria monocytogenes
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission;
China Food and Drug Administration.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Equipment and materials ... 4
3 Media and reagents ... 5
4 Testing procedures ... 6
5 Operation procedures ... 6
6 Test procedures ... 9
7 Operation procedures ... 9
8 Result count ... 11
9 Results report ... 12
10 Testing procedures ... 12
11 Operation procedures... 13
12 Results and reports ... 14
Appendix A Medium and reagents ... 15
Appendix B Listeria monocytogenes most probable number (MPN) search table
... 23
National food safety standard - Food microbiological
examination - Examination of Listeria monocytogenes
1 Scope
This standard specifies the test method for Listeria monocytogenes in food.
The first method of this standard is applicable to the qualitative test of Listeria
monocytogenes in food; the second method is applicable to the counting of
Listeria monocytogenes in foods with higher Listeria monocytogenes; the third
method is applicable to the count of Listeria monocytogenes in foods with low
Listeria monocytogenes (< 100 CFU/g) and high bacterial content, especially
milk, water and food containing particulate matter which interferes with the
counting of colonies.
2 Equipment and materials
In addition to routine sterilization and culture equipment in microbiology
laboratories, other equipment and materials are as follows.
2.1 Refrigerator. 2 °C ~ 5 °C.
2.2 Constant temperature incubator. 30 °C ± 1 °C, 36 °C ± 1 °C.
2.3 Homogenizer.
2.4 Microscope. 10 x ~ 100 x.
2.5 Electronic balance. the sensitivity is 0.1 g.
2.6 Conical flask. 100 mL, 500 mL.
2.7 Sterile pipette. 1 mL (with 0.01 mL scale), 10 mL (with 0.1 mL scale) or
micropipette and tip.
2.8 Sterile plate. 90 mm in diameter.
2.9 Sterile test tube. 16 mm × 160 mm.
2.10 Centrifuge tube. 30 mm × 100 mm.
2.11 Sterile syringe. 1 mL.
HOMOGENIZE it at 8000 r/min ~ 10000 r/min for 1 min ~ 2 min. CULTURE it at
30 °C ± 1 °C for 24 h ± 2 h, TAKE 0.1 mL, TRANSFER it to 10 mL of LB2
monocytogenes solution, CULTURE it at 30 °C ± 1 °C for 24 h ± 2 h.
5.2 Separation
TAKE the LB2 secondary monocytogenes solution, INOCULATE it onto the
Listeria chromogenic plate and the PALCAM agar plate in a line shape,
CULTURE it at 36 °C ± 1 °C for 24 h ~ 48 h, OBSERVE the colonies growing
on each plate. Typical colonies are small round gray-green colonies on
PALCAM agar plates with brown-black hydrolyzed circles around them, some
colonies have black depressions; colony characteristics on Listeria color-
developing plates are determined by reference to product specifications.
5.3 Initial screening
TAKE to five typical or suspect colonies from selective agar plates, respective
INOCULATE them into the xylose and rhamnose fermentation tubes, CULTURE
it at 36 °C ± 1 °C for 24 h ± 2 h, meanwhile MARK a line on the TSA-YE plate,
CULTURE it at 36 ° C ± 1 ° C for 18 h ~ 24 h, then SELECT the pure culture of
xylose-negative and rhamnose-positive for further identification.
5.4 Identification (or selection of biochemical identification kits or fully
automated microbial identification systems, etc.)
5.4.1 Staining microscopy. Listeria monocytogenes is a Gram-positive
Brevibacterium, the size is (0.4 μm ~ 0.5 μm) × (0.5 μm ~ 2.0 μm); the bacterial
suspension is made with physiological saline, which is observed in oil mirror or
under the phase contrast microscope, the bacteria shows slight rotation or
rolling-like motion.
5.4.2 Dynamic test. PICK a single suspicious colony to puncture semi-solid or
SIM dynamic medium in pure culture, CULTURE it at 25 °C ~ 30 °C for 48 h.
Listeria is motivated and has an umbrella shape growth above semi-solid or
SIM medium. If the umbrella growth is not obvious, it can continue to culture it
for 5 d, then observe the results.
5.4.3 Biochemical identification. PICK the single suspicious colony in pure
culture to perform catalase test. Colonies with positive catalase reaction is
continued to be subject to sugar fermentation test and MR-VP test. The main
biochemical characteristics of Listeria monocytogenes are as shown in Table 1.
