Application of luminous bacteria in preparation of index organisms for evaluating disinfectant effect and disinfectant effect evaluation method

Abstract

The invention discloses application of luminous bacteria in preparation of index organisms for evaluating the effect of a disinfectant and a rapid evaluation method for the effect of the disinfectant.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and in particular relates to the application of luminous bacteria in the preparation of indicator organisms for evaluating the effect of disinfectants and an evaluation method. Background Art

[0002] Spray disinfection and pest control is a common method of quarantine treatment in animal and plant quarantine work. It can be used on vehicles carrying incoming or transiting animals, animal quarantine isolation sites, and sites contaminated by animal and plant epidemics. As a key department for "preventing imported infections," customs has a heavy responsibility in preventing and controlling infectious diseases and preventing the introduction and export of animal and plant pests. To implement the requirements of "simultaneous prevention of people, objects, and the environment," preventive disinfection and pest control measures are important links in epidemic prevention and control. Failure of disinfection to achieve the expected results is a major factor in the spread of epidemics. Therefore, disinfection effectiveness evaluation is also a key link and needs to be carried out simultaneously with disinfection work to ensure that the disinfection effect is achieved.

[0003] At present, the biological indicator strains used in my country's national standards for spray disinfection effect evaluation are pathogenic microorganisms such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, all of which need to be cultured for 48 hours before evaluation, which is time-consuming and complicated to operate. Summary of the Invention

[0004] The present invention aims to overcome the deficiencies of the prior art and provides an application of luminescent bacteria in the preparation of indicator organisms for evaluating the effectiveness of disinfectants and a method for evaluating the effectiveness of disinfectants.

[0005] In order to achieve the above object, the technical solution provided by the present invention is:

[0006] The present invention provides the use of luminous bacteria in preparing indicator organisms for evaluating the effect of disinfectants.

[0007] Preferably, the luminescent bacteria is Photobacterium luminescens T3 variant.

[0008] The present invention also provides a method for quickly evaluating the effect of a disinfectant based on the above-mentioned luminescent bacteria, the method comprising the following steps:

[0009] (1) Select luminescent bacteria as indicator microorganisms and prepare a viable bacterial count of 1×10 8 ~5×10 8 cfu / mL of fresh bacterial suspension (visible luminescence under dark conditions) or freeze-dried bacterial powder with a luminescence intensity of more than 50,000 RLU within 5 minutes of recovery;

[0010] (2) Apply the bacterial suspension or freeze-dried bacterial powder resuscitation solution to the surface to be tested. Preferably, apply 100 μL of bacterial suspension or freeze-dried powder resuscitation solution to a 5 cm × 5 cm surface to be tested, spray the disinfectant, and use the disinfection time specified in the disinfectant instructions;

[0011] (3) using a neutralizer corresponding to the disinfectant to terminate the disinfection reaction and measuring the luminescence intensity of the sample; preferably, the luminescence intensity of the sample is measured after mixing with an electric mixer for 30 seconds or tapping on the palm of the hand 80 times;

[0012] (4) Calculate the microbial killing rate K based on the luminous intensity and determine the disinfection effect. When K ≥ 99.9%, it is considered qualified.

[0013] Preferably, the luminescent bacteria is Photobacterium luminescens T3 variant.

[0014] Preferably, the disinfectant is a chlorine-containing disinfectant or a 75% ethanol disinfectant.

[0015] More preferably, the chlorine-containing disinfectant includes 84 disinfectant.

[0016] Preferably, the base liquid of the fresh bacterial suspension dilution solution of the luminescent bacteria and the freeze-dried bacterial powder resuscitation solution is 2216E liquid culture medium.

[0017] Preferably, the disinfection effect evaluation test should be completed within 90 minutes after the freeze-dried bacterial powder is revived.

[0018] Preferably, the neutralizer of the 84 disinfectant is 0.2% sodium thiosulfate, and the neutralizer of the 75% ethanol is 2% Tween 80.

[0019] The present invention will be further described below:

[0020] Luminescent bacteria are a class of non-pathogenic, Gram-negative, anaerobic bacteria that, under normal culture conditions, emit visible fluorescence at wavelengths between 450 and 490 nm, visible to the naked eye in darkness. Due to their unique bioluminescent properties, they are widely used in water and environmental toxicity monitoring.

