CN104390948A - Method for detecting bacterioplankton in air - Google Patents
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- CN104390948A CN104390948A CN201410680798.9A CN201410680798A CN104390948A CN 104390948 A CN104390948 A CN 104390948A CN 201410680798 A CN201410680798 A CN 201410680798A CN 104390948 A CN104390948 A CN 104390948A
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- 238000000034 method Methods 0.000 title claims abstract description 35
- 239000007788 liquid Substances 0.000 claims abstract description 30
- 239000002504 physiological saline solution Substances 0.000 claims abstract description 11
- 241000894006 Bacteria Species 0.000 claims description 58
- 239000003153 chemical reaction reagent Substances 0.000 claims description 15
- 239000002131 composite material Substances 0.000 claims description 13
- 238000005259 measurement Methods 0.000 claims description 6
- 230000003749 cleanliness Effects 0.000 claims description 4
- 239000005089 Luciferase Substances 0.000 claims description 3
- 108060001084 Luciferase Proteins 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 230000009089 cytolysis Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 abstract description 4
- 238000012545 processing Methods 0.000 abstract description 4
- 238000005070 sampling Methods 0.000 abstract description 2
- 230000029918 bioluminescence Effects 0.000 abstract 1
- 238000005415 bioluminescence Methods 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 description 11
- 238000001514 detection method Methods 0.000 description 5
- 238000013178 mathematical model Methods 0.000 description 5
- 238000010998 test method Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 239000001967 plate count agar Substances 0.000 description 2
- 238000007492 two-way ANOVA Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003116 impacting effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a method for detecting bacterioplankton in air. The method comprises the following steps: (1) putting a sterile plate containing a sterile physiological saline solution into a bacterioplankton sampler, and collecting air from a plurality of sampling points; (2) determining the bacterial content in collecting liquid; (3) absorbing the collecting liquid and determining a fluorescence value by virtue of an ATP fluorescence detector; (4) obtaining a standard curvilinear equation between a logarithmic value of the bacterial content and the logarithmic value of the fluorescence value according to the results in the steps (2) and (3); and (5) determining the fluorescence value of to-be-detected air by referring to the steps (1) and (3), and substituting the fluorescence values into the standard curvilinear equation so as to obtain the content of the bacterioplankton in the to-be-detected air. According to the method disclosed by the invention, a conventional impact type air collection method is combined with an ATP bioluminescence method for the first time; the bacterial content in air can be quickly determined through the ATP fluorescence value; the microculture time is greatly saved; the efficiency of detecting the bacterioplankton is improved; and the exterior microorganism pollution in the food processing process is reduced.
Description
Technical field
The invention belongs to microorganism detection field, relate to a kind of method detecting planktonic bacteria in air.
Background technology
In the process of food production processing, the planktonic bacteria in air is one of main source of food microorganisms pollution.Monitor and forecast is carried out to the quantity of planktonic bacteria, significant to guarantee food safety and sanitation.According to " GB/T18204-2000 public place air microbe detection method total number of bacteria mensuration ", the method of collection microbes in air general is at present mainly impingement method and natural sedimentation, the planktonic bacteria constant temperature culture 48h to collecting all is needed after air sample collection, and then carry out plate count, the method has obvious hysteresis quality, the food enterprises cannot according to the method to starting material storage, food production process workshop, finished product and semi-manufacture store air purity in place and monitor in real time, this also brings safe and sanitary hidden danger to food production, therefore, food production processing industry needs the Fast Detection Technique of planktonic bacteria in a kind of air badly, improve the monitoring efficiency of food production overall process relevant environment hollow air supporting trip bacterial contamination.
Summary of the invention
An object of the present invention is to provide a kind of method detecting planktonic bacteria in air.