5.4.4 Hemolysis test. DIVIDE the bottom surface of fresh sheep blood agar
plate into 20 ~ 25 small cells, PICK a single suspicious colony of pure culture,
INOCULATE it onto the blood plate by puncture, one colony per cell, and
INOCULATE the positive control bacteria by puncture (Listeria monocytogenes,
Listeria ivanovii and Listeria seeligeri) and negative control bacteria (Listeria
without additives, HOMOGENIZE it continuously for 1 min ~ 2 min on a tapping
homogenizer, or HOMOGENIZE it for 1 min ~ 2 min at 8000 r/min ~ 10000 r/min.
SHAKE and MIX the liquid sample uniformly, to make it into 1.10 sample
homogenate.
7.1.2 USE a 1 mL sterile pipette or micropipette to pipette 1 mL of 1.10 sample
homogenate, along the tube wall, INJECT it slowly into a sterile test tube which
contains 9 mL of buffered peptone or LB broth without additives (note avoiding
the pipette or tip end from touching the dilution solution surface), SHAKE the
test tube or USE another 1 mL sterile pipette to repeatedly mix it uniformly, to
prepare it into 1.100 sample homogenate.
7.1.3 PREPARE 10 times serial dilution sample homogenate in accordance with
the procedure of 7.1.2. For each incremental dilution, replace one 1 mL sterile
pipette or tip.
7.2 Inoculation of samples
In accordance with the estimation of the contamination status of the sample,
SELECT 2 ~ 3 sample homogenate of appropriate continuous dilution (the liquid
sample may include the original solution), respectively TAKE 1 mL of each
dilution of the sample homogenate, at the inoculation amount of 0.3 mL, 0.3 mL,
0.4 mL, respectively ADD it onto three Listeria chromogenic plates, USE the
sterile L bar to cover it over the entire plate, PAY attention to avoiding touching
the plate edge. Before use, if there is water drops on the surface of the agar
plate, it can be dried in an incubator at 25 °C ~ 50 °C until the water drops on
the surface of the plate disappear.
7.3 Culture
7.3.1 Under normal circumstances, after coating, ALLOW the plate to stand for
10 min. If the sample solution is not easily absorbed, it may place the plate in
an incubator at 36 °C ± 1 °C for 1 h; after the sample homogenate is absorbed,
INVERT the plate, PLACE it into the incubator upside down, to culture it at 36 °C
± 1 °C for 24 h ~ 48 h.
7.4 Typical colony count and confirmation
7.4.1 Colony characteristics of Listeria monocytogenes on Listeria chromogenic
plates are subject to product description.
7.4.2 SELECT a plate which has typical Listeria monocytogenes colonies, and
the total number of colonies in the same dilution of 3 plates between 15 CFU
and 150 CFU, COUNT the number of typical colonies. In case.
a) The plate colony count of only one dilution is between 15 CFU ~ 150 CFU
and there is typical colony, it shall count the typical colonies on the plate
Appendix A
Medium and reagents
A.1 Tryptic soy broth (TSB-YE) containing 0.6% yeast extract
A.1.1 Ingredients
Tryptone 17.0 g
Polyad tryptone 3.0 g
Yeast extract 6.0 g
Sodium chloride 5.0 g
Dipotassium hydrogen phosphate 2.5 g
Glucose 2.5 g
Distilled water 1000 mL
A.1.2 Preparation method
HEAT and STIR the above components to dissolve it, ADJUST the pH to 7.2 ±
0.2, CONTAIN it separately, AUTOCLAVE it at 121 °C for 15 min, PREPARE for
use.
A.2 Tryptic soy agar (TSA-YE) containing 0.6% yeast extract
A.2.1 Ingredients
Tryptone 17.0 g
Polyad tryptone 3.0 g
Yeast extract 6.0 g
Sodium chloride 5.0 g
Dipotassium hydrogen phosphate 2.5 g
Glucose 2.5 g
Agar 15.0 g
Distilled water 1000 mL
Glucose 0.5 g
Esculin 0.8 g
Ammonium ferric citrate 0.5 g
Mannitol 10.0 g
Phenol red 0.1 g
Lithium chloride 15.0 g
Casein trypsin digest 10.0 g
Heart trypsin digest 3.0 g
Corn Starch 1.0 g
Meat and stomach enzyme digest 5.0 g
Sodium chloride 5.0 g
Agar 15.0 g
Distilled water 1000 mL
A.4.2 Preparation method
HEAT and STIR the above components to dissolve it, ADJUST the pH to 7.2 ±
0.2, CONTAIN it separately, AUTOCLAVE it at 121 °C for 15 min, PREPARE for
use.
