Jiangsu Province "Safety Technical Specification for Public Use Textiles" Standard

Preface
 

Chapter 4 of this standard contains mandatory provisions. This standard is formulated to eliminate hygiene and quality hazards in textiles used in public places, protect consumers' health and hygiene, and regulate the market demand for textiles used in public places.
This standard is prepared in accordance with GB / T1.1 —2000 "Standardization Work Guidelines Part 1: Structure and Writing Rules of Standards".
Appendices A, B, C, and D of this standard are normative appendices, and Appendix E is an informative appendix.
This standard is proposed by Jiangsu Fiber Inspection Institute (Jiangsu Textile Product Quality Supervision and Inspection Testing Center). It is drafted by Jiangsu Fiber Inspection Institute (Jiangsu Textile Product Quality Supervision and Inspection Testing Center), Nanjing Jinlong Textile Industry Co., Ltd., Jiangsu Carnation Weaving Co., Ltd., and Nantong Stedeford Textile Decoration Co., Ltd.
Main drafters of this standard: Li Hui, Shi Qing, Wei Changsheng, Zhou Guanlin.

Safety Technical Specification for Public Use Textiles
 

1 Scope
This specification defines the terms and definitions, requirements, and test methods for the safety technical specification of public use textiles.
This specification applies to the requirements for textiles used in public places within Jiangsu Province.
2 Normative References
The provisions in the following documents become provisions of this standard through reference. For dated references, all subsequent amendments (excluding errata) or revised versions do not apply to this standard; however, parties agreeing to this standard are encouraged to study the possibility of using the latest versions of these documents. For undated references, the latest version applies.
GB 251—1995 Gray scale for assessing staining
GB/T5455—1997 Textile burning performance test vertical method
GB/T7573—2002 Determination of pH value of textile water extract
GB/T817—1987 Rules for rounding off numerical values
GB18383 — 2007 General technical requirements for wadding fiber products
GB 18401 — 2003 National basic safety technical specification for textile products
FZ / T62006 —2004 Towels
3 Terms and Definitions
The following terms and definitions apply to this standard.
3.1 Public use textiles linen supply
Textiles used repeatedly by different people in public places, such as bedding, towel products, clothing, restaurant textiles, curtains, etc.
3.2 Medical public use textiles hospital textiles
Public use textiles used within medical institutions, such as ward and treatment textiles, towel products, patient clothing, etc.
3.3 Non-medical public use textiles hotel textiles
Public use textiles not used within medical institutions.
3.4 Body secretion
Substances secreted, excreted, or shed from the human body.
4 Requirements
4.1 Classification
Public use textiles are divided into two categories based on usage type: medical and non-medical.
4 . 2
Safety indicators for public use textiles are shown in Table 1.

4 . 3 Towel products, bed sheets, quilt covers, pillowcases, and clothing in public use textiles should not undergo antibacterial or flame retardant treatment.
4 . 4 Non-medical public use textiles should not be washed using washing equipment inside medical institutions; medical public use textiles should not
be washed using washing equipment inside hotels, restaurants, and similar units; companies providing external washing services should separately handle medical public use textiles and non-medical public use textiles (including equipment, environment, and personnel) when conducting washing operations.
4 . 5 The filling materials in public use textiles shall comply with the provisions of GB18383.
5 Test Methods
5.1 Bacteria and Fungi
5.1.1 Sampling
Using sterile operation, moisten 3 sterile medical cotton swabs with sterile physiological saline or PBS, and evenly smear three times on three 25cm² (5cm×5cm) sampling areas respectively. Immediately cut off the part of the swab handle touched by hand with sterile scissors, place the swabbed parts of the 3 cotton swabs into containers containing 30ml sterile physiological saline or PBS, seal them. Each container is one sample, stored at (1—5)°C, and tested within (4—6) hours. Shake the container with the cotton swabs thoroughly; this liquid is a 1:10 dilution.
5.1.2 Total colony count
Conducted according to Appendix A.
5.1.3 Coliform bacteria
Conducted according to Appendix B.
5.1.4 Pathogenic bacteria
Conducted according to Appendix C.
5.1.5 Total fungi count
Conducted according to Appendix A.
5.2 Extract pH value
5.2.1 Sampling
Samples are soaked at room temperature in 0.1 mol/l potassium chloride solution (prepared with distilled or deionized water) at a bath ratio of 1:50 for 1 hour ± 5 minutes, turning every 10 minutes. After soaking, 3 liquid samples are taken for testing.
5.2.2 Test method
Conducted according to GB/T 7573.
5.3 Odor
Conducted according to clause 6.7 of GB18401—2003, with additional odor types including chlorine, sweat, and blood-like smells.
5.4 Color stains
Conducted according to GB251.
5.5 Human debris
Conducted according to Appendix D.
5.6 Water absorption
Conducted according to Appendix A of FZ/T 62006—2004.
5.7 Flame retardancy
Conducted according to GB/T 5455.
       