In detection air provided by the present invention, the method for planktonic bacteria, specifically can comprise the steps:
(1) sterilized petri dishes filling stroke-physiological saline solution is put into plankton bactgeria sampler, gather air from some sampled points (more than 10);
(2), after gathering, the bacteria content (CFU/mL) gathering liquid in the plate of each sampled point is measured respectively;
(3) draw described collection liquid and put into ATP fluorescence detector sample cup, add composite fluorescence reagent, repeatedly aspirate 4 times with liquid-transfering gun, after mixing, described sample cup is placed in ATP fluorescence detector and measures fluorescent value (RLU);
(4) according to the measurement result of step (2) and step (3), the typical curve equation between the logarithm value of the bacteria content of described collection liquid and the logarithm value of described fluorescent value is obtained;
(5) sterilized petri dishes filling stroke-physiological saline solution is put into described plankton bactgeria sampler, gather air to be measured; The collection liquid to be measured drawn in plate puts into described ATP fluorescence detector sample cup, adds described composite fluorescence reagent, described sample cup is placed in ATP fluorescence detector and measures fluorescent value, be designated as fluorescent value to be measured;
(6) logarithm of the fluorescent value described to be measured step (5) obtained substitutes into the described typical curve equation of step (4), calculate the bacteria content in described collection liquid to be measured, and then obtain the content of planktonic bacteria in described air to be measured.
In the process, described composite fluorescence reagent is primarily of bacteria lysis agent and luciferase composition.In one embodiment of the invention, described composite fluorescence reagent is specially the composite fluorescence reagent that the production code member of middle matter match good fortune (Beijing) tech equipment company limited production is ZF0011.
In the present invention, the consumption of described collection liquid or described collection liquid to be measured is specially 100 μ L, and the consumption of described composite fluorescence reagent is specially 25 μ L.
In the step (1) and step (5) of described method, the consumption of described stroke-physiological saline solution is 20mL.Described sterilized petri dishes is aseptic microbial culture dish.
In the step (1) and step (5) of described method, the collection capacity of described air is all with reference to " method of testing of GB/T 16293-2010 medical industry toilet (district) flcating germ ", air acquisition amount is determined according to concrete purity requirements: if the cleanliness factor of described air is 100 grades, then the collection capacity of described air is 1000L; If the cleanliness factor of described air is 10000 grades, then the collection capacity of described air is 500L; If more than 100000 of described air grades and 100000 grades, then the collection capacity of described air is 100L.
In the step (2) of described method, gather the bacteria content of liquid in the plate of each sampled point of described mensuration, measure according to GB GB4789.2-2010.
In the step (6) of described method, after calculate the bacteria content in described collection liquid to be measured according to described typical curve equation, be obtain the content of planktonic bacteria in described air to be measured according to the volume computing of the air described to be measured collected.
In the present invention, the model of described plankton bactgeria sampler is FKC-1 type, can therefrom matter match good fortune (Beijing) tech equipment company limited buy.
In the present invention, the described typical curve equation that step (4) obtains is specially Y=0.9189X+1.4651, wherein, Y is the logarithm value of the bacteria content (CFU/mL) gathered in liquid, and X is the logarithm value of fluorescent value (RLU); Linearly dependent coefficient R
2be 0.8762.
In practical operation, sample when gathering described air to be measured in the described some sampled points in step (1) and step (5) is in same building or same on-site.
Another object of the present invention is to provide a kind of suit for detecting planktonic bacteria in air.
Suit for detecting planktonic bacteria in air provided by the present invention, containing plankton bactgeria sampler, ATP fluorescence detector and the instructions recording method described above.
The described application be sleeved in quantitative detection air to be measured in planktonic bacteria content also belongs to protection scope of the present invention.
The impacting type air acquisition method of routine combines with ATP biloluminescence method by the present invention first, after Mathematical Models, the total number of bacteria in air is determined fast by ATP fluorescent value, greatly save the time that microorganism is cultivated, improve the efficiency detecting planktonic bacteria, can in time the air planktonic bacteria of food production relevant environment be detected and be assessed, instruct food enterprise to the clean of environment and disinfection, the sanitary condition of microbes in air is fed back to food enterprise and supervision department timely and accurately, reduce the generation of external source microbial contamination in food processing process.Meanwhile, the present invention does not relate to poisonous harmful reagent, safety and sanitation, environmental protection.