[0021] Based on the physiological properties of luminescent bacteria, such as their sensitivity to disinfecting factors and rapid autofluorescence testing, this invention uses them as indicator strains to evaluate the pest control and disinfection efficacy of various disinfectants. This replaces traditional pathogenic indicator strains and achieves the goal of rapidly and accurately evaluating disinfection efficacy at disinfection sites. This invention offers the advantages of safety, rapid detection, high sensitivity, and low cost. It addresses the difficulties and obstacles of time-consuming disinfection efficacy evaluation, delayed results, and inadequate adaptation to the rapid and timely response to epidemics. It can be applied to various preventive disinfection, sanitary treatment, and pest control treatment for animal and plant epidemics, providing a new technical guarantee for disinfection efficacy evaluation.

[0022] In short, the present invention is based on the luminescence principle of luminescent bacteria, and uses the bright photobacterium (Photobacteriumphosphoreum) T3 variant as a specific microorganism to detect the spray disinfection and pest control effects of common disinfectants. The luminescence conditions, screening of neutralizers, etc., are studied, and a method for quickly evaluating the on-site disinfection effect by a luminescent bacteria method is established. The results show that commonly used spray disinfectants such as 84 disinfectant and 75% ethanol can inhibit the growth of bright photobacteria, and the relative luminescence intensity can be measured by smearing method to effectively evaluate the spray disinfection effect, which is basically consistent with the evaluation results of the current disinfection effect evaluation indicator strains Staphylococcus aureus and Escherichia coli. This method can objectively, quickly and accurately detect the spray disinfection effect, which can solve the problem that the current disinfection and pest control effect evaluation operation is cumbersome and time-consuming, and has broad application prospects in port health quarantine and animal and plant epidemic prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Luminescence of Photobacterium luminescens T3 cultured in different culture media for 24 hours (from left to right: 2216E, Photobacteria, and culture media from existing literature);

[0024] Figure 2 :Changes in luminescence intensity of T3 in different culture media;

[0025] Figure 3 : Luminescence of T3 cultured at different inoculum sizes for 24 hours (from left to right, inoculum sizes are 1%, 2%, 3%, 4%, and 5%);

[0026] Figure 4 :The effect of inoculum size on the luminescence of Photobacterium luminescens T3;

[0027] Figure 5 : Luminescence of T3 cultured at different temperatures for 24 hours (from left to right, the culture temperatures are 15°C, 18°C, 20°C, 22°C, and 25°C);

[0028] Figure 6 :The effect of temperature on the luminescence intensity of Photobacterium luminescens T3;

[0029] Figure 7 : Live bacteria count of T3 freeze-dried powder resuscitation direct smear spray sterilization test (positive control dilution was 10 -4 ). DETAILED DESCRIPTION

[0030] 1 Materials and Methods

[0031] 1.1 Materials

[0032] 1.1.1 Instruments

[0033] Microplate bioluminescence photometer (Spark 10M, TECAN, Switzerland), constant temperature incubator, sprayer, electric mixer, colony counter, biosafety cabinet, freeze dryer, etc.

[0034] 1.1.2 Reagents

[0035] 84 disinfectant, 75% ethanol, anhydrous disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, diammonium hydrogen phosphate, trypsin, yeast powder, sodium chloride, sodium thiosulfate, Tween 80, glycerol, etc.

[0036] 1.1.3 Strains

[0037] Photobacterium phosphoreum T3 variant was purchased from Shanghai Guyan Industrial Co., Ltd. Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, and Pathogen Pseudomonas aeruginosa NCPPB 1511 were commercial strains.

[0038] 1.1.4 Culture medium

[0039] 2216E agar medium, 2216E liquid medium, photobacterial medium, tryptone soy agar medium (TSA), tryptone soy broth medium (TSB), 3% bovine serum albumin stock solution (BSA), nutrient agar medium, nutrient broth medium, 0.03 mol / L phosphate buffer (PBS), etc.