Warning — Personnel performing microbial tests under this standard should have microbiological knowledge and practical experience in formal laboratory work. This standard does not address all possible safety issues. Users are responsible for taking appropriate safety and health measures and ensuring compliance with relevant national regulations.

Appendix A
(Normative Appendix)
Microbial total count test method

A.1 Bacterial total colony count test method
A.1.1 Under sterile (class 100) conditions, take 2.0 ml of 1:10 diluted sample and inoculate into two sterilized petri dishes, 1.0 ml per dish. If the estimated bacterial count is too high (more than 300 colonies per dish), perform serial tenfold dilutions before inoculation, i.e., take 1.0 ml and add to 9.0 ml sterilized physiological saline, mix well; this is the 1:100 dilution. Two plates are prepared for each dilution.
A.1.2 Pour melted nutrient agar cooled to about 45 ℃ into petri dishes, about (15–20) ml per dish, gently swirl to mix the sample, allow agar to solidify, then invert dishes and incubate at (36 ± 1) ℃ for 48 hours.
A.2 Colony count calculation
Colonies on plates can be counted by naked eye; use a magnifier if necessary to avoid missing colonies. Record colony counts for each plate and calculate the average for the same dilution. If colonies form confluent patches, that plate is not counted; if patches cover less than half the plate and the rest is evenly distributed, count colonies on the half plate and multiply by 2 to estimate total colonies.
Total colony count is calculated by formula (A.1):
M = k × n ...................................................................................... (A.1)
Where: m — total bacterial colony count, cfu/25cm²;
k — dilution factor;
n — average colony count.
A.3 Colony count report
A.3.1 Select the dilution with average colony count between 30 and 300, multiply by dilution factor for count (see example 1 in Table A.1).
A.3.2 If two dilutions have average colony counts between 30 and 300, the ratio of their total colony counts determines the report. If the ratio is less than or equal to 2, report the average; if greater than 2, report the count from the lower dilution plate (see examples 2 and 3 in Table A.1).
A.3.3 If the average colony counts of all dilutions are greater than 300, the report should be based on the highest dilution's average colony count multiplied by the dilution factor (see example 4 in Table A.1).
A.3.4 If the average colony counts of all dilutions are less than 30, the report should be based on the lowest dilution's average colony count multiplied by the dilution factor (see example 5 in Table A.1).
A.3.5 If the average colony counts of all dilutions are not between 30 and 300, with one dilution's average colony count greater than 300 and an adjacent dilution's average colony count less than 30, the report should be based on the average colony count closest to 30 or 300 multiplied by the dilution factor (see example 6 in Table A.1).
A.3.6 If there is no bacterial growth in all dilutions, the report number is less than 1 cfu/25cm² (see example 7 in Table A.1).
A.3.7 For colony count reports, when the colony count is within 100, report the actual value; when it is greater than or equal to 100, use two significant digits and round according to the GB/TS 170 "Rules for Numerical Rounding" standard. Scientific notation may be used (see the reporting method column in Table A.1). When the colony count is reported as "uncountable," the dilution of the sample should be noted.
A.4 Total Fungal Colony Count Detection Method
The fungal detection procedure is basically the same as the bacterial detection operation procedure, with the main difference being the culture medium used. Fungi are cultured on Sabouraud agar medium at a temperature of (25 ± 1) °C for 5 days. Do not open the petri dish lid casually to prevent fungal spores from spreading and contaminating the experimental environment. When counting colonies at the cultivation temperature and time, do not open the petri dish lid casually to avoid fungal spore contamination.