Accompanying drawing explanation
Fig. 1 is for gathering the typical curve between the logarithm value of bacteria content (CFU/mL) in liquid and the logarithm value of fluorescent value (RLU).
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Instrument and consumptive material:
ATP fluorescence detector (middle matter match good fortune (Beijing) tech equipment company limited, SF0012 type);
The special disposable sample cup of ATP fluorescence detector;
Plankton bactgeria sampler (middle matter match good fortune (Beijing) tech equipment company limited product, FKC-1 type);
Aseptic microbial culture dish;
Plate count agar nutrient culture media (PCA);
Composite fluorescence reagent (middle matter match good fortune (Beijing) tech equipment company limited, production code member is ZF0011), the principal ingredient of this composite fluorescence reagent is bacteria lysis agent and luciferase.
The mensuration of bacteria content in embodiment 1, Laboratory Air
1, the formulation of typical curve
(1) sterilized petri dishes filling 20ml stroke-physiological saline solution is put into plankton bactgeria sampler, gather air respectively from 55 selected sampled points, gather 500L air (air purity of sampled point is 10000 grades) with reference to " method of testing of GB/T 16293-2010 medical industry toilet (district) flcating germ " each sampled point;
(2) after gathering, the bacteria content (CFU/mL) gathering liquid in the plate of each sampled point is measured respectively according to GB GB4789.2-2010;
(3) draw and gather liquid described in 100 μ L and put into ATP fluorescence detector sample cup, add 25 μ L composite fluorescence reagent, repeatedly aspirate 4 times with liquid-transfering gun, after mixing, described sample cup is placed in ATP fluorescence detector and measures fluorescent value (RLU);
(4) linear regression analysis is carried out to the data of step (2) and (3), the logarithm value of the bacteria content of the described collection liquid obtained with step (2) is for ordinate, the logarithm value of the described fluorescent value obtained with step (3) is for horizontal ordinate, production standard curve, obtain typical curve equation Y=aX+b, wherein Y representative gathers the logarithm value of bacteria content (CFU/mL) in liquid, and X representative records the logarithm value of fluorescent value (RLU).
In the air of 55 selected sample point planktonic bacteria gather the ATP fluorescent value of liquid and bacteria content measurement result specifically as shown in table 1.
In the air of table 155 sampled point, planktonic bacteria gathers ATP fluorescent value and the bacteria content measurement result of liquid
| Sequence number | Fluorescent value (RLU) | The logarithm value of fluorescent value | GB4789.2 measures bacteria content | The logarithm value of bacteria content |
| (CFU/mL) | ||||
| 1 | 107 | 2.03 | 640 | 2.81 |
| 2 | 1112 | 3.05 | 6400 | 3.81 |
| 3 | 11328 | 4.05 | 64000 | 4.81 |
| 4 | 125115 | 5.10 | 640000 | 5.81 |
| 5 | 1010524 | 6.00 | 6400000 | 6.81 |
| 6 | 344 | 2.54 | 350 | 2.54 |
| 7 | 2307 | 3.36 | 3500 | 3.54 |
| 8 | 18699 | 4.27 | 35000 | 4.54 |
| 9 | 154950 | 5.19 | 350000 | 5.54 |
