Separation and purification experiments of microorganisms
Separation and purification experiments of microorganisms
The vast majority of different species of microorganisms live in mixed company in soil, water, air, or in human and animal and plant bodies, (1) for cell isolation (2) for cell purification (3) for cell proliferation culture.
Operation method
Isolation and purification of microorganisms
Principle
In order to obtain the pure culture of a certain microorganism, generally according to the microorganism on nutrition, pH, oxygen and other conditions require different, and supply it with suitable culture conditions, or add some kind of inhibitor to cause only conducive to the growth of this bacterium, but inhibit the growth of other bacteria environment, so as to eliminate some other unwanted microorganisms, and then dilute smear plate method or dilute mixing plate method or plate delineation separation method to separate, purify the microorganisms until the pure strain is obtained. microorganisms until a pure strain is obtained. Soil is the base camp of microorganisms, and the microorganisms living here are extremely diverse in number and species, therefore, soil is an important base for us to develop and utilize microbial resources, and we can isolate and purify a lot of useful strains from it.
Materials and Instruments
Soil Sample Move 1. Dilution-coated plate method Caveat 1. During inoculation, it is necessary to operate next to an alcohol lamp in order to maintain a relatively sterile environment. The inoculation ring and test tube spout should not be left arbitrarily on the table or in contact with other objects. 2. The plate or slant needs to be scratched quickly and gently, without scratching the medium. 3. After inoculation, pull out the inoculation ring, cauterize the mouth of the tube, and put on the silicone plug. Do not meet the silicone plug with the mouth of the test tube when putting on the silicone plug, so as not to incorporate unclean air into the test tube in the flowing air. 4. When doing plate partitioning and delineation separation, after delineating a plot, uniformly and thoroughly cauterize the inoculation ring and sterilize it, and then delineate the next plot after it cools down. 5. All culture vessels need to be strictly sterilized. Common Problems 1. A colony is a group of daughter cells visible to the naked eye formed by the growth and multiplication of a single or a few cells on the surface of a solid culture medium or inside it. The morphology of the colony may vary to different degrees depending on the strain or the same strain but with different conditions such as medium, incubation temperature, pH value and time. If the culture medium, culture temperature and time conditions are the same, the colony morphology formed by the same bacteria has relative stability and uniformity. 2. 2. The determination of pure culture in addition to the observation of its colony characteristics, but also combined with the microscope to detect the individual morphological characteristics can be decided, some microorganisms pure culture after a series of isolation and purification process and the identification of diversity characteristics can be determined. For more product details, please visit Aladdin Scientific website.
Koch 1 agar medium Peptone agar medium Martin's agar medium Phenol Streptomycin
Triangular flasks Test tubes Glass beads Sterile glass applicators Sterile pipettes Inoculation rings
(1) Pour the plate will be meat paste peptone medium, Gao's No. 1 agar medium, Martin's agar medium dissolved, to be cold to 55 ~ 60 ℃, to the Gao's No. 1 agar medium to add a few drops of 10% phenol, to the Martin's medium to add streptomycin solution, so that each milliliter of medium containing streptomycin 30 ug.
Then pour the plate respectively, each kind of medium poured three dishes, the method is to hold the test tube or triangular flask with medium in the right hand, set next to the flame, the left hand take the flat dish and loosen the test tube plug or bottle stopper, use the edge of the palm and the pinky finger, ring finger to clamp and pull out, if the test tube or triangular flask in the medium is available at one time, then the tube stopper or bottle stopper do not need to be clamped in the finger.
The mouth of the test tube (bottle) is sterilized on the flame, and then the petri dish lid is opened with the left hand near the flame to open a slit, and the medium is quickly poured into about 15 ml, and the lid is gently shaken to distribute the medium evenly, and then the petri dish is placed flat on the tabletop to be congealed to form a flat plate. Can also be placed on the tabletop near the flame, with the index finger and middle finger of the left hand to clamp the tube plug and open the petri dish, and then injected into the culture medium, shaking well to make a plate. It is best to put the plate at room temperature for 2-3 days, or 37 ℃ culture for 24 hours, check the absence of bacterial droplets and the lid of the dish without condensation before use.
(2) Prepare soil diluent by weighing 10 g of soil sample into a triangular flask containing 90 ml of sterile water with glass beads, and shaking for about 20 minutes to mix the soil sample with the water sufficiently to disperse the bacteria. A 1 ml sterile pipette was used to draw up 1 ml of the soil suspension into a test tube containing 9 ml of sterile water and blown three times to mix well. Then a 1 ml sterile pipette is used to inject 1 ml of the soil suspension into a test tube containing 9 ml of sterile water, and so on to make 10-1, 10-2, 10-3, 10-4, 10-5, 10-6 soil solutions at various dilutions.
(3) Spread the three plates of each culture medium with a marker on the bottom surface of 10-4, 10-5 and 10-6 three dilutions, and then use three 1 ml sterile pipettes to suck 0.2 ml of soil dilution from 10-4, 10-5 and 10-6 tubes respectively, and put them into the plates with written dilutions, and then gently spread evenly on the surface of the medium with the sterile glass coating rod.
(4) Cultivate the plates of Gao's No.1 medium and Martin's medium by placing them upside down in the greenhouse at 28℃ for 3~5 days, and the plates of meat paste peptone by placing them upside down in the greenhouse at 37℃ for 2~3 days.
(5) pick bacteria will be cultured after the growth of a single colony were picked and inoculated to the above three media on the slant, were placed at 28 ℃ and 37 ℃ greenhouse culture, to be the bacterial moss grows, check whether the moss is simple, but also can be used to microscope smear staining to check whether a single micro-organisms, if there is a mix of other stray bacteria, it is necessary to once again for the isolation, purification, until the acquisition of a pure culture.
2. Dilution mixed plate methodThis method is basically the same as the dilution of coated plate method, aseptic operation is the same, the difference is that the first to draw 0.5 ml 10-4, 10-5, 10-6 dilution of soil suspension into the flat plate, and then poured into the melted and cooled to about 45 ℃ culture medium, pouring the edge of the shaking, so that the microorganisms in the samples are mixed evenly with the medium, to be condensed into a flat plate, were inverted in the 28 ℃ and After condensing into a plate, it was placed in the greenhouse at 28℃ and 37℃ for culture, and then single colonies were picked until pure culture was obtained.
3. Plate delineation isolation method
(1) Pour the plate according to the method of dilution coated plate, and mark the name of the culture medium with a marker.
(2) Scribe the line in the near flame, the left hand to take the bottom of the dish, the right hand to take the inoculation ring, pick the above 10-1 of the soil suspension of a ring on the plate line. There are many ways to draw a line, but no matter which method to draw a line, its purpose is to dilute the sample on the plate by drawing a line, so that the formation of a single colony.
The following two methods of delineation are commonly used:
① use the inoculation ring to aseptically pick a ring of soil suspension, the first side of the plate medium for the first parallel line 3~4, and then rotate the Petri dish at an angle of about 70 degrees, and will be inoculated with the ring on the remainder of the burning, to be cooled down through the first part of the parallel line for the second parallel line, and then with the same method through the second part of the parallel line for the third parallel line and through the third part of the parallel line for the fourth parallel line. Scribe. After the lines were drawn, the dish was covered and placed upside down in the greenhouse for incubation.
(ii) The inoculation loop with the sample picked out is continuously scribed on the plate culture medium. After the line is drawn, cover the dish with a lid and place it upside down in the greenhouse for incubation.
(3) Pick the bacteria in the same way as the diluted smear plate method, until the bacteria are pure.