CN102321729A - Fluorescent microscopic counting method for detecting bacterial count in soil and sediment - Google Patents

Abstract

The invention relates to a fluorescent microscopic counting method for detecting the number of bacteria in soil and sediment, which belongs to the field of environmental microbiology. The detection method comprises the following steps: (1) preparing a sample diluent and an anti-fading agent; (2) adding the sample to the sample diluent for processing to prepare a sample counting fluid. Take the sample counting solution into a centrifuge tube, add 10× SYBR Green I fluorescent dye, and add anti-fading agent after dark staining to make a stained sample; (3) Cover the clean cover glass on a clean hemocytometer for counting On the chamber, vortex to mix the stained sample, take the stained sample with a pipette, add the sample along the edge of the coverslip, and make the stained sample fill the counting chamber. Let the loaded hemocytometer stand for 5 minutes, place it on the stage of a fluorescence microscope, excite it with a light wave of 480 nm, observe it under a 40 times objective lens, and count the number of bacterial particles in the 5 squares. Finally, the number of bacteria in 1 g of the sample is calculated according to the formula Y=31.25×A×10 ⁶ , wherein Y represents the number of bacteria in 1 g of the sample, and A represents the average number of bacteria in a middle grid. The method is time-saving, labor-saving, low-cost, anti-fading, and can quickly and accurately detect the number of bacteria in soil and sediment.

Description

A fluorescence microscopic counting method for the detection of bacterial populations in soil and sediment

technical field

The invention belongs to the field of environmental microbiology and relates to a detection technology for the total number of bacteria in the environment, in particular to a reagent combination and detection method for sample pretreatment and fluorescence microscopic observation of the total number of bacteria in soil and sediments.

Background technique

Fluorescent staining direct counting method is a new method for bacterial counting developed in the last 30 years. It has the characteristics of rapidity and accuracy, and is widely used in the determination of bacterial counts in environmental samples such as water samples, soil and sediments. In 1973, Francisco et al first established a fluorescent microscope staining and counting method based on the AO staining system for the determination of the total number of bacteria in natural water bodies. In 1980, Porter et al. began to use DAPI to stain bacteria. On the basis of Porter et al., Velji et al. used ultrasonic waves to pre-treat bacteria and seaweed samples in seawater and sediments, and then counted them by DAPI staining method, and achieved good results. Lunau et al. used SYBR Green I fluorescent dye staining method to measure the number of bacteria in seawater and sediments, and compared with DAPI and AO staining and counting, they found that SYBR Green I had higher specificity in binding to DNA, and the effect of staining and counting was better.

In the traditional membrane method to count the total number of bacteria in soil or sediment, the pretreatment of samples is very important. Because the soil or sediment contains a large number of bacteria combined with matrix particles, the distribution is extremely uneven, and some parts may be affected by the absorption of fluorescent dyes by opaque mineral particles, making the imaging under the fluorescence microscope not obvious, and due to microbial cells. The extracellular polymers (extracellular polymeric substances, EPS) are tightly combined with the particles to form aggregates, so they may not be counted. In order to obtain accurate measurement results, the separation of bacteria and matrix particles has become the key technology for the pretreatment of membrane fluorescence microscope observation and determination. These separation methods are mainly divided into physical methods (including centrifugal sedimentation, oscillation, ultrasonic treatment, dilution, etc.), chemical methods (using sodium pyrophosphate, surfactants, methanol, and other dispersants) and enzymatic treatment methods (EPS enzyme). The current common practice is to combine several pretreatment methods to make up for the deficiency of a single treatment method. But in general, the pretreatment method that meets the requirements of membrane fluorescence microscopic counting is time-consuming and laborious, and is not suitable for the rapid detection of a large number of samples. For example, Amalfitano et al. used the chemical method of sodium pyrophosphate and polysorbate treatment and the physical method of shaking and ultrasonic treatment combined with density gradient centrifugation to treat the samples when separating bacteria in riverbed sediments, although about 93% of the bacterial cells were eventually destroyed Recovery, but the entire pre-treatment takes 2.5 hours.

There are two types of filter membranes used in traditional membrane detection: organic and inorganic. Organic filter membranes have been used earlier and are relatively cheap, but the filter membranes have to be dyed, which is time-consuming and not suitable for rapid detection, such as the one used in the patent "Fluorescence Microscopic Observation and Counting Method of Live Bacteria in a Non-Cultivable State" by Li Ying et al. The filter membrane must be hydrophobically treated, dyed and dried before it can be used for fluorescence microscopy. The commonly used filter membrane is alumina inorganic filter membrane. Inorganic membranes do not need to be dyed and are easy to use, but they are more expensive. In addition, the anti-fading treatment of the membrane method is carried out on the membrane. The main disadvantage of this treatment process is that the amount of anti-fading agent attached to the film is small, the anti-fading effect is poor, and the fluorescence of the dye is quenched quickly, which is not conducive to observation and counting. .