[0040] 1.2 Methods

[0041] 1.2.1 Selection of Luminescent Bacteria Culture Medium

[0042] Because Photobacterium luminescens is a marine photobacterium, commercially available marine bacterial enrichment medium 2216E, Photobacterium isolation medium, and a literature-optimized medium formula were used as screening media in this study. The literature-based medium formula for Photobacterium luminescens consisted of 5.0 g of tryptone, 5.0 g of yeast extract, 5.0 g of Na₂HPO₄, 1.0 g of KH₂PO₄, 30.0 g of NaCl, and 3.0 g of glycerol, dissolved in deionized water to a volume of 1000 mL and adjusted to pH 7.0. The Photobacterium was inoculated into each of the three liquid media and incubated in the dark at 18°C ​​to measure luminescence intensity to determine the optimal luminescence medium formula.

[0043] 1.2.2 Optimization of luminescent bacteria culture conditions

[0044] 1.2.2.1 Inoculation volume

[0045] Inoculate 1%, 2%, 3%, 4%, and 5% of fresh bacteria solution of Photobacterium luminescens (concentration of about 10 8 cfu / mL) in 2216E liquid culture medium and cultured at 18°C ​​for 24 h. The luminescence intensity was measured to determine the optimal inoculum size.

[0046] 1.2.2.2 Culture temperature

[0047] Using the optimal culture medium and inoculum size, the bacterial solution was cultured at 15, 18, 20, 22, and 25°C for 24 hours, and the luminescence intensity was measured to determine the optimal culture temperature.

[0048] 1.2.3 Identification of Neutralizers

[0049] 1.2.3.1 Primary selection of neutralizer

[0050] Prepare 1×10 3 ~1×10 4 The test bacterial suspension with a concentration of cfu / mL was tested according to the neutralizer preliminary selection test procedure in the "Technical Specifications for Disinfection (2002)" of the National Health Commission. 0.2% sodium thiosulfate was used as the neutralizer for 84 disinfectant, and 2% Tween 80 was used as the neutralizer for 75% ethanol. The viable bacterial culture count was adjusted to a 0.1 mL suspension coated on a 2216E plate and cultured for 48 hours. The luminescence intensity of the suspension was measured while counting the viable bacteria.

[0051] 1.2.3.2 Identification of Neutralizers

[0052] Prepare 1×10 8 ~5×10 8 cfu / mL test bacterial suspension. Add 2.0 mL of the test bacterial suspension to 2.0 mL of the organic interfering substance to create a bacterial suspension containing organic interfering substances. Conduct a formal neutralizer identification test according to the National Health Commission's "Technical Specifications for Disinfection" for quantitative identification of neutralizer suspensions. The viable bacterial count is adjusted to a 0.1 mL suspension plate coated on a 2216E plate for 48 hours.

[0053] 1.2.4 Quantitative killing test of luminescent bacteria

[0054] According to the results of neutralizer identification, Photobacterium luminescens can be used as a specific microorganism to carry out quantitative killing tests of luminescent bacteria.

[0055] 1.2.4.1 Suspension quantitative bactericidal test

[0056] Use sterile hard water to prepare 84 disinfectant and ethanol disinfectant at a concentration of 1.25 times the concentration required by the product manual. Take the third generation bacterial culture to prepare a concentration of 1×10 8 cfu / mL~5×108 cfu / mL bacterial suspension. First, add 0.5mL of bacterial suspension to a sterile test tube, then add 0.5mL of the organic interfering substance BSA, mix well, and then pipette 4.0mL of the prepared disinfectant into the tube. Mix quickly and disinfect for 2 minutes, 5 minutes, and 10 minutes (the shortest action time specified in the product manual). Add the corresponding neutralizer and let it act for 10 minutes. Use the diluent instead of the disinfectant for simultaneous testing as a positive control. Draw samples for viable bacterial counts and simultaneously draw 100μL of sample liquid to measure the luminescence intensity for comparison with the viable bacterial count method.

[0057] 1.2.4.2 Vector preparation and testing

[0058] Prepare fresh content of approximately 1 × 10 8 ~5×10 8 A luminescent bacteria suspension (cfu / mL) was prepared using filter paper and metal discs as carriers. 10 μL of the suspension was dripped onto each carrier. After drying, the number of recovered bacteria on each disc was determined using the viable bacterial count method. Escherichia coli and Staphylococcus aureus were also prepared for viable bacterial counts and compared to the luminescent bacteria.