Appendix B
(Normative Appendix)
Coliform Test Method

B.1 Operating Steps
Under sterile (Class 100) conditions, inoculate 5 ml of 1:10 dilution into a 50 ml lactose bile salt fermentation tube, incubate at (36 ± 1) °C for 24 h. If no acid or gas is produced, report as coliform negative.
If acid and gas are produced, streak inoculate onto eosin methylene blue agar plates, incubate at (36 ± 1) °C for (18–24) h, and observe colony morphology on the plate. Typical coliform colonies are black-purple or red-purple, round, with neat edges, smooth and moist surfaces, often with a metallic luster; some may appear purple-black without or with slight metallic luster, or pink with a darker center.
Take 1–2 suspected colonies for Gram staining microscopic examination, simultaneously inoculate lactose fermentation tubes, incubate at (36 ± 1) °C for 24 h, and observe gas production.
B.2 Result Reporting
If the lactose bile salt fermentation tube produces acid and gas, and typical coliform colonies appear on eosin methylene blue plates, and Gram staining shows negative non-spore-forming bacilli, the tested sample can be reported as positive for coliforms.

Appendix C
(Normative Appendix)
Pathogen Test Method

C.1 Pseudomonas aeruginosa Detection Method
C.1.1 Operating Steps
Under sterile (Class 100) conditions, add 5 ml of 1:10 dilution to 50 ml SCDLP culture medium, mix thoroughly, incubate at (36 ± 1) °C for (18–24) h. If Pseudomonas aeruginosa grows, a thin film appears on the surface of the culture medium. The culture medium is usually yellow-green or blue-green. Pick culture from the thin film on the culture medium, streak inoculate onto cetyltrimethylammonium bromide agar plates, incubate at (36 ± 1) °C for (18–24) h, and observe colony characteristics. Pseudomonas aeruginosa grows well on this medium, colonies are flat, edges irregular, smooth and moist, gray-white, with water-soluble pigment diffusion around colonies. This medium is highly selective; Escherichia coli cannot grow, and Gram-positive bacteria grow poorly. If cetyltrimethylammonium bromide agar is unavailable, acetamide medium can be used for isolation. Pick culture from the thin film on the culture medium, streak inoculate onto acetamide agar plates, incubate at (36 ± 1) °C for (18–24) h, and observe colony characteristics. Pseudomonas aeruginosa grows well on this medium, colonies are flat, edges irregular, with slightly pink medium around colonies; other bacteria do not grow.
Take suspected colonies for smear and Gram staining; if Gram-negative bacteria are observed, the following tests should be performed:
Oxidase test: Place a small clean white filter paper in a sterile petri dish, pick suspected colonies with a sterile glass rod and smear on the filter paper, then add one drop of freshly prepared 1% dimethyl-p-phenylenediamine reagent. If pink or purple-red color appears within (15–30) seconds, the oxidase test is positive; no color change indicates negative.
Pseudomonas aeruginosa test: Take 2-3 suspected colonies and inoculate them separately on the Pseudomonas aeruginosa detection medium (PDP) slant, incubate at (36 ± 1) °C for (20-24) hours, add chloroform (trichloromethane) (3-5) ml, shake well to dissolve any possible Pseudomonas aeruginosa in the culture into the chloroform solution. When the chloroform extract turns blue, use a pipette to transfer the chloroform to another test tube and add 1 ml of 1 mol/l hydrochloric acid, shake and let stand for a moment. If a pink or purplish-red color appears in the upper layer, it is positive, indicating the presence of Pseudomonas aeruginosa in the tested sample.
Nitrate reduction gas production test: Pick a pure culture of the tested colony and inoculate it into nitrate peptone water medium, incubate at (36 ± 1) °C for 24 hours. The presence of gas in the small inverted tube of the nitrate peptone water medium is positive, indicating that the bacteria can reduce nitrate and decompose nitrite to produce nitrogen gas.
Gelatin liquefaction test: Take a pure culture of the suspected colony, stab inoculate into gelatin medium, incubate at (36 ± 1) °C for 24 hours, then place in a (4-10) °C refrigerator for 30 minutes. If it remains in a dissolved liquid state, it is positive; if it solidifies and does not dissolve, it is negative.
42 °C growth test: Take the suspected culture, inoculate on ordinary agar slant medium, incubate in an incubator at (41-42) °C for (24-48) hours. Growth of Pseudomonas aeruginosa is positive.
C.1.2 Result Report
