According to the nature of the sample to be tested, the purpose of culture and the type of medium used, different inoculation methods are used.
1. Plate streak separation culture method
For samples mixed with various bacteria, use streaks to separate and culture, so that the bacteria that were originally mixed together are separated on the surface of the agar plate along the streaks to obtain scattered single colonies to obtain pure species. There are many forms of streaking. A plate can be divided into four different areas for marking. The first zone (A zone) has the smallest area and is used as the source area of bacteria to be isolated. The second and third zones (B , C area) is the transition area for gradual dilution, and the fourth area (D area) is the key area, so that a large number of single colonies appear in this area for the selection of pure species. In order to obtain more typical single colonies, the area distribution of the four areas on the plate should be D>C>B>A.
2. Agar slant inoculation method
It is mainly used for colony transplantation to obtain pure species for identification and preservation of strains, etc. Use an inoculation loop (needle) to pick a single colony or culture, draw a straight line upward from the bottom of the slant of the culture medium, and then draw a continuous line from the bottom along the straight line until the slant is near the top.
3. Puncture inoculation method
This method is mostly used for the inoculation of semi-solid medium or medium with high layers such as trisaccharide iron and gelatin, and the puncture inoculation of semi-solid medium can be used to observe the dynamics of bacteria. When inoculating, use the inoculation needle to pick the colony, puncture vertically from the center of the culture medium to 0.4cm from the bottom of the tube, and then withdraw the inoculation needle along the puncture line. For mediums such as trisaccharide and iron, which are divided into high-layer and slope, puncture the layer part, withdraw the inoculation needle and directly inoculate the slope part.
4. Liquid medium inoculation method
Used for the inoculation of various liquid media such as broth, peptone water, sugar fermentation tubes, etc. Pick a single colony with an inoculation loop, tilt the liquid culture tube, and grind the inoculum at the junction of the liquid surface and the tube wall, so that the bacteria are evenly scattered in the liquid medium. This inoculation method should avoid too much contact between the inoculation loop and the liquid.
5. Pour plate method
This method is mainly used for counting bacteria in samples such as drinking water, beverages, milk and urine. Take 1ml of the pure culture dilution or the original sample into a sterile petri dish, then pour 15-20ml of agar medium that has been melted and cooled to about 45-50°C into the sterile petri dish, mix well, and wait until After coagulation, culture at 37°C, and count the colonies after the colonies grow, so as to calculate the number of bacteria contained in each milliliter of the sample.
6. Spreading inoculation method
The coating method inoculation is a commonly used inoculation method, which can not only be used to count the number of viable bacteria, but also can be used to detect the inhibitory effect of chemical factors on microorganisms by using the characteristics of growing and forming a lawn on the surface of the plate. Add a certain amount of bacterial solution or diluted solution to the surface of the agar medium, and then use a sterilized L-shaped glass rod or cotton swab to spread it repeatedly at different angles, so that the inoculated solution is evenly distributed on the surface of the agar medium, and then paste it on the surface of the agar medium. Drug-sensitive paper, or direct culture, the bacteria form a lawn after culture in this method.
Monitoring Method of Sterilization Effect of Autoclave
The sterilization effect of the pressure cooker is related to the success or failure of the final test results. Therefore, the laboratory should regularly monitor the sterilization effect of the pressure cooker. At present, there are mainly three monitoring methods:
1. Chemical indicator card
The chemical indicator card can only monitor the sterilization temperature of the sterilizer, but cannot accurately monitor the sterilization time, and achieves the monitoring effect through color changes.
The advantage is that it is convenient and quick to use, and the disadvantage is that the sterilization time cannot be accurately monitored.
If the pressure cooker is used frequently, it is recommended to use a chemical indicator card on the outside of the material that needs to be sterilized every time as an identification.
2. Keep a thermometer
The reserved thermometer can be used for the verification test only after it has passed the standardization. Before testing, the mercury column of the thermometer needs to be thrown below 40°C. After each monitoring, the temperature difference of the left-point thermometer should be between 1°C, which means that the temperature distribution in the sterilizer is uniform.
If the pressure cooker is used frequently, it is recommended to use a thermometer to monitor the sterilization effect of the pressure cooker every month.
3. Biological indicator
Biological indicators are a special kind of live microbial products, which can be used to confirm the performance of sterilization equipment, verify the sterilization process, and monitor the sterilization effect of the production process.
1) According to the structure, it can be divided into sheet biological indicators and self-contained biological indicators. Generally speaking, most of them are self-contained, that is, there are bacteria tablets inside and sealed ampoules outside.
2) According to the culture time, it can be divided into general biological indicators and rapid biological indicators. The general-purpose biological indicator is based on the pH change of the culture solution caused by the metabolism of the indicator bacteria after spore resuscitation, and is judged by the color change of the acid-base indicator. The time is generally more than 24 or 48 hours; Accelerated reaction is interpreted by fluorescence, and the time is generally 3-4 hours. At the same time, faster biological indicators are being developed abroad, and the results are expected to be obtained within 1 hour.
Biological indicators are also recommended in GB15981-1995 "Evaluation Methods and Standards for Disinfection and Sterilization Effects" to monitor the effect of pressure steam sterilization.
The advantage is that it can accurately monitor the sterilization temperature and time, but the disadvantage is that it takes a long time.
It is recommended to use biological indicators to monitor the sterilization effect of the autoclave every quarter.
Quantitative detection method (plate method) precautions
Food microbiological testing can be divided into quantitative testing and qualitative testing, of which the plate method and MPN method are the most common quantitative testing methods. Taking the plate method as an example, some common precautions in quantitative detection are briefly introduced for your reference in daily work.