[0059] 1.2.4.3 Freeze-drying and stability testing of luminescent bacteria

[0060] Prepare a vial of lyophilized powder in a 1:1 ratio of skim milk to bacterial suspension. Reconstitute the lyophilized powder with 1 mL of 3% NaCl solution and mix for 2 minutes. Observe in a dark room for visible luminescence. Simultaneously, pipette 0.1 mL of the reconstituted bacterial solution to measure luminescence intensity.

[0061] 1.2.4.4 Spray-based sterilization test

[0062] According to the actual situation that Photorhabdus luminescens is not suitable for preparing carriers for bacterial killing tests, the present invention adopts two methods, namely, fresh bacterial suspension smear spray method and freeze-dried bacteria direct smear spray method, to evaluate the disinfection effects of two disinfectants, 84 disinfectant and 75% ethanol.

[0063] Take freshly prepared solution at a concentration of 1×10 8 cfu / mL~5×10 8Evenly apply 100 μL of a bacterial suspension (cfu / mL) or a lyophilized powder resuscitation solution to a 5 cm x 5 cm area in a sterile dish. A disinfectant solution of a selected concentration (the minimum concentration specified in the product instructions) is then evenly sprayed onto the smeared area. A simultaneous positive control test is performed using the diluted solution instead of the disinfectant. After disinfection for 2, 5, and 10 minutes (the minimum duration specified in the product instructions), a sterile cotton swab is removed from the previously smeared area and immediately placed in a test tube containing 5 mL of a neutralizer of the corresponding concentration. After mixing for 10 minutes, viable bacteria are counted and 100 μL of the sample is simultaneously aspirated to measure luminescence intensity. Comparative tests are also performed with Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa for comparison with the luminescent bacteria method.

[0064] 1.2.4.5 Data Processing and Analysis Methods

[0065] Relative luminescence (X) (%) was calculated from the average luminescence as (RLU / RLU0) × 100%, where RLU represents the luminescence after disinfectant application and RLU0 represents the luminescence of the blank control (diluent). Microbial kill rate (K) (%) was calculated from the relative luminescence as (X0 - X1) / X0 × 100%, where X1 represents the relative luminescence after disinfectant application and X0 represents the luminescence of the positive control (diluent replacing disinfectant). The results were compared with the logarithmic killing values ​​of the viable bacteria enumeration method specified in the National Health Commission's "Technical Specifications for Disinfection (2002)." A logarithmic killing value ≥5.00 in the suspension quantitative killing test and ≥3.00 in the carrier quantitative killing test were considered acceptable disinfection. The differences between the luminescent bacteria method and the traditional culture enumeration method were analyzed to determine the disinfection efficacy.

[0066] 2 Results

[0067] 2.1 Selection of culture medium

[0068] The luminous bacteria were inoculated with commercial luminescent bacteria culture medium and literature method culture medium, and the changes in luminous intensity were shown in Figure 1 and Figure 2 The results showed that luminescent bacteria were more likely to grow and emit light in 2216E liquid medium. The luminescent bacteria reached their maximum luminescence intensity 22 to 24 hours after inoculation and then entered the decay phase.

[0069] 2.2 Optimal culture conditions for luminescence

[0070] 2.2.1 Inoculation volume

[0071] Different inoculum amounts have different luminescence intensities when cultured under the same conditions for 24 hours (see Figure 3), with time as the horizontal axis and the luminescence intensity of the bacteria as the vertical axis, the effects of different inoculation amounts on the luminescence and growth of luminescent bacteria were studied. The results are as follows Figure 4 It can be clearly seen that within the inoculum size range of 1% to 5%, the luminescence intensity of Photobacterium luminescens decreases with the increase of inoculum size.

[0072] 2.2.2 Culture temperature

[0073] Temperature is one of the key factors for the growth of microorganisms. The photobacterium used in the present invention originates from the ocean and is a low-temperature microorganism. Therefore, the present invention selects the range of 15 to 25°C to determine the optimal growth and luminescence temperature of the photobacterium. Figure 5 and Figure 6 It can be seen that the overall specific luminosity level of Photobacterium luminescens was the highest at 18°C, and the luminosity level of bacterial growth at 15°C was slightly lower than that at 18°C, followed by 20°C, and weaker at 22°C and 25°C.