After enrichment and isolation culture of the tested sample, if confirmed as Gram-negative bacilli, oxidase and Pseudomonas aeruginosa tests are positive, it can be reported that Pseudomonas aeruginosa is detected in the tested sample. When the Pseudomonas aeruginosa test is negative, but the gelatin liquefaction test, nitrate reduction gas production test, and 42 °C growth test are all positive, it can still be reported that Pseudomonas aeruginosa is detected in the tested sample.
C.2 Staphylococcus aureus Detection Method
C.2.1 Operation Steps
Under sterile (Class 100) operation, take 5 ml of 1:10 dilution, add to 50 ml SCDLP culture medium, mix well, and incubate at (36 ± 1) °C for 24 hours.
Take 1-2 inoculation loops from the above enrichment culture, streak on Baird-Parker medium; if this medium is not available, streak on blood agar medium, incubate at (36 ± 1) °C for (24-48) hours. On blood agar plates, the colonies appear golden yellow, large and raised, round, opaque, smooth surface, with hemolytic zones around. On Baird-Parker medium, colonies are round, smooth, moist, with a diameter of (2-3) mm, gray to black in color, with pale edges, surrounded by a turbid zone, and an outer transparent zone.
Staining microscopy: Pick typical colonies, prepare smears for Gram staining microscopy. Staphylococcus aureus is Gram-positive cocci arranged in grape-like clusters, without spores or capsules, diameter (0.5-1.0) µm. If microscopy matches the above, proceed with the following tests:
Mannitol fermentation test: Inoculate the above colonies into mannitol culture broth, incubate at (36 ± 1) °C for 24 hours. Acid production from mannitol fermentation is positive.
Plasma coagulase test ：
Slide method: Take a clean dry slide, add one drop of physiological saline at one end and one drop of rabbit plasma at the other end. Pick colonies and mix thoroughly with saline and plasma respectively. If clumps or granular coagula appear in the plasma-bacteria suspension within 5 minutes, while the saline drop remains uniformly turbid without coagulation, it is positive. If neither shows coagulation, it is negative. If the slide test is negative or both saline and plasma show coagulation, proceed to tube coagulase test.
Tube method: Take 0.5 ml of 1:4 fresh plasma into a sterile small test tube, add 0.5 ml of 24-hour broth culture of the tested bacteria. Mix well and incubate at (36 ± 1) °C in an incubator or water bath, observe every 30 minutes. Coagulation within 24 hours is positive. Use known plasma coagulase positive and negative strains' broth cultures (0.5 ml each) as positive and negative controls.
C.2.2 Result Report
If suspicious colonies grow on agar plates, microscopy shows Gram-positive cocci in clusters, can ferment mannitol producing acid, and plasma coagulase test is positive, it can be reported that Staphylococcus aureus is detected in the tested sample.
C.3 Hemolytic Streptococcus Detection Method
C.3.1 Operation Steps
Under sterile (Class 100) operation, take 5 ml of 1:10 dilution and add to 50 ml glucose broth, incubate at (36 ± 1) °C for 24 hours.
Inoculate the culture by streaking on blood agar plates, incubate at (36 ± 1) °C for 24 hours and observe colony characteristics. Hemolytic streptococci on blood plates appear gray-white, translucent or opaque, with needle-like projections, smooth surface, neat edges, and colorless transparent hemolytic zones around.
Staining microscopy: Pick typical colonies, prepare smears for Gram staining microscopy. They should be Gram-positive cocci arranged in chains. If microscopy matches the above, proceed with the following tests:
Streptokinase test: Take 0.2ml of potassium oxalate plasma (0.01g potassium oxalate mixed with 5ml rabbit plasma, centrifuged to precipitate, take the supernatant), add 0.8ml sterile saline, mix well, then add 0.5ml of 24h broth culture of the test bacteria and 0.25ml of 0.25% calcium chloride, mix well, place in a (36±1)°C water bath, observe once every 2 minutes (generally coagulates within 10 minutes), after plasma coagulation continue to observe and record melting time. If no melting occurs within 2 hours, continue to observe for 24 hours; if the clot completely melts it is positive, if no melting after 24 hours it is negative.
Bacitracin sensitivity test: Smear the test bacterial suspension on a blood agar plate, place a paper disc containing 0.04 units of bacitracin on the plate surface with sterile tweezers, simultaneously use a known positive strain as control, incubate at (36±2)°C for (18–24) hours, presence of an inhibition zone indicates positive.
C.3.1 Result Report
Microscopic examination shows Gram-positive chain-arranged cocci, hemolysis zones appear on blood agar plates, streptokinase and bacitracin tests are positive, the tested sample can be reported as containing hemolytic streptococci.

