Vaccination, separation and culture of microorganisms

, the purpose requires
1, master a variety of separation, vaccination methods.
2, master the basic link of sterile operation., the experiment shows thatmicroorganisms
exist in a mixed state in nature, in order to obtain the desired species, it is necessary to separate them. Accidental contamination when preserving strains also needs to be purified. There are many methods of microbial separation and purification, but the basic principles are similar, the sample to be separated to be diluted, and the microbial cells (or spores) as far as possible in a dispersed state, and then make it grow into a purebred monobacteria. However, the above work can not be separated from inoculation, the process of moving one microorganism to another sterilized
culture
the base., experimental materials
1,
thermostat
culture box.
2, vaccination rings, glass rods, straws, alcohol lamps.
3,
culture base
(the last experiment preparation).
4, E. coli, Staphylococcus aureus, Staphylococcus aureus,
and
bacteria.4, method step
(i) vaccination operation
1, slope vaccination (Staphylococcus austa)
(1) before the operation, first wipe hands with 75% alcohol, wait for alcohol evaporation after lighting the alcohol lamp.
(2) holds the strain tube and the slope between the left thumb and the other four fingers, so that the slope and the bacterial side up and in a horizontal position.
(3) rotate the strain and beveled cotton plugs first so that they can be easily pulled out when inoculated.
(4) take the inoculation ring in your left hand (as if you were holding a pen) and burn the ring end red on the flame to sterilise, and then spread the possibility of reaching into the rest of the test tube.
(5) Use the ring finger, little finger and palm of the right hand to pull out the bacterial tube and the cotton plug or test tube cap of the waiting oblique interview tube at the same time, and then let the test tube mouth slowly over-fire sterilization (do not burn too hot).
(6) Extend the burnt inoculation ring into the strain tube, touch the inoculation ring on the inner wall of the test tube or on the culture base of the unfilled moss, let it cool down sufficiently, then gently scrape a little moss, and then extract the inoculation ring from the strain tube.
(7) quickly extends the bacteria-stained vaccination ring into another waiting interview tube. Make a dense back-and-forth dash from the bottom of the bevel to the "Z" shape. Sometimes it is also possible to use a vaccination needle to pull only one line in the center of the culture for slope inoculation in order to observe the growth characteristics of the strain.
(7) quickly extends the bacteria-stained vaccination ring into another waiting interview tube. Make a dense back-and-forth dash from the bottom of the bevel to the "Z" shape. Sometimes it is also possible to use the inoculation needle to pull only a line in the center of the culture for slope inoculation in order to observe the growth characteristics of the strain.
(8) after vaccination, pull out the ring burning tube mouth, stuffed with cotton plugs.
(9) will be inoculated ring burning red sterilization. Put down the inoculation ring and plug the cotton tightly.
2, liquid inoculation
(1) by the bevel media into the liquid media, this method is used to observe the growth characteristics of bacteria and
biochemic
reaction determination, the operation method is the same as before, but make the test tube mouth tilt upward, so as to avoid the culture fluid flow into the bacteria, so that the inoculation ring and tube wall friction several times to help wash the ring upper body. After inoculation, plug the cotton plug and gently tap the test tube in the palm of your hand so that the bacteria are fully dispersed.
(2) by the liquid culture base inoculated liquid culture, bacteria are liquid, the joint in addition to the inoculation ring is still using sterile straws or dropper. Vaccination only need to pull out the cotton plug next to the flame, the tube mouth through the flame, with sterile straw to absorb the bacteria into the culture liquid, shake well.
3, tablet inoculation
bacteria on the plate to draw and coat.
(1) dash vaccination see separation dash method.
(2) coating inoculated with sterile straw to absorb bacteria into the plate, with sterilized glass rod on the surface of the plate for uniform coating.
4, puncture vaccination
inoculated the bacteria into solid deep medium, this method is used for anti-gas bacteria inoculation or for the identification of bacteria to observe physiological performance.
