Screening for the separation of bacteria produced by industrial microorganisms (III)

the concentration of
culture
after the sample, the purpose
microbials
to be multiplyed, dominant, other types of microorganisms in the number of relatively reduced, but did not die. There are still a variety of microorganisms mixed in the rich culture, even in the dominant class of microorganisms, is not purebred. For example, the same group of fat enzymes with grease as the carbon source produce bacteria, some bacteria, some mold, some spores, some do not produce spores, some have strong production capacity, some weak production capacity and so on. Therefore, after the rich culture of the sample, also need to be further through separation purification, the most needed strains directly separated from the sample.
I. The separation of gas microorganisms
there are many methods of separation, can be broadly divided into two categories: one is more extensive, can only achieve "pure bacteria", such as dilution coating method, line separation method, tissue separation method. The first two methods are applied more in industrial production because they are simple and effective. The method of tissue separation is usually to isolate strains from diseased or special tissues. The other is a finer single-cell or single-spore separation method, which can reach the level of "pure strain" or "pure cell". Such methods require specialized
-instrument equipment
, complex such as micro-operation devices, simple use of petri dishes or concave slides as separation chambers for separation. The following separation purification methods are introduced separately.
(i) Dilution coating method
the soil sample to ten times the grade difference, diluted with sterile water, take a certain amount of dilution of the suspension, applied to the separation of
frate
plate, after culture, grow a single bacterios, pick the required bacterios moved to the sloped culture base culture. The degree of dilution of soil samples depends on the number of bacteria in the sample, generally high organic matter content of vegetable garden soil, etc. , the sample contains a large amount of bacteria, the dilution multiplication is higher, and the opposite dilution multiplication is lower. Using this method, there is a greater chance of obtaining single bacterios on the flat plate culture base, which is especially suitable for isolating microorganisms that are prone to spread.
(ii) Line separation method
take part of the sample or bacteria with the inoculation ring, draw a line on a pre-prepared media plate, when a single bacterios grow out, move the droplets into the sloped media, culture after standby. The separation method is simple, fast and effective.
In cases where the sample contains less bacteria or a small number of microorganisms for a purpose, the thoroughbred separation method of the microorganism can be simplified as follows: The first method is to take a crude test tube or small triangular bottle containing 3 to 5 ml of sterile water, and take a few mixed samples. (0.5g or so) into it, fully oscillating dispersion, with sterilized dropper to take a drop of soil suspension on the
gascide
plate coating culture, or with inoculation ring to line culture on the plate. This method does not require a bacterial count and is simpler than the conventional dilution method above. The second method, take air-dried powdered soil samples a little (tens of milligrams) directly sprinkled on the selective separation medium plate or mixed into the medium to make a plate, set the temperature culture for a certain period of time, grow out of the bacteria. For example, the separation of small monosporidium bacteria can be used in this method, sampling from the river mud, air-dried and crushed, sample powder 20 to 50 mg directly added to the Tianmen dongamide medium, mixed evenly into a flat plate, cultured after the growth of fish ovary-like bacteria. This method is sometimes not sufficiently separated and can be further purified by dashing.
(iii) separation by using the
bio-chemical
reaction of flat dishes
which is a method of initial separation of a large number of mixed microorganisms using a special separation culture. The separation culture base is designed according to the specific physiological characteristics of the intended microorganism or the bio-chemical reaction of certain metabolites. By observing the growth of microorganisms on selective cultures or the separation of bio-chemical reactions, the efficiency of strain separation and purification can be significantly improved.
1. The transparent
method allows poorly soluble substrates to be added to the plate medium to cloud the medium. Microorganisms that break down the substrate will produce transparent rings around the sphere, the size of which initially reflects the strain's ability to utilize the substrate. The method is used to isolate hydrolyzed enzymes to produce bacteria, such as lipase, amylase, protease, nuclease-producing bacteria will contain substrates on the selective culture base plate to form a transparent circle visible to the naked eye. In the separation of amylase to produce bacteria, the culture base to starch as the only carbon source, to be coated to the sample to the plate, after culture to form a single bacteria backward, and then with iodine immersion, according to whether there is a transparent hydrolytic circle around the bacteria to distinguish the production of enzyme strains. If you want to isolate the acid hydrolyzed enzyme to produce bacteria, you can use the double-layer plate method, first of all, on the ordinary plate culture base to apply the suspension to culture, such as long-form bacteria behind cover a layer of nutrient agar, containing 3%
yeast
RNA, 0 .7% agar and 0.1mol/L
EDTA
, pH7.0, cultured at about 42 degrees C 2 to 4h, around the production of transparent circles of bacteria, that is, nucleic acid decomposition enzymes to produce bacteria.
when separating a strain that produces organic acids, a transparent ring method is also commonly used for primary screening. Add calcium carbonate to the selective medium, make the plate mixed, apply the sample suspension to the plate for culture, because the bacteria can be around the calcium carbonate hydrolysis, forming a clear transparent circle, can be easily identified. When the lactic acid is isolated to produce bacteria, because lactic acid is a strong organic acid, calcium carbonate added to the medium not only has a identifying effect, but also acid-in-the-middle effect.