Appendix D
(Normative Appendix)
Human Debris Inspection Method

D.1 Principle
Use visual inspection to detect whether the sample contains hair, skin flakes, body fluids, stains, etc.
D.2 Test Conditions
Inspectors should wash their face and hands first, and seal hair, hands, arms, etc., to prevent self-contamination affecting the test results.
D.3 Samples
Towel items, clothing, etc. can be either arranged ready for use or packaged before placement; bedding should be arranged ready for use.
D.4 Method
On-site inspection is mainly by visual observation, supplemented if necessary by a (1–2) times magnifying glass and pointed tweezers to detect hair, skin flakes, body fluids, stains, etc. Objects smaller than 1.0mm in maximum dimension are not judged. Detected substances should be collected and sealed for preservation.

Appendix E
(Informative Appendix)
Preparation of Culture Media and Reagents

E.1 Nutrient Agar Medium
Ingredients:
Peptone 10.0g
Beef Extract 3.0g
Sodium Chloride 5.0g
Agar 15g–20g
Distilled Water 1000ml
Preparation: Dissolve all ingredients except agar in distilled water, adjust pH to 7.2–7.4, add agar, heat to dissolve, dispense into test tubes, sterilize at 121°C under high pressure for 20 minutes and set aside.
E.2 Lactose Bile Salt Fermentation Tube
Ingredients:
Peptone 20.0g
Pig bile salt (or bovine, ovine bile salt) 5.0g
Lactose 10.0g
0.04% Bromocresol Purple Aqueous Solution 25ml
Distilled Water Make up to 1000ml
Preparation: Dissolve peptone, bile salt, and lactose in water, adjust pH to 7.4, add indicator, dispense 50ml per tube, insert a small inverted tube, sterilize at 115°C under high pressure for 15 minutes and set aside.
E.3 Lactose Fermentation Tube
Ingredients:
Peptone 20.0g
Lactose 10.0g
0.04% Bromocresol Purple Aqueous Solution 25 ml
Distilled Water Add to 1000ml
Preparation: Dissolve peptone and lactose in water, adjust pH to 7.4, add indicator, dispense 10ml per tube, place a small inverted tube inside, sterilize at 115°C under high pressure for 15 minutes, and set aside.
E.4 Eosin Methylene Blue Agar (EMB)
Ingredients:
Peptone 20.0g
Lactose 10.0g
Dipotassium Phosphate 2.0g
Agar 15g to 20g
2% Eosin Y Solution 20ml
0.65% Methylene Blue Solution 10ml
Distilled Water Add to 1000ml
Preparation: Dissolve peptone, phosphate, and agar in distilled water, adjust pH to 7.1, dispense into flasks, sterilize at 121°C under high pressure for 15 minutes and set aside. Before use, heat to dissolve agar, add lactose (sterilized by filtration), cool to 55°C, add eosin and methylene blue solutions, mix well, and pour plates.
E.5 SCDLP Liquid Medium
Ingredients:
Casein Peptone 17.0g
Soy Peptone 3.0g
Sodium Chloride 5.0g
Dipotassium Phosphate 2.5g
Glucose 2.5g
Lecithin 1.0g
Tween 80 7.0g
Distilled Water 1000ml
Preparation method: Mix various ingredients (if no casein peptone and soybean peptone are available, Japanese polyvalent peptone can be used as a substitute), heat to dissolve, adjust pH to 7.2 - 7.3, dispense, sterilize at 121 ℃ under high pressure for 20 minutes, shake well to avoid Tween 80 settling at the bottom, cool to 25 ℃ before use.
E.6 Hexadecyltrimethylammonium Bromide Culture Medium
Ingredients:
Beef extract 3.0g
Peptone 10.0g
Sodium chloride 5.0g
Hexadecyltrimethylammonium bromide 0.3g Agar 15g—20g
Distilled water 1000ml
Preparation method: Except for agar, mix and heat dissolve the above ingredients, adjust pH to 7.4 - 7.6, then add agar, sterilize at 115 ℃ under high pressure for 20 minutes, cool to about 55 ℃, and pour into petri dishes.
E.7 Acetamide Culture Medium
Ingredients:
Acetamide 10.0g
Sodium chloride 5.0g
Anhydrous dipotassium phosphate 1.39g
Anhydrous monopotassium phosphate 0.73g
Magnesium sulfate (MgSO4•7H2O) 0.5g
Phenol red 0.012g
Agar 15g to 20g
Distilled water 1000ml
Preparation method: Except for agar and phenol red, add other ingredients to distilled water, heat to dissolve, adjust pH to 7.2—7.4, then add agar and phenol red, sterilize at 121 ℃ under high pressure for 20 minutes, and prepare plates for use.
E.8 Pseudomonas aeruginosa Antibiotic Susceptibility Test Medium Slant
Ingredients:
Peptone 20.0g
Magnesium chloride 1.4g
Potassium sulfate 10.0g
Agar 15g—20g
Glycerol (chemically pure)                             10.0g
Distilled Water Add to 1000ml
