CN103969207A - Method for rapidly measuring total number of bacteria in poultry-derived pasteurella multocida culture - Google Patents

Abstract

The invention relates to a method for rapidly measuring the total number of bacteria in poultry-derived pasteurella multocida culture. The method comprises the steps of (1) preparing washing liquid; (2) sampling; (3) carrying out sample treatment; (4) detecting: measuring the outside diameter (OD) value of the treated sample at the wave length of 600nm by an ultraviolet spectrophotometer, and taking the washing liquid obtained in the step (1) as reference; and (5) calculating: when the OD value is within the range of 0.4-1.2, obtaining the number of the bacteria in the sample of (4129.4*OD600+509.31)*106 colony forming unit (CFU)/ mL; when the OD value is less than 0.4, properly concentrating the sample and measuring; when the OD value is more than 1.2, properly diluting the sample and measuring, and calculating the total number of the bacteria in the poultry-derived pasteurella multocida culture. The invention aims at providing the method capable of rapidly measuring the total number of the bacteria in the poultry-derived pasteurella multocida culture. The method has the advantages of being simple, rapid and small in error.

Description

A kind of method of total number of bacteria in Fast Measurement Pasteurella multocida culture

Technical field

The present invention relates to the method for total number of bacteria in a kind of Fast Measurement Pasteurella multocida culture.

Background technology

Cholera fowl, has another name called fowl hueppe's disease, fowl pasteurellosis or the fowl pest etc. of shaking the head, and is the contagious disease of the various birds of infringement that caused by pasteurella multocida (Pasteurella multocida).This sick epidemic season is mainly late summer and autumn, mainly by alimentary canal and respiratory tract infection.The equal susceptible of various ages in days, but be multiplely born in sexually matured bird.When the acute attack of this disease, the course of disease is very brief, death is fast, often cannot see morbidity dead.When acute attack, sick fowl hyperthermia, feather pine is random, down in spirits, a solitary a corner, closes order drowsiness.Mouthful, nasal discharge increases and causes expiratory dyspnea, shakes the head and attempt to throw away larynx mucus.Typically cuing open inspection pathology is acute sepsis, is full of the yellow exudate of transparent orange in pericardium, on heart hat fat, has blutpunkte.Liver, splenomegaly, quality becomes fragile, surface is densely covered with the downright bad point of circular canescence that a large amount of needle points are large.Intestinal bleeding, congested and hemorrhage the most serious with small intestine leading portion and colorectal mucosa, small intestine back segment and caecum are lighter.Intestinal contents is gel-shaped, and intestinal mucosa comes off, intestines aggregate nodules ring-type enlargement, hemorrhage.When chronic outbreak, sick fowl shows as and becomes thin, diarrhea, rhinitis, arthritis.The slightly visible local arthroncus of elder of the course of disease, affects the walking of disease fowl.This disease is distributed widely in all over the world, has brought huge economic loss to aviculture.

Count of bacteria is the basis of bacterium being carried out to each side research.The method of plate culture count is the most frequently used method.The principle of the method for plate culture count is that each bacterium alive can form macroscopic bacterium colony by growth under suitable nutrient culture media and better growing conditions.Can be by bacteria suspension serial dilution, get a certain amount of dilution and carry out flat board cultivation, according to the clump count of turning out, calculate the viable count in bacteria suspension.The method is applicable to the mensuration of the educable bacterium number of the work in sample, highly sensitive, higher to operator's competency profiling, and the time of expending is longer.

Summary of the invention

The object of the present invention is to provide the method for total number of bacteria in a kind of Fast Measurement Pasteurella multocida culture.

