Microorganisms are cultured on slopes, liquids
Purpose requirements
1. To understand the characteristics of different microbial cultures on inclined surfaces and in liquid and semi-solid media.
2. Further proficiency and mastery of aseptic inoculation techniques for microorganisms.
II. Basic principles
microorganismThe culture characteristics of microorganisms refer to the population morphology and growth of microorganisms cultured on the medium. Generally, the culture characteristics of different microorganisms can be tested by inclined, liquid and semi-solid media. They are cultured on inclined media and can be filamentous, hairy, beaded, sparse, dendritic or pseudoroot-like (Fig. VII-6). Grown in liquid medium, it can be cloudy, flocculent, mucous, form a bacterial film, the upper layer is clear and the bottom is precipitated. Puncture culture in semi-solid medium can spread around along the inoculation line; or only grow along the line; or the upper layer can grow well, or even form a piece, and the bottom rarely grows; or the bottom grows well, and the upper layer does not even grow. Using the culture characteristics of microorganisms can be used as a reference for their species identification and identification of whether pure culture is contaminated.
When testing the culture characteristics of microorganisms or conducting other microbiological experiments, the inoculation process must ensure that it is not contaminated by other microorganisms. Therefore, in addition to the requirements of the working environment to avoid or reduce bacterial contamination as much as possible, it is important to be skilled in various aseptic inoculation techniques.
III. Equipment
Bacillus subtilis, Escherichia coli,Staphylococcus aureusGeo-trichum candidum, Bacillus mycoides, Serratia marcescens;
Meat paste peptone bevel medium, meat paste peptone liquid medium, semi-solid meat paste peptone medium, inoculation ring, inoculation needle, sterile straw, alcohol lamp, etc.
IV. Operation steps
1. Inclined vaccination
(1) On the inclined test tube of meat paste peptone, write the name of the bacteria to be inoculated, the date and the vaccinator with a marker.
(2) Ignite an alcohol lamp or gas lamp.
(3) Hold the strain test tube and the inclined test tube to be inoculated in the left hand with the thumb, index finger, middle finger, and ring finger, and clip the middle finger between the two test tubes, so that the inclined surface is upward and in a horizontal state, as shown in Figure VII-7. Loosen the tube plug with the right hand by the flame to facilitate extraction during inoculation.
(4) Hold the inoculation ring in your right hand and sterilize it through flame cauterization (refer to Experiment 1), clamp the tampon (or test tube cap) with the palm edge of your right hand and your little finger, little finger, and ring finger respectively by the flame, remove it, and quickly cauterize the nozzle.
(5) Extend the sterilized inoculation ring into the strain test tube, first contact the ring with the inner wall of the test tube or the medium that has not grown bacteria to achieve the purpose of cooling, and then pick a little moss. Exit the inoculation ring from the strain test tube, quickly extend it into the inclined surface test tube to be inoculated, and use the ring on the inclined surface to gently draw a straight line from the bottom of the test tube to the top. Do not cut the medium, nor do you let the ring contact the tube wall or nozzle.
(6) The inoculation ring exits the inclined tube, burns the nozzle with flame, and plugs the tube on the side of the flame. Gradually approach the inoculation ring to the flame, and then cauterize. If there are many bacteria stained on the inoculation ring, the ring should be dried by the flame first, and then cauterized to prevent the unburned bacteria from splashing out of the polluted environment. Pay more attention to this when inoculating pathogens.
2. Inoculation in liquid medium
Meat cream peptone liquidmediumWhen inoculating a small amount of bacteria, the operation steps are basically the same as when inoculating on the inclined surface. The difference is that after picking the inoculation ring of the bacteria and putting it into the liquid medium, it should be gently rubbed on the inner wall of the tube at the liquid surface to make the bacteria fall off from the ring and mixed into the liquid medium. After plugging the test tube plug, shake the liquid to distribute the bacteria evenly in the liquid, or mix with a test tube shaker.
When inoculating a large amount of liquid medium or requiring quantitative inoculation, sterile water or liquid medium can be injected into the strain test tube, the moss can be scraped off with an inoculation ring, and then the strain suspension can be quantitatively aspirated and added with a sterile straw, or directly poured into the liquid medium. If the strain is a liquid culture, it can be quantitatively aspirated and added with a sterile straw or directly poured into the liquid medium. The entire inoculation process requires aseptic operation.
3. Puncture vaccination
Pick the strain with the inoculation needle (the needle must be straight), pierce the semi-solid medium vertically from the center of the medium until it is close to the bottom of the tube, but do not penetrate, then pull the needle out along the original puncture line, plug the test tube plug, and cauterize the inoculation needle, as shown in Figure VII-9.
The aseptic operation of the above-mentioned inoculation methods shall be operated according to the first experiment. The tester should practice the aseptic operation inoculation technique repeatedly until he is more proficient.
4. Place the inoculated inclined, liquid and semi-solid media in a incubator at 28-30 ° C, and take out the observation results after 2-3 days of incubation.