Method: Add peptone, magnesium chloride, and potassium sulfate to distilled water, heat to dissolve, adjust pH to 7.4, add agar and glycerol, heat to dissolve, dispense into test tubes, sterilize at 115 ℃ under high pressure for 20 minutes, prepare slants for later use.
E.9 Gelatin Medium
Ingredients:
Beef extract                                                                                             3.0g
Peptone                                                                                                     5.0g
Gelatin                                                                                                     120g
Distilled water                                                                                             1000ml
Method: Soak each component in distilled water for 20 minutes, heat and stir to dissolve, adjust pH to 7.4, dispense 5ml into test tubes, sterilize at 115 ℃ under high pressure for 20 minutes, stand upright to prepare deep layers for later use.
E.10 Nitrate Peptone Water Medium
Ingredients:
Peptone                                                                                                     10.0g
Yeast extract                                                                                                3.0g
Potassium nitrate                                                                                         12.0g
Sodium nitrite                                                                                             0.5g
Distilled water                                                                                             1000ml
Method: Add peptone and yeast extract to distilled water, heat to dissolve, adjust pH to 7.2, boil and filter, make up volume, add potassium nitrate and sodium nitrite and dissolve evenly, dispense into test tubes with small side tubes, sterilize at 115 ℃ under high pressure for 20 minutes, keep for later use.
E.11 Baird Parker Medium
Ingredients:
Tryptone                                                                                                    10.0g
Beef extract                                                                                                 5.0g
Yeast extract                                                                                                 1.0g
Sodium pyruvate                                                                                            10.0g
Glycine                                                                                                     12.0g
Lithium chloride (LiCl • 6H2O)                                                                           5.0g
Agar                                                                                                        20g
Distilled Water                                                                     1000ml
Preparation of enrichment agent: Mix 30% egg yolk saline 50ml with sterilized filtered 1% potassium tellurite solution 10ml, store in refrigerator for later use.
Preparation: Add all components to distilled water, heat to boil until completely dissolved, cool to 25 ℃ and adjust pH to 7.2. Dispense 95ml per bottle, sterilize at 121 ℃ under high pressure for 20 minutes and keep for later use. Before use, heat to dissolve agar, add 5ml of egg yolk potassium tellurite enrichment agent preheated to 50 ℃ per 95ml, shake well and pour onto plates. The medium should be dense and opaque, and should not be stored in the refrigerator for more than 18 hours before use.
E.12 Blood Agar Medium
Ingredients:
Nutrient Agar                                                                     100ml
Defibrinated Sheep Blood (or Rabbit Blood)                                     10ml
Preparation: Heat sterilized nutrient agar to dissolve, cool to about 55 ℃, add 10ml defibrinated blood aseptically, mix well, pour into plates and store in refrigerator for later use.
E.13 Mannitol Fermentation Medium
Ingredients:
Peptone                                                                             10.0g
Beef Extract                                                                       5.0g
Sodium Chloride                                                                   5.0g
Mannitol                                                                           10.0g