A.4.2.1 PALCAM selective additives
Polymyxin B 5.0 mg
Acriflavine HCl 2.5 mg
Ceftazidime 10.0 mg
Sterile distilled water 500 mL
A.4.2.2 Preparation method
DISSOLVE the PALCAM base medium, COOL it to 50 °C, ADD 2 mL of
PALCAM selective additive, MIX it uniformly, POUR it into a sterile plate,
PREPARE for use.
A.5 Gram staining solution
After dissolving it, ADJUST the pH to 7.0 ± 0.2, CONTAIN it in different test
tubes, 1 mL per tube, AUTOCLAVE it at 121 ° C for 15 min, PREPARE for use.
A.7.3 Methyl red (MR) test
A.7.3.1 Methyl red reagent
A.7.3.1.1 Ingredients
Methyl red 10 mg
95% ethanol 30 mL
Distilled water 20 mL
A.7.3.1.2 Preparation method
DISSOLVE 10 mg of methyl red in 30 mL of 95% ethanol, then ADD 20 mL of
distilled water.
A.7.3.1.3 Test method
TAKE an appropriate amount of agar culture, INOCULATE it into buffered
glucose protein water, CULTURE it at 36 °C ± 1 °C for 2 d ~ 5 d. ADD one drop
of methyl red reagent, immediately OBSERVE it. Bright red is positive and
yellow is negative.
A.7.4 V-P test
A.7.4.1 6% α-naphthol-ethanol solution
Ingredients and preparation method. Take 6.0 g of α-naphthol, ADD absolute
ethanol to dissolve it, MAKE its volume reach to 100 mL.
A.7.4.2 40% potassium hydroxide solution
Ingredients and preparation method. TAKE 40 g of potassium hydroxide, ADD
distilled water to dissolve it, MAKE its volume reach to 100 mL.
A.7.4.3 Test method
TAKE an appropriate amount of agar culture, INOCULATE it into buffered
glucose protein water, CULTURE it at 36 °C ± 1 °C for 2 d ~ 4 d. ADD 0.5 mL
of 6% α-naphthol-ethanol solution and 0.2 mL of a 40% potassium hydroxide
solution, SHAKE the test tube completely, OBSERVE the results. If red color
appears immediately or after several minutes, it is positive reaction; if it is
negative reaction, it shall be cultured continuously at 36 °C ± 1 °C for 1 h and
observed.
meanwhile AUTOCLAVE it. ADD 5 mL of the sugar solution into 100 mL of the
medium, aseptically DISPENSE it into a small tube containing the inverted tube.
Or PREPARE other sugar fermentation tubes in accordance with the
preparation method of A.9.2.1 glucose fermentation tube.
A.9.3 Test methods
TAKE appropriate amount of pure culture, INOCULATE it into the sugar
fermentation tube, CULTURE it at 36 °C ± 1 °C for 24 h ~ 48 h. OBSERVE the
results, the blue color indicates negative and yellow color indicates positive.
A.10 Catalase test
A.10.1 Reagent
3% hydrogen peroxide solution. PREPARE it at the time of use.
A.10.2 Test methods
USE a thin glass rod or a disposable inoculation needle to pick a single colony,
PLACE it into a clean glass plate, ADD 2 drops of 3% hydrogen peroxide
solution, OBSERVE the result.
A.10.3 Results
Those which have bubbles in half a minute are positive, and those which do not
have bubbles are negative.
A.11 Buffered protein water (BPW)
A.11.1 Ingredients
Peptone 10.0 g
Sodium chloride 5.0 g
Disodium hydrogen phosphate (Na2HPO4 • 12H2O) 9.0 g
Potassium dihydrogen phosphate 1.5 g
Distilled water 1000 mL
A.11.2 Preparation method
HEAT and STIR to dissolve it, ADJUST the pH to 7.2 ± 0.2, AUTOCLAVE it at
121 °C for 15 min.
......
GB 4789.30-2010
GB
National Standard of the People’s Republic of China
National food safety standard
Food microbiological examination.