| 10 | 1082821 | 6.03 | 3500000 | 6.54 |
| 11 | 104 | 2.02 | 1900 | 3.28 |
| 12 | 1073 | 3.03 | 19000 | 4.28 |
| 13 | 12471 | 4.10 | 190000 | 5.28 |
| 14 | 141123 | 5.15 | 1900000 | 6.28 |
| 15 | 1168734 | 6.07 | 19000000 | 7.28 |
| 16 | 95 | 1.98 | 340 | 2.53 |
| 17 | 970 | 2.99 | 3400 | 3.53 |
| 18 | 10522 | 4.02 | 34000 | 4.53 |
| 19 | 121174 | 5.08 | 340000 | 5.53 |
| 20 | 1149938 | 6.06 | 3400000 | 6.53 |
| 21 | 30 | 1.48 | 2900 | 3.46 |
| 22 | 472 | 2.67 | 29000 | 4.46 |
| 23 | 9076 | 3.96 | 290000 | 5.46 |
| 24 | 218433 | 5.34 | 2900000 | 6.46 |
| 25 | 1128907 | 6.05 | 29000000 | 7.46 |
| 26 | 55 | 1.74 | 3500 | 3.54 |
| 27 | 944 | 2.97 | 35000 | 4.54 |
| 28 | 9641 | 3.98 | 350000 | 5.54 |
| 29 | 119935 | 5.08 | 3500000 | 6.54 |
| 30 | 1219592 | 6.09 | 35000000 | 7.54 |
| 31 | 49 | 1.69 | 1500 | 3.18 |
| 32 | 1527 | 3.18 | 15000 | 4.18 |
| 33 | 11508 | 4.06 | 150000 | 5.18 |
| 34 | 236276 | 5.37 | 1500000 | 6.18 |
| 35 | 2110160 | 6.32 | 15000000 | 7.18 |
| 36 | 19 | 1.28 | 1600 | 3.20 |
| 37 | 443 | 2.65 | 16000 | 4.20 |
| 38 | 7638 | 3.88 | 160000 | 5.20 |
| 39 | 64911 | 4.81 | 1600000 | 6.20 |
| 40 | 226821 | 5.36 | 16000000 | 7.20 |
| 41 | 384 | 2.58 | 1800 | 3.26 |
| 42 | 4835 | 3.68 | 18000 | 4.26 |
| 43 | 60630 | 4.78 | 180000 | 5.26 |
| 44 | 444076 | 5.65 | 1800000 | 6.26 |
| 45 | 1864358 | 6.27 | 18000000 | 7.26 |
| 46 | 141 | 2.15 | 5000 | 3.70 |
| 47 | 2114 | 3.33 | 50000 | 4.70 |
| 48 | 27087 | 4.43 | 500000 | 5.70 |
| 49 | 290377 | 5.46 | 5000000 | 6.70 |
| 50 | 1704678 | 6.23 | 50000000 | 7.70 |
| 51 | 44 | 1.64 | 9000 | 3.95 |
| 52 | 770 | 2.89 | 90000 | 4.95 |
| 53 | 16888 | 4.23 | 900000 | 5.95 |
| 54 | 217533 | 5.34 | 9000000 | 6.95 |
| 55 | 1472439 | 6.17 | 90000000 | 7.95 |
Regretional analysis is carried out to above data, obtains mathematical model (Fig. 1):
Y＝0.9189X+1.4651
Wherein, Y is the logarithm value gathering bacteria content (CFU/mL) in liquid, and X is the logarithm value of fluorescent value (RLU); Linearly dependent coefficient R
2be 0.8762.
2, sample tests and Accuracy Verification
(1) sterilized petri dishes filling 20ml stroke-physiological saline solution is put into plankton bactgeria sampler, choose 10 sampled points and gather air to be measured respectively, 500L air (air purity of sampled point is 10000 grades) is gathered with reference to " method of testing of GB/T 16293-2010 medical industry toilet (district) flcating germ " each sampled point, and the air gathered is numbered, be numbered 1-10;
(2) 10 parts of collection liquid to be measured, get 100 μ L for every part, put into ATP fluorescence detector sample cup respectively, add 25 μ L composite fluorescence reagent, described sample cup are placed in ATP fluorescence detector and measure fluorescent value, be designated as fluorescent value to be measured;
(3) logarithm (X) of the fluorescent value described to be measured step (2) obtained substitutes in the mathematical model Y=0.9189X+1.4651 of step 1 acquisition, calculate Y value, bacteria content in this sample can be calculated according to extension rate, and then converse the quantity of planktonic bacteria in air according to stroke-physiological saline solution volume and air sampling volume.
