To do an experiment on microorganisms, how do you take soil samples?

Go to a representative place to take it, then beat it with a glass ball in sterile water and coat the plate.

There are many microorganisms that can break down cellulose. There are both aerobic and anaerobic microorganisms; There are bacteria, actinomycetes, and fungi.

Aerobic cellulolytic bacteria: Fibrous spp. and Sporeus fibrous spp. are common aerobic cellulolytic bacteria found in soil. Polycystic spp., Sickle spp., and Vibrio fibrosus spp.

Many actinomycetes are able to break down cellulose. Soil actinomycetes can break down cellulose, including Streptomyces alba, Streptomyces gray, Streptomyces rubrum, etc. Actinomycetes have a weaker ability to break down cellulose than bacteria and fungi.

Many fungi have a strong ability to break down cellulolysis. Among them, there are mainly some species of Trichoderma, Fusarium, Penicillium, Aspergillus, Mucor, and Botryspora of the genus. In the litter of forests, the dominant cellulolytic bacteria are basidiomycetes.

In moist soils, fungi are also the dominant flora for cellulolysis.

Anaerobic cellulolytic microorganisms are mainly some species of Clostridium spores, such as Clostridium aussenii, and some thermophilic species that are very different from Clostridium aussenii, such as Clostridium thermofibrilla, Clostridium lyticum, etc.

Microorganisms can be isolated using the Congo red staining method.

(1) Collect plants, remove loose soil around the roots, and those that are still attached to the surface of the root system are regarded as rhizosphere soil. Place the plants in an incubator with ice packs or dry ice, protected from light conditions, and transport them back to the laboratory below 0;

2) Prepare a sterile container, place the plant root part in buffer (PBS or normal saline)**, adjust the time and intensity according to the actual situation of the sample, and wash off the rhizosphere soil;

3) The washing solution is centrifuged at high speed to collect soil sediment, if the precipitation is less, the membrane can also be filtered, and the samples sampled from the same square can be mixed at multiple points and in equal amounts;

4) Aliquot the samples into centrifuge tubes, seal them, label them with sample information, freeze them with liquid nitrogen, store them in a -80 freezer, and send them on dry ice.

To isolate a single colony microorganism from soil, the protocol is as follows:

1. Preparation of beef paste peptone medium: preparation of beef paste peptone agar medium 500ml (for 12 plates and 10 test tube bevels). Pour 12 plates and 10 test tubes into an inclined plane, bandage, and sterilize for 121 min for 20min

2. Preparation of soil diluent: weigh 10g of soil sample, put it into a triangular bottle with glass beads filled with 100ml of sterile water, shake and shake for 10min to fully mix soil and water.

Then use a pipette to aspirate 1ml from the triangular bottle (this operation requires aseptic operation), add it to another test tube containing 9ml of sterile water, mix well, and so on0001 soil solutions of different dilutions.

3. Coating culture: take two concentrations of soil dilution as the object of coating plate culture, and coat it in 3 beef paste peptone mediums, a total of 6 mediums, labeled, and cultured in 37 °C incubator for 48 h.

4. Scribing separation (scribing culture): the microbial culture medium of the two soil solutions was observed, and the two distinct bacterial traits were recorded. These two bacteria were streaked and cultured in a new medium (3 Petri dishes for each bacterial culture), labeled, and incubated at 37°C.

5. Preliminary identification: simple staining, Gram staining and spore staining were carried out on the two bacteria, and the results were recorded.

6. Test tube inclined re-culture: the two pure strains were inoculated in 5 test tubes, a total of 10 test tubes. Incubate at 37 °C (18 for 24 h).

7. Physiological and biochemical identification of the two bacteria cultured in the test tube.

1. Select representative soils according to the research design and determine the sampling sites. Understand the bioclimatic conditions of the area, determine the sampling time, and avoid sampling during the rainy season.

2. Select the area that has not been disturbed by human disturbance, and the cultivated land sample should be collected before fertilization.

3. When sampling, it is necessary to investigate and record environmental factors such as soil, biology and climate, such as topography, vegetation, soil profile structure, soil hydrothermal conditions, pH, organic matter content, etc

4. All tools, plastic bags or other items should be sterilized in advance or wiped with the soil taken during sampling.

5. The sampling procedure is as follows: remove ground vegetation and litter; Remove the topsoil about 1cm of the surface; Take soil of equal weight for mixing at multiple points, remove impurities such as gravel, and then take a certain amount of soil to bag; The sampling depth is determined according to the study design, and when sampling in layers in the same profile, the lower layer of soil samples should be taken first, and then the upper layer of soil samples should be taken after the profile is dug to avoid mixing up and down.