Object of the present invention is achieved through the following technical solutions: a kind of method of total number of bacteria in Fast Measurement Pasteurella multocida culture, and it comprises the following steps:

1. cleansing solution preparation: preparation is applicable to the fluid nutrient medium of Pasteurella multocida growth as cleansing solution, room temperature preservation;

2. sampling: the testing sample of getting certain volume;

3. sample preparation: by centrifugal sample 5000g 5min, sucking-off supernatant, adds and remove the isopyknic cleansing solution of sample last after supernatant, piping and druming mixes gently, the centrifugal 5min of 5000g again, sucking-off supernatant, add and remove the isopyknic cleansing solution of sample last after supernatant, piping and druming mixes gently;

4. detect: utilize ultraviolet spectrophotometer, measure the OD value of sample after treatment in the time of 600nm wavelength, taking step 1. middle cleansing solution as reference;

5. calculate: when in the scope of OD value at 0.4-1.2, sample to contain bacterium number be (4129.4 × OD ₆₀₀+ 509.31) × 10 ⁶cFU/mL, in the time of OD value < 0.4, by suitably concentrated rear mensuration of sample, in the time of OD value > 1.2, by mensuration after suitable sample dilution, and calculating contains bacterium sum.

Compared to prior art, the invention has the advantages that:

1, simple.Compared with the method for plate culture count, save the step of dilution, cultivation, artificial counting.

2, quick.Compared with the method for plate culture count, save the time of microbe growth.Fowl source killing property Pasteurella forms the obvious visible bacterium colony of naked eyes and approximately takes 16h-18h on flat board, and the method only needs 30min to obtain a result.

3, error is little.The method of plate culture count is higher to operating personnel's competency profiling, and it is larger that different personnel operate the data difference obtaining, repeatable poor.And the method utilizes ultraviolet spectrophotometer to carry out reading, favorable repeatability.

Brief description of the drawings

Fig. 1 is sample OD ₆₀₀value and the linear relationship chart that contains bacterium number.

Fig. 2 is sample segment OD in Fig. 1 ₆₀₀value and the linear relationship chart that contains bacterium number.

Embodiment

Below in conjunction with embodiment, content of the present invention is elaborated:

1, the selection of cleansing solution

The cleansing solution of conventional bacterium is selected sterile distilled water or physiological saline, but Pasteurella multocida is dead rapidly in these two kinds of liquid, so can only select the fluid nutrient medium that is applicable to Pasteurella multocida growth as martin's bouillon, LB nutrient culture media, pancreas peptone soybean broth, brain heart infusion meat soup etc.Martin's bouillon is selected in the foundation of the technical program.

2, Pasteurella multocida CVCC44801 strain (purchased from national veterinary microorganism DSMZ), streak inoculation is in Martin's agar plate, cultivate 20h for 37 DEG C, 10 single colony inoculations of picking are in 400mL martin's bouillon, in 37 DEG C of shaken cultivation casees, 200rpm shaken cultivation 5h.Now also in the exponential phase in pasteurella multocida, the viable count in nutrient solution is total bacteria count.

3, nutrient solution is taken out to water-bath 30min in 2 DEG C of-8 DEG C of cold water.

4, sample by table 1, by centrifugal sample 5000g 5min, sucking-off supernatant, adds the aseptic martin's bouillon of equal-volume, and piping and druming mixes gently.The centrifugal 5min of 5000g again, sucking-off supernatant, adds the aseptic martin's bouillon of corresponding final volume in table, and piping and druming mixes gently.Taking aseptic martin's bouillon as reference, measure different multiples dilution or concentrated after the OD of sample in the time of 600nm wavelength ₆₀₀value, each sample is surveyed 3 times, records its mean value.

The dilution of table 1 nutrient solution or concentrated

5, sterile sampling 1mL, is carried out 10 times of serial dilutions with martin's bouillon, gets 10 ^-6, 10 ^-7, 10 ^-8dilution bacterium liquid paving Martin agar plate, 3 flat boards of each dilutability paving, each dull and stereotyped consumption is 200 μ L, 16h-18h after cultivating in 37 DEG C of incubators, read the clump count on flat board, same dilution 3 blocks of plates are taken the mean, and choose the dilutability of clump count between 30-300 in table 2 10 ^-6dilutability, the viable count of extrapolating every milliliter of nutrient solution is 1105 × 10 ⁶cFU/mL.

