ATP biological luminous method use for rapid estimating effect of antiseptics

A technology of bioluminescence and disinfectant, which is applied in the direction of chemiluminescence/bioluminescence, and analysis through chemical reaction of materials, etc., can solve the problems of little improvement and achieve the effect of improving efficiency and shortening evaluation time

Inactive Publication Date: 2005-10-12
GUANGDONG INST OF MICROORGANISM +1
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Problems solved by technology

These rapid evaluation methods have their practical side, however thes

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  • ATP biological luminous method use for rapid estimating effect of antiseptics

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Effect test

Embodiment 1

[0032] Embodiment 1: the mensuration of the conversion coefficient (K) of standard bacterial strain ATP content and cell number

[0033] (1) Prepare 4 kinds of standard indicator strain bacterial suspensions

[0034] At 37°C, culture Escherichia coli 8099, ATCC 6538, and ATCC 9372 standard strains with tryptone (TSA) medium for 20 hours, and culture ATCC 10231 strains with Sandburger’s agar medium for 45 hours. For moss, make bacterial suspension with physiological saline, centrifuge at room temperature at 8000r / min for 5min, discard the supernatant and suspend the bacterial sludge with raw saline, so that the number of indicator bacteria is 1×10 8 ~5×10 8 cells / mL, the number of indicator fungi is 1×10 7 ~5×10 7 cells / mL, as the standard bacterial suspension (referred to as liquid A) for testing.

[0035] (2) Plate culture count of live bacteria content of bacterial suspension

[0036] Perform pouring plate culture counting according to the 10-fold dilution method, and t...

Embodiment 2

[0048] Embodiment 2: Determination of bioluminescent method control value after disinfectant treatment

[0049] Treat the ATCC10231 and 8099 bacterial suspensions with peracetic acid disinfectant, after plate culture verification 100% kill, the treatment solution is vacuum filtered with a sterile microporous membrane (diameter is 47mm, pore size is 0.22~0.45μm) After removing the vacuum, use the ATP scavenger AE to treat the filter membrane in situ for 5 minutes, remove the filter membrane and use the ATP extractant Ec to extract the ATP in the residual cells, absorb 0.1mL of the ATP extract, and add 0.8mL of pH7. 8. 25mmol / L Tricine buffer solution and 0.1mL luciferase-luciferin reagent, after shaking well, do luminescence detection immediately, and use a series of standard ATP and sterile pure water for preparing ATP to perform the same luminescence detection of ATP extraction . Through the series of standard ATP and the corresponding CPM value to take the logarithmic regre...

Embodiment 3

[0053] (1) Preparation of bacteria suspension for evaluation of disinfection effect

[0054] At 37°C, use tryptone (TSA) medium to cultivate Escherichia coli 8099 strain for 20 hours, and culture ATCC 10231 strain with Sandcastle weak agar medium for 45 hours. Use an inoculation needle to scrape off the bacterial lawn on the slope and prepare it in normal saline. The resulting bacterial suspension was centrifuged at 8000r / min for 5min at room temperature, and the supernatant was discarded, and the bacterial sludge was suspended with buried saline, so that the number of bacterial indicator bacteria was 1×10 8 ~5×10 8 cells / mL, the number of fungal indicator bacteria is 1×10 7 ~5×10 7 cells / mL, directly used as the bacterial solution for evaluation without adding organic matter (abbreviated as liquid A). For the bacteria suspension with organic matter, the centrifuged bacteria sludge is suspended in physiological saline and then mixed with 30g / L BSA (bovine serum albumin) ste...

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Abstract

A method for evaluating sterilization effect of disinfectant by means of ATP bioluminescence includes preparing standard bacterial suspension, processing it with cell ATP release agent Ec and adding luciferase - luciferin reagent to detect out luminous value or filtering it with microhole film in combination with ATP scavenger and detecting out luminous value of filtering film, then carrying out equation calculation and utilizing conversion coefficient K to derive out live bacteria number in suspension for evaluation.

Description

[Affiliated technical field] [0001] The invention relates to a method and a reagent for rapidly evaluating the disinfecting effect of a disinfectant, in particular to the application of an ATP bioluminescent method in rapidly evaluating the disinfecting effect of a disinfectant. [Background technique] [0002] According to the "Evaluation Methods and Standards for Disinfection and Sterilization" (GB15981-1995) and the "Technical Specifications for Disinfection" promulgated by the Ministry of Health in 2002, the test for evaluating or testing the ability of disinfectants to kill microorganisms generally includes three steps. The test microorganisms are first treated with disinfectant, then neutralized to remove the effect of the disinfectant, and finally transferred to liquid culture medium to observe the change of turbidity (qualitative) or to count colonies after culture in solid medium (quantitative), so as to calculate sterilizing efficiency. Since the culture method is ...

Claims

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Application Information

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IPC IPC(8): G01N21/76
Inventor 吴清平吴慧清张菊梅李程思
Owner GUANGDONG INST OF MICROORGANISM
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