[0074] 2.3 Neutralizer Identification

[0075] 2.3.1 Neutralizer Preliminary Selection Results

[0076] A preliminary selection of neutralizers for Photobacterium luminescens was conducted for 84 disinfectant and ethanol disinfectant, and luminescence intensity was measured simultaneously. Specific data are shown in Table 1. The results showed that the colony counts of Photobacterium luminescens, a specific microorganism, on the three plates were very similar and within ±50% of the positive control group. The selected neutralizers were suitable for formal neutralizer identification testing. However, the 2% Tween 80 neutralizer in the ethanol disinfectant significantly affected the luminescence intensity of the Photobacterium.

[0077] Table 1 Live bacteria count and luminescence intensity of neutralizer preliminary test

[0078]

[0079] 2.3.2 Neutralizer identification results

[0080] Based on the preliminary neutralizer selection results in 2.3.1, the present invention selected 0.2% sodium thiosulfate and 2% Tween 80 as neutralizers for 84 disinfectant and ethanol disinfectant, respectively, to conduct neutralizer identification tests. Specific data are shown in Table 2. The results showed that Photobacterium luminescens, as a specific microorganism, could be determined as an acceptable neutralizer for 84 disinfectant and ethanol disinfectant.

[0081] Table 2 Live bacteria counts in neutralizer identification test

[0082]

[0083] 2.4 Quantitative killing test of luminescent bacteria

[0084] 2.4.1 Suspension quantitative bactericidal test

[0085] According to the results of the neutralizer identification in 2.3.2, the suspension quantitative sterilization test operation procedure in the "Technical Specifications for Disinfection (2002)" of the National Health Commission was used to conduct a bacterial killing test on Photobacterium luminescens using 84 disinfectant and ethanol disinfectant. The bacterial suspension concentration was controlled at 1×10 8 cfu / mL~5×10 8 The luminescence intensity was measured simultaneously with the viable bacteria count. Specific data are shown in Table 3. The results showed that the logarithmic kill values ​​(KL) for both 84 disinfectant and 75% ethanol disinfectant were >7.00, indicating that disinfection was qualified. The luminescence intensity data was processed according to the method in 1.2.4.5. The microbial elimination rates (K) after 10 minutes of disinfectant application were both >99.9%.

[0086] Table 3 Comparison of the effects of T3 suspension method, live bacteria counting method and luminescent bacteria method

[0087]

[0088] 2.4.2 Vector preparation and test results

[0089] According to the test results in 2.4.1, the present invention prepared bright photobacterium bacterial slices on filter paper and metal carriers in accordance with the "Disinfection Technical Specifications" of the National Health Commission. At the same time, Staphylococcus aureus and Escherichia coli were used for comparison, and bacterial slice recovery tests were carried out. The specific results are shown in Table 4. The results showed that the recovery of live Staphylococcus aureus and Escherichia coli was 5×10 5 cfu /

[0090] slices~5×10 6 cfu / plate, which meets the standard requirements; the recovery rates of Photobacterium luminescens live bacteria on both carriers were both between 5×10 5 The number of cells per plate was less than 100 cfu / plate, and the recovery rate decreased with longer storage time. Therefore, Photobacterium luminescens is not suitable for preparing carriers for bacterial killing experiments.

[0091] Table 4 Recovery of live bacteria in specific microbial carriers

[0092]

[0093] 2.4.3 Freeze-drying and stability testing of Photobacterium luminescens

[0094] Based on the results of the carrier test in 2.4.2 and the principle that Photobacterium luminescens can be used for rapid water toxicity testing, the present invention prepared Photobacterium luminescens as a lyophilized powder in vials. The luminescence intensity of the same batch of lyophilized powder was measured over a 1.5-hour period over a period of 1 to 60 days. The specific results are shown in Table 5. The results showed that the luminescence intensity of the Photobacterium luminescens lyophilized powder did not change much within 1 hour after resuscitation, making it suitable for field testing. The luminescence intensity of the lyophilized powder was also relatively stable over 30 days.

