Medium sensitivity verification scheme

First, the purpose of verification

Increase the credibility of sterility testing by verifying the sensitivity of the medium

Second, the principle

A known strain containing the growth conditions was added to the different kinds of culture medium for growth experiments, and the sensitivity of the medium was evaluated according to the growth condition of the added quantitative bacteria. The smaller the amount of bacteria, the higher the sensitivity of the medium.

Third, equipment

Sterile room (purification level less than 10000 in the operation room, partial purification less than 100, room temperature 18-26 ° C, relative humidity 40-60%), constant temperature incubator (30-35 ° C), constant temperature incubator (20-25 ° C ), autoclave, dry oven (250 ° C), phase contrast microscope, etc.

Medium: air-reducing bacteria, anaerobic culture medium (thioglycolate medium); fungal medium (modified Martin medium) Shanghai Chuangsai Technology provides modified Martin medium, product number: C015222-250g, price 80 yuan .

Test strains: Staphylococcus aureus, Bacillus subtilis, Candida albicans, Clostridium sporogenes. Shanghai Chuangsai Technology provides Staphylococcus aureus chromogenic medium, BR, commodity number: D16-1028481-500g, inquiry.

Fourth, verification implementation

1. Medium configuration:

Weigh 7.5 g of thioglycolate medium, 7.0 g of modified Martin medium, add 250 ml of distilled water, dissolve hot, dispense 10 ml/tube, autoclave at 116 ° C for 20 minutes. Shanghai Chuangsai Technology provides liquid thioglycolate medium, product number: C015190-250g, price 115 yuan.

2. Training of the test:

In the sterile operation platform, take a small amount of Staphylococcus aureus, Bacillus subtilis, and Clostridium sporogenes, inoculate in thioglycolate fluid medium (10ml / tube), and culture at 30-35 ° C for 24 hours. A small amount of Candida albicans solution was inoculated in modified Martin medium and cultured at 20-25 ° C for 24 hours. The culture solution at this time was called the original bacterial solution. Shanghai Chuangsai Technology provides protease (Bacillus subtilis), 9014-01-1, BR, 150u/mg, product number: D23-RM1036-1g, price 480 yuan.

3, test bacteria count: using direct dilution of the original culture medium, counting plate counting method

1 Take 1 ml of the cultured original bacterial solution and dilute it 10 times with physiological saline.

2 Use a clean cover slip on a clean count slide

3Suck a small amount of bacteria-containing medium to contact the edge of the coverslip

4 Observe under a phase contrast microscope at 400 times, and calculate the bacterial concentration for approximately 2 x 107 bacteria/ml per bacteria as seen in a small square.

4, the original bacterial solution dilution:

The above-mentioned original bacterial solution was diluted with physiological saline to 10 to 100 bacteria/ml according to the bacterial count result.

5. Sensitivity experiment:

1 ml of 4 diluted bacterial solution was inoculated into 1 ml of 10 ml/tube medium, each medium was inoculated with 3 tubes of medium, and the uninoculated medium was used as a negative control, cultured for 5 days according to the specified conditions, and recorded daily. . Shanghai Chuangsai Technology provides Candida BCG agar base, Candida BCG Agar Base, product number: BD-283510-500g, price 3070.06 yuan.

6, the result of the judgment:

A medium inoculated with each bacterial solution shall be no less than 2 tubes positive. That is, the sensitivity of the medium meets the requirements.

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