Table 2 is cultivated the clump count of rear each flat board

6, with OD ₆₀₀value is horizontal ordinate, to contain bacterium number divided by 10 ⁶value be ordinate, curve plotting, is shown in Fig. 1, linearly dependent coefficient is 0.9934.In the time that OD600 value is between 0.402 and 1.210, linearly dependent coefficient is 0.9989, sees Fig. 2, and confidence level is higher.

7, conclusion: work as OD ₆₀₀value is between 0.402 and 1.210 time, sample containing bacterium number and OD ₆₀₀the linearly dependent coefficient of value is 0.9989, and regression equation is y=4129.4x+509.31, and sample is (4129.4 × OD containing bacterium number ₆₀₀+ 509.31) × 10 ⁶cFU/mL.As the OD of sample ₆₀₀while being worth not between 0.402 and 1.210, for guaranteeing to contain the accurate of bacterium number, can not directly utilize this formula to calculate.As the OD of sample ₆₀₀when < 0.4, sample is suitably concentrated; As the OD of sample ₆₀₀when value > 1.2, sample is suitably diluted, after measuring, calculate containing bacterium number.

Embodiment 1:

Pasteurella multocida CVCC44801 strain (purchased from national veterinary microorganism DSMZ), streak inoculation, in Martin's agar plate, is cultivated 20h for 37 DEG C, and the single colony inoculation of picking is in 20mL martin's bouillon, add 50% glucose injection 2mL, cultivate 15h for 37 DEG C.Measure in this nutrient solution containing the concrete operation method of bacterium number as follows:

1, sampling: get respectively 4mL nutrient solution and add in two 5mL EP pipes.

2, sample preparation: open TGL-15B type hydro-extractor (Anting Scientific Instrument Factory, Shanghai), two EP pipe symmetries are put into, the centrifugal 5min of 5000g, sucking-off supernatant, discards.Add the aseptic martin's bouillon of 4mL, piping and druming mixes gently.The centrifugal 5min of 5000g, sucking-off supernatant, discards.Add the aseptic martin's bouillon of 4mL, piping and druming mixes gently.

3, detect: open UV1600 type ultraviolet spectrophotometer (Beijing Rayleigh Analytical Instrument Co.,Ltd), adjusting wavelength value is 600nm.Draw aseptic martin's bouillon 3mL in glass cuvette, put into reference position, draw sample 3mL after treatment in glass cuvette, put into sample position, record OD ₆₀₀value is 0.648.

4, calculate: OD ₆₀₀value, between 0.40 and 1.20, is directly pressed formula (4129.4 × OD ₆₀₀+ 509.31) × 10 ⁶cFU/mL calculates, and the content that obtains the Pasteurella multocida of this nutrient solution is 3.185 × 10 ⁹cFU/mL.

Embodiment 2:

Pasteurella multocida CVCC44801 strain (purchased from national veterinary microorganism DSMZ), streak inoculation, in Martin's agar plate, is cultivated 20h for 37 DEG C, washes lower bacterium colony with 8.5%NaCl, makes containing bacterium suspension.Measure in this suspension containing the concrete operation method of bacterium number as follows:

1, sampling: get respectively 4mL suspension and add in two 5mL EP pipes.

2, sample preparation: open TGL-15B type hydro-extractor (Anting Scientific Instrument Factory, Shanghai), two EP pipe symmetries are put into, the centrifugal 5min of 5000g, sucking-off supernatant, discards.Add the aseptic martin's bouillon of 4mL, piping and druming mixes gently.The centrifugal 5min of 5000g, sucking-off supernatant, discards.Add the aseptic martin's bouillon of 4mL, piping and druming mixes gently.

3, detect: open UV1600 type ultraviolet spectrophotometer (Beijing Rayleigh Analytical Instrument Co.,Ltd), adjusting wavelength value is 600nm.Draw aseptic martin's bouillon 3mL in glass cuvette, put into reference position, draw sample 3mL after treatment in glass cuvette, put into sample position, record OD ₆₀₀value is 1.834.