[0095] Table 5 Luminescence recovery of Photobacterium luminescens freeze-dried powder

[0096]

[0097] 2.4.4 Evaluation of the bactericidal effect of smear spray method

[0098] 2.4.4.1 Bacterial suspension smear spray method

[0099] Bacterial killing tests were conducted on freshly cultured Photobacterium luminescens T3 suspensions, while changes in luminescence intensity were measured. Simultaneous bacterial killing and live bacteria count tests were conducted using two specific microorganisms, Staphylococcus aureus and Escherichia coli, as well as Psoralea oryzae. The results showed that after 10 minutes of exposure, the logarithmic kill values ​​(KL) for Photobacterium luminescens were both >5.00, and the luminescence intensity kill rates (K) were both above 99.95%. Based on the viable bacteria counts, disinfection was considered qualified, consistent with the disinfection efficacy evaluation results for Staphylococcus aureus, Escherichia coli, and Psoralea oryzae. No pattern was observed in the viable bacteria counts and luminescence intensity across the three replicates. However, within each group, the number of viable bacteria decreased, and the luminescence intensity also weakened, with prolonged disinfection time. The average luminescence intensity kill rate (K) was 99.9% for groups with KL values ​​between 5.00 and 6.00. Therefore, when the luminescence intensity killing rate K≥99.9%, it can be preliminarily judged that the live bacteria killing logarithm value KL≥5.00.

[0100] Table 6 Comparison of the effects of T3 suspension smear spray sterilization live bacteria counting method and luminescent bacteria method

[0101]

[0102] Table 7 Results of simultaneous quantitative smear spraying sterilization tests on other microbial suspensions

[0103]

[0104] 2.4.4.2 Freeze-dried bacteria direct application spray method

[0105] In order to facilitate the rapid evaluation of disinfection effect at the port, a quantitative smear spray evaluation test was carried out within 1 hour using freeze-dried Photobacterium luminescens powder for on-site resuscitation. The test results are shown in Figure 7and Table 8. The results showed that after 10 minutes of exposure, the luminescence intensity of the two disinfectants decreased sharply compared to the positive control. The logarithmic values ​​of the viable bacteria kill (KL) were all above 6.00, and the average luminescence intensity kill rate (K) was 99.9%. However, the results of three repeated tests showed that luminescence intensity cannot directly determine the number of colonies. Only the changes in luminescence intensity of the same bacterial powder can be used for preliminary screening of on-site disinfection effectiveness.

[0106] Table 8 Comparison of T3 freeze-dried bacteria resuscitation smear spray sterilization live bacteria counting method and luminescent bacteria method

[0107]

[0108] 3 Conclusion

[0109] As can be seen from the test results, different culture media, culture temperatures, and inoculum sizes have a significant impact on the luminescence of Photobacterium luminescens. The optimal luminescence culture medium for Photobacterium luminescens is 2216E, the optimal culture temperature is 18°C, and the optimal inoculum size is 1%. Photobacterium luminescens is a specific microorganism, and the neutralizer 0.2% sodium thiosulfate in 84 disinfectant and the neutralizer 2% Tween 80 in ethanol disinfectant have both passed the neutralizer identification test. Therefore, the present invention uses Photobacterium luminescens as a specific microorganism to carry out disinfection effect evaluation tests. In the quantitative sterilization test of its bacterial suspension, the logarithmic values ​​KL of the live bacteria kill of 84 disinfectant and 75% ethanol disinfectant are all ≥5.00, which meets the requirements of the "Technical Specifications for Disinfection (2002)" of the National Health Commission. The calculation results of its luminescence intensity data show that the microbial elimination rate K of each time is >99.8%. However, the recovery of both filter paper and metal carrier bacterial sheets does not meet the requirements, and the recovery rate decreases as the storage time increases.