Contents of the invention

The invention aims to overcome the defects of the prior art and provide a method which saves time and labor, is low in cost, has better anti-fading effect, and can quickly and accurately detect the number of bacteria in soil and sediment.

The present invention is realized through the following technical solutions:

(1) Buffer preparation: Weigh 0.446g sodium pyrophosphate and 0.15g EDTA, dissolve in 100ml sterile distilled water to make sample dilution; weigh 0.1g p-phenylenediamine, dissolve in 1ml PBS-glycerol buffer In, make anti-fading agent.

(2) Sample pretreatment: Weigh 0.1 g of mud sample into a sterile Eppendolf tube, add 1 ml of sample diluent, and vortex for 5 minutes to make a uniform suspension. Take 0.1ml of the suspension into 0.9ml of the sample diluent, vortex for 5 minutes, and prepare the sample counting solution. Take 80 μl of sample counting solution into a 200 μl centrifuge tube, add 10 μl of 10× SYBR Green I fluorescent dye, stain in the dark for 1 min, and then add 10 μl of anti-fading agent to prepare a stained sample.

(3) Fluorescence microscopic counting: Cover the clean coverslip on the counting chamber of a clean hemocytometer, vortex and mix the stained sample, take 20 μl of the stained sample with a pipette, add the sample along the edge of the coverslip, and make The stained sample fills the counting chamber. Let the loaded hemocytometer stand for 5 minutes, place it on the stage of a fluorescence microscope, excite it with a light wave of 480 nm, observe it under a 40 times objective lens, and count the number of bacterial particles in the 5 squares. Finally, the number of bacteria in 1 g of the sample is calculated according to the formula Y=31.25×A×10 ⁶ , wherein Y represents the number of bacteria in 1 g of the sample, and A represents the average number of bacteria in a middle grid.

The main advantages of the present invention are:

1. The present invention does not require cumbersome sample pretreatment work, greatly shortens sample pretreatment time, improves detection efficiency, saves time and labor compared with traditional fluorescence microscopy methods, and can quickly and accurately detect the number of bacteria in soil and sediment.

2. The present invention does not need to use expensive inorganic filter membranes in the detection process, which reduces the detection cost.

3. The present invention changes the anti-fading treatment method and significantly improves the anti-fading effect.

Description of drawings

Figure 1: It is the effect diagram of the stained sediment bacteria observed through the 40X objective lens in the hemocytometer.

Figure 2: The total number of bacteria in the pond sediment obtained by the fluorescent microscopic counting method of the hemocytometer.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited.

The comparison of three kinds of counting methods of embodiment 1

Escherichia coli DH5α (gifted by Teacher Yang Jiaoyan of Central China Normal University) was cultivated in LB liquid medium until the absorbance value at 600 nm was 0.2-0.3, and diluted with 0.9% normal saline.

Plate counting method: select an appropriate dilution gradient, draw 0.2 ml of coating plate, so that the number of colonies growing on each plate is between 30 and 300. After culturing at 37°C for 36 hours, count the number of colonies on the plate, and calculate the concentration of the original bacterial solution based on the number of colonies. The number of bacteria per milliliter of bacterial liquid = the average number of colonies repeated three times at the same dilution × the dilution factor.

Hemocytometer fluorescent microscopic counting method: Take 80 μl of the diluted sample into a 200 μl centrifuge tube, add 10 μl of 10× SYBR Green I fluorescent dye, and add 10 μl of anti-fading agent after staining for 1 minute. Cover the clean cover glass on the clean counting chamber of the hemocytometer, vortex to mix the stained sample, take 20 μl of the stained sample with a pipette, add the sample along the edge of the coverslip, and make the stained sample fill the counting chamber. Let the loaded hemocytometer stand for 5 minutes, put it on the stage of a fluorescence microscope, excite it with 480nm light wave, and observe it under a 40x objective lens. When counting, use a counting board with 25 squares, and count the 5 middle squares in the upper left, lower left, upper right, lower right and middle according to the diagonal orientation. The number of bacteria per milliliter of bacterial liquid is according to the formula Y=31.25×A×C×10 ⁴ , where Y is the number of bacteria per milliliter of bacterial liquid, A is the average number of bacteria per grid, and C is the dilution factor.