4, sample is processed and is detected: because OD ₆₀₀value > 1.2, then process.Draw aseptic martin's bouillon 1.5mL in glass cuvette, then draw sample 1.5mL after treatment and mix in same glass cuvette, 2 times of dilutions, put into sample position, record OD ₆₀₀value is 1.016.

5, calculate: first press formula (4129.4 × OD ₆₀₀+ 509.31) × 10 ⁶cFU/mL calculates, and obtains 4.7 × 10 ⁹cFU/mL, then be multiplied by extension rate 2, the Pasteurella multocida content of this suspension is 9.4 × 10 ⁹cFU/mL.

Embodiment 3:

Pasteurella multocida CVCC44801 strain (purchased from national veterinary microorganism DSMZ), streak inoculation, in Martin's agar plate, is cultivated 20h for 37 DEG C, and the single colony inoculation of picking is in 20mL martin's bouillon, add 50% glucose injection 10mL, cultivate 6h for 37 DEG C.Measure in this nutrient solution containing the concrete operation method of bacterium number as follows:

1, sampling: get respectively 4mL nutrient solution and add in two 5mL EP pipes.

2, sample preparation: open TGL-15B type hydro-extractor (Anting Scientific Instrument Factory, Shanghai), two EP pipe symmetries are put into, the centrifugal 5min of 5000g, sucking-off supernatant, discards.Add the aseptic martin's bouillon of 4mL, piping and druming mixes gently.The centrifugal 5min of 5000g, sucking-off supernatant, discards.Add the aseptic martin's bouillon of 4mL, piping and druming mixes gently.

3, detect: open UV1600 type ultraviolet spectrophotometer (Beijing Rayleigh Analytical Instrument Co.,Ltd), adjusting wavelength value is 600nm.Draw aseptic martin's bouillon 3mL in glass cuvette, put into reference position, draw sample 3mL after treatment in glass cuvette, put into sample position, record OD ₆₀₀value is 0.103.

4, sample is processed and is detected: because OD ₆₀₀value < 0.4, then process.Get respectively 4mL nutrient solution and add in 8 5mL EP pipes, symmetry is put into hydro-extractor, the centrifugal 5min of 5000g, and sucking-off supernatant, discards.Respectively add the aseptic martin's bouillon of 4mL, piping and druming mixes gently, the centrifugal 5min of 5000g, and sucking-off supernatant, discards.By the aseptic martin's bouillon Eddy diffusion of 3.2mL for the bacterium mud of 8 pipes, draw sample 3mL after treatment in glass cuvette, put into sample position, record OD ₆₀₀value is 0.983.

5, calculate: first press formula (4129.4 × OD ₆₀₀+ 509.31) × 10 ⁶cFU/mL calculates, and obtains 4.6 × 10 ⁹cFU/mL, then divided by cycles of concentration 10, the Pasteurella multocida content of this suspension is 4.6 × 10 ⁸cFU/mL.

Claims (1)

1. a method for total number of bacteria in Fast Measurement Pasteurella multocida culture, is characterized in that, it comprises the following steps:

1. cleansing solution preparation: preparation is applicable to the fluid nutrient medium of Pasteurella multocida growth as cleansing solution, room temperature preservation;

2. sampling: the testing sample of getting certain volume;

3. sample preparation: by centrifugal sample 5000g 5min, sucking-off supernatant, add with step 2. the isopyknic cleansing solution of the testing sample of getting, piping and druming mixes gently, the centrifugal 5min of 5000g again, sucking-off supernatant, add again with step 2. the isopyknic cleansing solution of the testing sample of getting, gently piping and druming mix;

4. detect: utilize ultraviolet spectrophotometer, measure the OD value of sample after treatment in the time of 600nm wavelength, taking step 1. middle cleansing solution as reference;

5. calculate: when in the scope of OD value at 0.4-1.2, sample to contain bacterium number be (4129.4 × OD ₆₀₀+ 509.31) × 10 ⁶cFU/mL, in the time of OD value < 0.4, by suitably concentrated rear mensuration of sample, in the time of OD value > 1.2, by mensuration after suitable sample dilution, and calculating contains bacterium sum.