Soil sampling is usually done at 9 a.m., at a suitable location, with a shovel to remove the surface soil, and remove the soil 10 to 15 centimeters from the ground. Then put it in a sterile plastic bag, seal it, and mark the time, place and other basic conditions of sampling with a marker pen for later use!

1.Sampling, select the sampling location, use 5-point sampling, and add 2 centers to the four cornersSample processing, mashing, add an appropriate amount of distilled water, stir, and let stand for 30 min3Aspirate the supernatant, which is the bacterial suspension.

4.Serial dilution of the bacterial solution.

5.Coated flat plates.

6.Culture, observe colonies, count, etc.

1) This experiment is a control experiment, it is not difficult to see from the figure that the only difference between jar A and jar B is that the soil in bottle A has undergone strong heat, and the soil in bottle B has not undergone strong heat

2) Due to the presence of microorganisms in the soil of jar B, the microorganisms will respirate and produce carbon dioxide, which makes the clarified lime water in bottle B turbid; The soil in jar A is heat-treated to kill microorganisms, so no changes can be found in bottle A

3) The intent of this experiment is that if there are microorganisms in the soil, microbial respiration will produce carbon dioxide, which can make the clarified lime water turbid, which can prove the presence of microorganisms in the soil

Therefore, the answer is: (1) the soil in the test tube A is heated, and the soil in the test tube B is not heated;

2) There is no change in the test tube A, and the clarified lime water in the test tube B becomes turbid;

3) The clarified lime water became turbid, indicating that there were microorganisms in the soil that respirated and released carbon dioxide; Swift Calendar.

Generally, physiological and biochemical tests and 16SDNA tests are combined, and the first is to carry out preliminary staining of the obtained microorganisms to determine the basic morphological characteristics of microorganisms.

1. Physiological and biochemical tests.

1) Sugar fermentation and oxidation test: In the classification and identification of bacteria, the determination of sugar fermentation and oxidation is an important basis. In particular, the identification of bacteria is particularly important.

Commonly used fermented and oxidized sugars include monosaccharides, disaccharides, and polysaccharides: glucose, sucrose, starch, etc. Alcohols include pentacanol, hexacanol; Such as calendula alcohol, mannitol, etc.; Glycosides mainly include salicins and so on.

Most bacteria can use sugars as energy and carbon sources, but due to the differences in enzymes in different bacteria, the ability to ferment and oxidize sugars is different. Some can take advantage of this and can't, and some are just the opposite; Some can decompose sugars in the fermentation and oxidative metabolism of sugars, produce acid and gas, and some can only produce acid but not gas. Whether the acid is produced or not, whether it is produced by fermentation or oxidation, can be represented by an indicator (bromothymol blue aqueous solution) that is pre-added to the medium.

In this way, the medium of the strain is cultured under anaerobic or good conditions, and the medium changes from green to yellow for acid production, and whether the gas is produced is determined by observing whether there are bubbles or medium breaks in the medium, such as breaking or bubbles to prove the existence of gas.

2) Catalase assay The main basis for distinguishing lactic acid bacteria and many anaerobic bacteria from other bacteria is the determination of the presence or absence of catalase. Catalase, also known as contact enzyme, can decompose hydrogen peroxide into water and oxygen, and oxygen molecules become bubbles and run out, which is catalase positive. Lactic acid bacteria and many anaerobic bacteria are negative in the hydrogen peroxide test.

3) Cytochrome oxidase assay Cytochrome oxidase is one of the oxidases, sometimes people are accustomed to calling cytochrome oxidase oxidase, so the oxidase in the classification and identification of bacteria refers to cytochrome oxidase. In the presence of right molecular oxygen and cytochrome C, cytochrome oxidase can oxidize dimethyl-p-bendimine (aminodimethylaniline) to make it appear rose red to dark red, and on this basis, it can also be combined with A-phenol to form indolephenol blue, and blue appears.

2,16SDNA identification.

Compared with physiological and biochemical tests, 16SDNA identification is very fast and simple, through the preliminary determination of the biological species, the design of conservative PCR primers, PCR cloning of the microorganism, and the analysis of the cloned gene sequence can quickly determine which kind of microorganism is specific.