Listeria monocytogenes
ISSUED ON. MARCH 26, 2010
IMPLEMENTED ON. JUNE 01, 2010
Issued by. Ministry of Health of the People’s Republic of China
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Apparatuses and materials ... 4
3 Culture medium and reagents ... 5
4 Inspection procedure ... 5
5 Operation steps ... 6
6 Results and report ... 8
Appendix A ... 9
Foreword
This standard will replace GB/T 4789.30-2008 Microbiological examination of food
hygiene - Examination of listeria monocytogenes.
Compared with GB/T 4789.30-2008, the main changes of this standard are as follows.
- Modify this standard’s Chinese and English names;
- Delete “the second method - Automatic enzyme-linked fluorescence
immunoassay analyzer screening method”;
- Delete “the third method - Automatic screening method of pathogen detection
system”.
This standard’s appendix A is normative.
The previous versions replaced by this standard are.
- GB 4789.30-1994, GB/T 4789.30-2003, GB/T 4789.30-2008.
National food safety standard
Food microbiological examination.
Listeria monocytogenes
1 Scope
This standard specifies the test methods of Listeria monocytogenes in foods.
This standard applies to test of Listeria monocytogenes in foods.
2 Apparatuses and materials
In addition to the conventional sterilization and cultivation apparatuses, other
apparatuses and materials are as follows.
2.1 Refrigerator. 2°C~5°C.
2.2 Thermostatic incubator. 30°C±1°C, 36°C±1°C.
2.3 Homogenizer.
2.4 Microscope. 10x~100x.
2.5 Electronic balance. Sensitivity is 0.1g.
2.6 Erlenmeyer flasks. Capacity 100mL and 500mL.
2.7 Sterile pipette. 1mL (with 0.01mL scale), 10mL (with 0.1mL scale).
2.8 Sterile petri dish. Diameter is 90mm.
2.9 Sterile test tube. 16mmx160mm.
2.10 Centrifuge tube. 30mmx100mm.
2.11 Sterile syringe. 1mL.
2.12 Staphylococcus aureus (ATCC25923).
2.13 Rhodococcus equi.
2.14 Mus musculus albus. 16g - 18g.
2.15 Automated microbial and biochemical identification system.
3 Culture medium and reagents
3.1 Tryptone soya broth containing 0.6% yeast extract (TSB-YE). see appendix A.1.
3.2 Trypticase soy agar containing 0.6% yeast extract (TSA-YE). see appendix A.2.
3.3 Listeria enrichment broth LB (LB1, LB2). see appendix A.3.
3.4 1% acriflavine HCL solution. see appendix A.3.2.1.
3.5 1% of naladixic acid solution. see appendix A.3.2.1.
3.6 PALCAM agar. see appendix A.4.
3.7 Gram dye. see appendix A.5.
3.8 SIM motility culture medium. see appendix A.6.
3.9 Buffer glucose peptone water [for Methyl Red (MR) and V-P tests]. see appendix
A.7.
3.10 5%~8% sheep blood agar. see appendix A.8.
3.11 Sugar fermentation tube. see appendix A.9.
3.12 Catalase test. see appendix A.10.
3.13 Listeria chromogenic medium.
3.14 Biochemical identification kit.
4 Inspection procedure
The inspection procedure of Listeria monocytogenes is shown in Figure 1.
A.9.2 Preparation method
A.9.2.1 Allocate glucose fermentation tubes according to above compositions. Add
glucose according to proportion 0.5%. Pack into the small test tubes which have
inverted tubules. Adjust pH to 7.4. Autoclave it for 15min at 115°C. Preserve for spare
use.
A.9.2.2 Allocate other glucose fermentation tubes according to above compositions.
Pack every bottle for 100mL. Autoclave it for 15min at 115°C. Allocate all types of
carbohydrate to make 10% solution. Autoclave it simultaneously. Add 5mL of sugar
solution to 100mL of culture medium. Use sterile technique to pack into small tubes.
A.9.3 Test method
Use appropriate pure cultures to inoculate in sugar fermentation tube. Cultivate it for
24h~48h at 36°C±1°C. Observe the result. Blue represents negative. Yellow
represents positive.
A.10 Catalase test
A.10.1 Reagents
3% Hydrogen peroxide solution. Prepare it when use.
A.10.2 Test method
Use small glass rod or disposable inoculation needle to pick single colony. Place it in
clean test tube. Add 2mL of 3% Hydrogen peroxide solution. Observe the result.