0.2% Bromcresol Purple Solution                                                 12ml
Distilled Water                                                                   1000ml
Preparation: Add peptone, sodium chloride, and beef extract to distilled water, heat to dissolve, adjust pH to 7.4, add mannitol and bromcresol purple and mix well, dispense into tubes, sterilize at 115℃ under high pressure for 20 minutes and keep for later use.
E.14 Glucose Broth
Ingredients:
Peptone                                                                                                     10.0g
Beef Extract                                                                       5.0g
Sodium Chloride                                                                   5.0g
Glucose                                                                            10.0g
Distilled Water                                                                   1000ml
Preparation: Dissolve the above components in distilled water, adjust pH to 7.2–7.4, heat to dissolve, dispense into tubes, sterilize at 121℃ under high pressure for 15 minutes and keep for later use.
E.15 Rabbit Blood Serum
Preparation: Mix 1 part sterilized 3.8% sodium citrate with 4 parts whole rabbit blood, mix well and let stand, centrifuge at 3000r/min for 5 minutes, collect supernatant and discard blood cells.
E.16 Sabouraud Agar Medium
Ingredients:
Peptone                                                                             10.0g
Glucose                                                                            40.0g
Agar                                                             15g —20g
Distilled Water                                                 1000ml
Preparation: Dissolve agar in 700ml distilled water, dissolve glucose and peptone in 300ml distilled water, mix the two parts, shake well, dispense, sterilize at 115℃ under high pressure for 15 minutes and set aside. Before use, add 0.1g/l chloramphenicol or 0.03g/l streptomycin by filtration sterilization method.
Qualitative test uses Sha's culture solution, with the same components and preparation method as above except without agar.
E.17 Nutrient Broth Culture Medium
Ingredients:
Peptone                                                           10.0g
Sodium Chloride                                                   5.0g
Beef Extract                                                      3.0g
Distilled Water                                                 1000ml
Preparation: Adjust pH to 7.2—7.4, dispense, sterilize at 115℃ under high pressure for 20 minutes and set aside.
E.18 Bromocresol Purple Glucose Peptone Water Medium
Ingredients:
Peptone                                                           10.0g
Glucose                                                            5.0g
Distilled Water                                                 1000ml
Preparation: Adjust pH to 7.0—7.2, add 0.6ml 2% bromocresol purple alcoholic solution, sterilize at 115℃ under high pressure for 20 minutes and set aside.
E.19 Gram Staining Solution
Crystal Violet Staining Solution:
Crystal Violet                                                     1.0g
95% Ethanol                                                     20ml
1% Ammonium Oxalate Aqueous Solution                             80ml
Dissolve crystal violet in ethanol, then mix with ammonium oxalate solution.
Gram's Iodine Solution
Iodine                                                             1.0g
Potassium Iodide                                                 2.0g
Distilled Water                                                 300ml
Decolorizer
95% Ethanol
Counterstain Solution:
(1) Safranin Counterstain Solution:
Sahhuang 0.25g
95% Ethanol 10ml
Distilled Water 90ml
Dissolve Sahhuang in ethanol, then dilute with distilled water.
(2) Diluted Carbol Fuchsin Solution:
Weigh 10g of alkaline fuchsin, grind finely, add 100ml of 95% ethanol, let stand overnight, filter with filter paper. Take 10ml of this solution, add 90ml of 5% carbolic acid aqueous solution and mix to obtain carbol fuchsin solution. Then take 10ml of this solution, add 90ml of water to obtain diluted carbol fuchsin solution.
E.20 0.03mol / L Phosphate Buffered Saline (PBS, pH7.2)
Ingredients:
Disodium Hydrogen Phosphate 2.83g
Potassium Dihydrogen Phosphate 1.36g
Distilled Water 1000ml