Filter fluorescence microscopic counting method: Take 80 μl of an appropriately diluted sample, stain and anti-fade the bacteria as described in the hemocytometer method, then dilute to 25ml with sterile normal saline, and finally filter through a 0.22 μm inorganic filter membrane . The filter was placed on a glass slide, excited by a 480nm light wave under a fluorescence microscope, observed under a 40x objective lens, and the number of bacterial particles in 5 visual fields was randomly counted. The number of bacteria per milliliter of bacterial liquid is calculated according to the formula Y=1.25×P×K×C, where Y is the number of bacteria per milliliter of bacterial liquid, P is the average number of bacteria in each field of view, K is the number of fields of view of the filter membrane, and C is Dilution factor.

The above three methods were used to count the three DH5α cultures, and the results are shown in Table 1. The results prove that the detection rate of the hemocytometer fluorescence microscopic counting method is higher than the other two methods.

Table 1. Comparison of 3 counting methods for counting Escherichia coli DH5α

Example 2 Investigation of total number of bacteria in Chonghu fishery pond sediment

In October 2010, 200g of surface sediment was excavated from the center of 21 ponds in Chonghu Fishery, packed into sterile ziplock bags, and transported back to the laboratory in a refrigerated incubator. Use a 1.5ml centrifuge tube to weigh 0.1g of different sediment samples, add 1ml sodium pyrophosphate-EDTA buffer, vortex for 5min, take 0.1ml suspension, add it to 0.9ml sodium pyrophosphate-EDTA buffer, and then Vortex for 5 minutes. Take 80 μl of the diluted suspension into a 200 μl centrifuge tube, add 10 μl of 10× SYBR Green I dye, stain in the dark for 1 min, and then add 10 μl of anti-fading agent. Cover the clean coverslip on the counting chamber of a clean hemocytometer, vortex to mix the stained sample, take 20 μl of the stained sample with a pipette, add the sample along the edge of the coverslip, and make the stained sample fill the counting chamber. The added hemocytometer was left to stand for 5 minutes, placed on the stage of a fluorescence microscope, excited with 480nm light waves, observed and counted under a 40x objective lens. When counting, use a counting board with 25 squares, and count the 5 middle squares in the upper left, lower left, upper right, lower right and middle according to the diagonal orientation. The number of bacteria per gram of sample is according to the formula Y=31.25×A×10 ⁶ , where Y is the number of bacteria per gram of sample, and A is the average number of bacteria per grid. The effect of the stained sediment bacteria observed through a 40X objective lens in the hemocytometer is shown in Figure 1. The counting results are shown in Figure 2. The bacterial counts in the sediments of the 24 fish ponds were basically on the order of 10 ⁸ cell/g, but there were still significant differences in the counts among different ponds.

Claims (4)

1. fluorescence microscopy method of counting that detects bacterial number in soil and the settling is characterized in that may further comprise the steps:

(1) preparation of damping fluid: be dissolved in the sterile distilled water with trisodium phosphate and EDTA, be mixed with sample diluting liquid; Be dissolved in the PBS-glycerine damping fluid with Ursol D, be mixed with anti-decolourant;

(2) sample pre-treatments: take by weighing 0.1g mud appearance in aseptic Eppendolf pipe, add the 1ml sample diluting liquid, vortex vibration 5min; Process even suspension liquid, get the 0.1ml suspension liquid in the 0.9ml sample diluting liquid, vortex vibration 5min; Process sample counting liquid, get 80 μ l samples counting liquid in the centrifuge tube of 200 μ l, add 10 μ l 10 * SYBR Green I optical dye; Add the anti-decolourant of 10 μ l behind the lucifuge dyeing 1min, process stained specimens;

(3) fluorescence microscopy counting: the deckglass of cleaning is covered on clean blood counting chamber nucleonics, and vortex mixing stained specimens is got 20 μ l stained specimens with pipettor; The application of sample along the deckglass edge makes stained specimens be full of nucleonics, and the blood counting chamber of application of sample is left standstill 5min; Put on the fluorescent microscope Stage microscope, excite with the 480nm light wave, 40 times of object lens are observed down; Count the quantity of bacteria particles in 5 medium squares, at last Y=31.25 * A * 10 by formula ⁶Calculate number of bacteria in the 1g sample, wherein Y representes number of bacteria in the 1g sample, and A representes the mean number of bacterium in the medium square.

2. a kind of fluorescence microscopy method of counting that detects bacterial number in soil and the settling as claimed in claim 1 is characterized in that said sample diluting liquid is with 0.446g trisodium phosphate and 0.15g EDTA, is dissolved in the 100ml sterile distilled water formulated.

3. a kind of fluorescence microscopy method of counting that detects bacterial number in soil and the settling as claimed in claim 1 is characterized in that said anti-decolourant is dissolved in the 1ml PBS-glycerine damping fluid 0.1g Ursol D formulated.

4. a kind of fluorescence microscopy method of counting that detects bacterial number in soil and the settling as claimed in claim 1, the application of its bacterial number in detecting soil and settling.

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