(1) operation method is the same as above, but the vaccination needle used should be straight.
(2) will be the inoculation needle from the center of the culture base, straight to close to the bottom of the tube, but do not penetrate, and then the original puncture path slowly pulled out.
(ii) separation operation method
1, dilution separation method
through continuous dilution of the isolated samples scattered to a minimum, and then absorb a certain amount of injection plate and temperature suitable for melting
agarline
culture mix, so that the dispersed bacteria are fixed in place to form a single bacterium.
(1) make e. coli or yeast with sterile water to make bacterial suspension.
(2) to take a number of sterile test tubes, each containing 9 ml of sterile water.
(3) absorbs 1 ml of prepared bacterial suspension and places it in the first test tube containing 9 ml of sterile water, which dilutes it 10 times, or 10-2.
(4) draws 1 ml from the first test tube (10-2) and injects it into the second test tube containing sterile water, which dilutes it 100 times, or 10-2.
(5) in the same way until diluted to 10-5-10-6.
(6) to accurately absorb 10-5-10-6 dilution bacteria liquid 0.2 ml added to the numbered empty sterile flat dish, the same dilution repeated to do three flat dishes.
(7) pours the agar medium, which has dissolved and cooled to 45 degrees C, into each of the above flat dishes, gently rotates to mix the medium with the bacteria suspension, and places it upside down in a temperature tank at 37 degrees C or 38 degrees C for 24-48 hours to observe the growth and distribution of the bacteria on the plate.
2, plate dash separation method
flat dash separation method is a method to inoculate the ring on the surface of the flat plate culture base through zoning dashing to achieve the separation of microorganisms. The principle is to dilute the microbial sample from point to line several times on the surface of the solid culture base for separation purposes.
(1) inverted flat plate Dissolved beef paste protein agar culture base poured flat plate, water calmly to be condensed.
(2) burn the inoculation ring on the alcohol light flame, to be cold, take an inoculated ring Staphylococcus acetabacter, E. coli mixed bacteria liquid.
(3) The left hand grip agar plate slightly lifts the lid of the dish, while close to the flame, the right hand inoculation ring into the dish, in an area on the plate as a shaped backline, when drawing the inoculation ring and the surface of the plate into 30-40 degrees angle light contact, with wrist force on the surface for a light slide, do not make the surface of the plate cut or embedded in the base.
(4) burning inoculation cup, in order to kill the remaining bacteria on the inoculation ring, to be cooled, and then the inoculation ring into the dish, in the first area of the cross-line of the place after a little contact, turn 90 degrees, in the second area continue to draw the line.
(5) and then burn the inoculation cup, cool down and line up in other areas in the same way.
(6) are all crossed, the bacteria, date, group and name are indicated with special crayons at the bottom of the flat dish. Place the entire Petri dish upside down in a thermostat to culture.
(7) 37 degrees C after 24-48 hours of culture to remove observation. Pay attention to the switches, size, color, edges, surface structure, transparency and other symptoms of the fall.
a sterile operation link
(1) inoculation room should be kept clean, with coal powder soap liquid scrubbing the table and walls, regularly fumigated with lactic acid or formaldehyde. UV lamp sterilization is applied before each use. Regularly check the inoculation room for sterile levels.
(2) before entering the inoculation room, you should do a good job of personal hygiene, in the buffer room to replace the work shoes and hats, work clothes, wearing
masks
. Work clothes, work shoes and masks are only allowed in the inoculation room. Do not wear to other places, and regular replacement, disinfection.
(3) inoculated test tubes, triangular bottles, etc. should be marked, indicating the name and date of the culture base, strains. All items moved into the inoculation room must be cleaned with 70% alcohol in the buffer room.
(4) Before vaccination, the hands with 70% alcohol or neo-Jie disinfection, the operation process does not leave the alcohol lamp flame;
(5) culture box should be cleaned and disinfected frequently.