[0110] Based on the low recovery rate of live bacteria in Photobacterium luminescens carriers and the good stability of luminescence intensity within 1 hour after the lyophilized bacterial powder is revived, the present invention uses a bacterial suspension spraying method and a lyophilized powder revived direct spraying method for on-site disinfection effect evaluation. The bacterial suspension spraying method requires the use of fresh culture 24 hours after the concentration of about 10 8cfu / mL bacterial suspension, and the freeze-dried powder direct smear method is to resuscitate the freeze-dried powder in the vial (luminescence intensity is above 50,000 RLU) with bacterial solution within 1 hour. In the bacterial suspension smear spray method, as a specific microorganism, the number of live bacteria of Photobacterium luminescens decreased continuously with the extension of the action time of 84 disinfectant and 75% ethanol disinfectant, and its luminescence intensity also weakened. After the minimum action time specified by the disinfectant, the logarithmic value KL of the live bacteria killing was >5.00 in each time, which was not much different from the logarithmic value KL of the live bacteria killing of specific microorganisms Staphylococcus aureus and Escherichia coli for 10 minutes of disinfection specified in the "Technical Specifications for Disinfection (2002)" of the National Health Commission. The calculation results of its luminescence intensity data showed that the microbial elimination rate K was ≥99.95% in each time. The average calculated luminescence intensity killing rate K was 99.9% when the logarithmic value KL of the live bacteria killing was between 5.00 and 6.00. The logarithmic value KL of the live bacteria killing of the plant pathogen rice bacterial leaf streak fungus carried out simultaneously was also ≥6.00, which was consistent with the evaluation results of the live bacteria of Photobacteria. When Photobacterium luminescens freeze-dried powder is revived and applied directly to a disinfectant spray for the minimum exposure time specified by the disinfectant, the logarithmic kill value (KL) of viable bacteria remains above 6.00, and the average luminescence intensity kill rate (K) is 99.9%. Therefore, both the bacterial suspension spray method and the freeze-dried powder revived and applied directly to a spray method can be used to quickly evaluate on-site disinfection effectiveness. However, Photobacterium luminescens is a marine bacterium, and its luminescence intensity is easily affected by various factors such as temperature, salinity, and neutralizers. It is not directly proportional to the number of viable bacteria and cannot be directly determined by colony counts. Therefore, the luminescence bacteria method can only be used for preliminary screening of on-site disinfection effectiveness. A luminescence intensity kill rate (K) ≥ 99.9% can be preliminarily determined to be satisfactory. If quantitative evaluation of disinfection effectiveness is required, viable bacteria culture and enumeration should be performed in a laboratory.

Claims

1. The application of luminescent bacteria in the preparation of indicator organisms for evaluating the effectiveness of disinfectants.

2. The use according to claim 1, characterized in that The luminescent bacteria is a Photobacterium luminescens T3 variant.

3. A method for rapidly evaluating the disinfectant effect of luminescent bacteria according to claim 1 or 2, characterized in that: The method comprises the following steps: (1) Select luminescent bacteria as indicator microorganisms and prepare a viable cell count of 1×10 8 ~5×10 8 cfu / mL of fresh bacterial suspension or freeze-dried bacterial powder with a luminescence intensity of more than 50,000 RLU within 5 minutes after recovery; (2) Apply the bacterial suspension or freeze-dried bacterial powder resuscitation solution to the surface to be tested, spray the disinfectant and disinfect for the time specified in the disinfectant instructions; (3) Use the neutralizer corresponding to the disinfectant to terminate the disinfection reaction and measure the luminescence intensity of the sample; (4) Calculate the microbial killing rate K based on the luminescence intensity and determine the disinfection effect. When K ≥ 99.9%, it is considered qualified.

4. The disinfectant effect evaluation method of luminescent bacteria according to claim 3, wherein: The luminescent bacteria is a Photobacterium luminescens T3 variant.

5. The disinfectant effect evaluation method of luminescent bacteria according to claim 3, wherein: The disinfectant is a chlorine-containing disinfectant or a 75% ethanol disinfectant.

6. The disinfectant effect evaluation method of luminescent bacteria according to claim 5, wherein: The chlorine-containing disinfectant includes 84 disinfectant.

7. The disinfectant effect evaluation method of luminescent bacteria according to claim 3, wherein: The base liquid of the fresh bacterial suspension dilution liquid and the freeze-dried bacterial powder resuscitation liquid of the luminescent bacteria is 2216E liquid culture medium.

8. The disinfectant effect evaluation method of luminescent bacteria according to claim 3, wherein: The disinfection effect evaluation test should be completed within 90 minutes after the freeze-dried bacterial powder is revived.

9. The method for evaluating the disinfectant effect of luminescent bacteria according to claim 6, wherein: The neutralizer of the 84 disinfectant is 0.2% sodium thiosulfate, and the neutralizer of 75% ethanol is 2% Tween 80.

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