A.10.3 Results
If it produces bubbles within half minute, the result is positive. If not, the result is
negative.
......
GB/T 4789.30-2008
Microbiological examination of food hygiene.Examination of listeria monocytogenes
ICS 07.100.30
C53
National Standards of People's Republic of China
Replacing GB/T 4789.30-2003
Microbiological examination of food hygiene
Listeria monocytogenes bacteria test
Posted 2008-11-21
2009-03-01 implementation
People's Republic of China Ministry of Health
Standardization Administration of China released
Foreword
The first law revision of this standard used by the US FDA "bacterial test Handbook" (Bacteriologicalanalyticalmanual, Chapter
10,2002); second method is equivalent to using "monocytogenes Listeria test methods" international analyst Institute (AOAC-RIPer-
The standard method first main difference compared with the US FDA/BAM as follows.
--- LB broth enrichment medium to replace BLEB broth;
--- Increased screening step;
--- Culture temperature to 36 ℃ ± 1 ℃ instead of 35 ℃.
This standard replaces GB/T 4789.30-2003 "Microbiological examination of food hygiene monocytogenes Listeria test."
This standard compared with GB/T 4789.30-2003 The main changes are as follows.
--- Remove the normative references.
--- Amend the law first. Including selective isolation medium to PALCAM instead of MMA, an increase of CHROMagar
--- Adds a second method, enzyme-linked fluorescence immunoassay analyzer automatic screening method.
--- Adds a third method, automated pathogen detection screening system.
Appendix A of this standard is a 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.
Participated in the drafting of this standard. Disease Prevention and Control Center of Fujian Province, Shaanxi Provincial Disease Prevention and Control Center, Hubei Provincial Disease Prevention and Control
center.
The main drafters of this standard. Xiumei, Yang Yang, Fletch, Guo Yunchang, Tian Jing, SOUTH - Ma Yi.
The standard version of the calendar year instead of the standard release of case.
--- GB/T 4789.30-1994, GB/T 4789.30-2003.
Microbiological examination of food hygiene
Listeria monocytogenes bacteria test
1 Scope
This standard applies to food and food-borne illness in the sample monocytogenes Listeria test.
2 Equipment and Materials
In addition to conventional microbiological laboratory culture and sterile equipment, other equipment and materials as follows.
2.1 Refrigerator. 2 ℃ ~ 5 ℃.
2.2 incubator. 30 ℃ ± 1 ℃, 36 ℃ ± 1 ℃.
2.3 homogenizer.
2.4 Microscope. 10 × ~ 100 ×.
2.5 Electronic balance. a sense of the amount of 0.1g.
2.6 conical flask. 100mL, 500mL.
2.7 sterile pipette. 1mL (with 0.01mL scale), 10mL (with 0.1mL scale).
2.8 sterile petri dish. diameter of 90mm.
2.9 sterile tube. 16mm × 160mm.
2.10 tube. 30mm × 100mm.
2.11 sterile syringes. 1mL.
2.12 Staphylococcus aureus (ATCC25923).
2.14 mice. 16g ~ 18g.
2.15 automated microbial identification system (VITEK) 1).
2.16 ELFA automatic immunoassay analyzer (miniVIDAS or VIDAS) 1).
2.17 automated pathogen detection system (BAX system, including BAX System Q7) 2).
3 media and reagents
3.1 containing 0.6% yeast extract of trypticase soy broth (TSB-YE). See Section A. Chapter 1.
1) Product name supplied by the French company bioMérieux products. This information is given for the convenience of users of this standard does not mean that the product
Recognition. If other equivalent product has the same effect, you can use these equivalent products.
2) provided by the trade name of DuPont products. This information is given for the convenience of users of this standard does not imply endorsement of the product.
If other equivalent product has the same effect, you can use these equivalent products.
3.2 containing 0.6% yeast extract of trypticase soy agar (TSA-YE). See Section A. 2.
3.3 Lee enrichment broth LB (LB1, LB2). See Section A. 3.
3.4 1% hydrochloric acid acridine yellow (acriflavineHCl) Solution. See A. 3.2.1.
3.5 1% nalidixic acid sodium salt (naladixicacid) Solution. See A. 3.2.1.
3.6 PALCAM Agar. see Section A. 4.
3.7 Gram stain solution. See Section A. 5.
......
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