In order to verify operability and the accuracy of this method, the present inventor has carried out following experiment: on the one hand, adopt the fluorescent value of these 10 collection points (see table 2) in the step measurements of as above (1)-(3) laboratory, and the bacteria content (CFU/mL) gathering liquid is calculated according to mathematical model Y=0.9189X+1.4651, be designated as prediction bacteria content (CFU/mL).On the other hand, the bacteria content of these 10 collection points (see table 2) in laboratory is determined respectively: the sterilized petri dishes filling 20ml stroke-physiological saline solution is put into plankton bactgeria sampler by (a) according to following classic method, gather air respectively from these 10 sampled points, gather 500L air (air purity of sampled point is 10000 grades) with reference to " method of testing of GB/T 16293-2010 medical industry toilet (district) flcating germ " each sampled point; B () measures the bacteria content (CFU/mL) gathering liquid in the plate of each sampled point after gathering respectively according to GB GB4789.2, be designated as actual measurement bacteria content (CFU/mL).
Concrete outcome data are see table 2:
10, table 2 sampled point to be measured gathers predicted value and the actual value of planktonic bacteria (CFU/mL) in liquid
Whether there is significant difference for verifying between the predicted value of mathematical model bacteria content and actual value, the data in his-and-hers watches 2 are carried out nothing and are repeated two-way analysis of variance, and result is as follows:
The nothing of table 3 pair bacteria content predicted value and actual value repeats two-way analysis of variance
As shown in Table 3, F
row=0.17 < F crit=5.12, P=0.69 > 0.05, namely the predicted value of bacteria content and actual value difference not remarkable.Therefore, prove to use ATP biloluminescence method provided by the present invention can the quantity of planktonic bacteria in Accurate Prediction air.
Claims (7)
1. detect a method for planktonic bacteria in air, comprise the steps:
(1) sterilized petri dishes filling stroke-physiological saline solution is put into plankton bactgeria sampler, gather air from some sampled points;
(2), after gathering, prior art is utilized to measure the bacteria content gathering liquid in the plate of each sampled point respectively;
(3) draw described collection liquid and put into ATP fluorescence detector sample cup, add composite fluorescence reagent, described sample cup is placed in ATP fluorescence detector and measures fluorescent value;
(4) according to the measurement result of step (2) and step (3), the typical curve equation between the logarithm value of the bacteria content of described collection liquid and the logarithm value of described fluorescent value is obtained;
(5) sterilized petri dishes filling stroke-physiological saline solution is put into described plankton bactgeria sampler, gather air to be measured; The collection liquid to be measured drawn in plate puts into described ATP fluorescence detector sample cup, adds described composite fluorescence reagent, described sample cup is placed in ATP fluorescence detector and measures fluorescent value, be designated as fluorescent value to be measured;
(6) logarithm of the fluorescent value described to be measured step (5) obtained substitutes into the described typical curve equation of step (4), calculate the bacteria content in described collection liquid to be measured, and then obtain the content of planktonic bacteria in described air to be measured.
2. method according to claim 1, is characterized in that: described composite fluorescence reagent is primarily of bacteria lysis agent and luciferase composition.
3. method according to claim 1 and 2, is characterized in that: in step (1) and step (5), the consumption of described stroke-physiological saline solution is 20mL.
4. according to described method arbitrary in claim 1-3, it is characterized in that: in step (1) and step (5), the collection capacity of described air all meets following condition: if the cleanliness factor of described air is 100 grades, then the collection capacity of described air is 1000L; If the cleanliness factor of described air is 10000 grades, then the collection capacity of described air is 500L; If more than 100000 of described air grades and 100000 grades, then the collection capacity of described air is 100L.
5., according to described method arbitrary in claim 1-4, it is characterized in that: in step (2), gather the bacteria content of liquid in the plate of each sampled point of described mensuration, measure according to GB GB4789.2-2010.
6. for detecting the suit of planktonic bacteria in air, containing plankton bactgeria sampler, ATP fluorescence detector and the instructions recording arbitrary described method in claim 1-5.
7. described in claim 6, be sleeved on the application quantitatively detected in air to be measured in planktonic bacteria content.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113403202A (en) * | 2021-05-28 | 2021-09-17 | 重庆市中药研究院 | Method for rapidly breeding gastrodia elata armillaria mellea |
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