1. The effect of homogeneity on the test results. The homogenization process is the process of mixing the sample with the diluent. If the homogenization effect is not good, the microorganisms in the sample cannot be fully mixed into the diluent, and the test results cannot reflect the real situation of the sample. Generally, the test values are smaller than the real value. The current homogenization methods recommended by the national standard are the homogenization cup method of the rotary knife homogenizer and the homogenization bag method of the slap homogenizer. You can choose according to the actual situation of your samples.
2. The influence of ten-fold serial dilution on the test results. The ten-fold serial dilution has a great influence on the quantitative detection method, because it can multiply the experimental error. For example, when diluting from 10-1 to 10-2, if only 0.98mL is taken, then when it reaches 10-5, the error will be magnified by 1000 times. Therefore, in this step, we must pay attention to the accurate amount of liquid taken, and after each dilution, the diluted liquid should be mixed evenly before the next step of dilution.
3. Precautions for plate inoculation. Whether it is coating or pouring inoculation, the microorganisms should be distributed evenly, and should be as close to the middle as possible, away from the edge, because when the colony grows on the edge, it is not easy to read, and it is easy to connect into pieces. When coating, it should be in the middle of the plate. Add the thinner to start coating, trying not to spread the liquid to the edges. When pouring, also add the dilution to the middle of the plate, then pour the medium and swirl the plate gently to mix well.
Frequently Asked Questions about the Coliform MPN Method
① Standards on which the test is based
First, check the unit of the required MPN value, whether it is MPN/g or MPN/100g. If it is MPN/g, test according to GB4789.3-2016; if it is MPN/100g, according to the notification requirements issued by the Ministry of Health, it can be tested according to GB4789.3-2003.
②Inoculation of double materials and search method of MPN table
If the prepared dilutions are 10-1, 10-2, 10-3. Among them, 10-1 inoculated 10 mL into 3 tubes of double-material primary fermentation medium, another 10-11 mL was inoculated into 3 tubes of single-material primary fermentation medium, 10-2 inoculated 1 mL into 3 tubes of single-material primary fermentation culture Base. Complete the primary fermentation inoculation of the MPN method.
When finally judging the result, the number of tubes that were positive in the initial fermentation should be deduced based on the results of the re-fermentation, and then combined with the dilution of the initial fermentation inoculation to be 1, 0.1, 0.01. Retrieve the MPN table for judgment, and note that the MPN value in the table changes with the dilution.
③How to judge gas production
In the MPN method, the main basis for judging the positive tubes of primary fermentation and re-fermentation is gas production, but when doing sample testing, in many cases, the gas production of coliform bacteria is not much, and it is difficult to see in small tubes, so it cannot be directly judged at this time is negative. Shake the test tube slightly, and then tilt the test tube at a certain angle. If there are a large number of small bubbles rising from the bottom and gradually increasing, it can be judged as positive.
Purification of suspicious bacteria in biochemical tests
During the detection of pathogenic bacteria, it is generally required to purify the colonies of suspicious bacteria on a specific nutrient plate after biochemical tests or preliminary screening and before confirmatory tests, such as single-increase Lister test using TSA-YE for suspicious bacteria purification, Salmonella and Shigella Bacterial detection uses nutrient agar for purification. The purpose of purifying suspicious bacteria is as follows:
1. According to the size and shape of the colony grown on the plate after purification, it can be judged whether the picked suspicious bacteria is a single pure colony, and whether there is any superposition of colonies.
2. There are many general items in the biochemical test, and the suspicious bacteria on the selective separation plate are generally colored, so it is impossible to make a bacterial suspension. Suspicious single colonies with small growth cannot meet the inoculation volume requirements, and the inoculation volume of each project cannot be guaranteed to be the same. Experimental results will vary. However, all the colonies on the plate after purification come from the same single colony, and there are a large number of bacteria for biochemical tests.
3. Selective separation plates generally have complex components and have certain inhibitory properties, which may contain certain substances that affect individual biochemistry. The plates used for purification are all nutritional and will not affect biochemical projects.
Frequently Asked Questions about Serological Tests in Salmonella Testing
Serological identification is an important identification method in Salmonella testing, including bacterial antigen (0) identification, flagellar antigen (H) identification and Vi antigen identification. Since serological tests involve a lot of content, here are some frequently asked questions for your reference in your daily work.
1. When determining Salmonella, should the results of biochemical tests or serological tests be the main ones?
When we judge, we should focus on the results of biochemical tests, and conduct serological judgments on the basis of biochemical tests. Some experimenters will directly use polyvalent serum for testing without biochemical testing, which is absolutely not advisable. Because the principle of serology is antigen-antibody binding, non-Salmonella microorganisms may also carry Salmonella-like antigens. Therefore, when we do identification, we must first conduct biochemical tests, and conduct serological identification on the basis of biochemical tests.
2. To what extent should serological tests be performed?
If we only need to identify whether a certain strain belongs to Salmonella, we only need to make 0 polyvalent and H polyvalent serum. If it is necessary to identify what kind of Salmonella a certain strain is, such as whether it is Salmonella typhimurium or whether it is Salmonella enteritidis, then a serological typing test is required, from multivalent serum to the single factor of 0 antigen and H antigen, and then refer to Look up the corresponding Salmonella name in the table in Appendix B of the GB4789.4-2016 Salmonella Inspection Standard.
3. If zero polyvalent serum does not agglutinate, must it be negative for zero antigen?
When we make 0 polyvalent serum, if there is no agglutination, it cannot be directly judged as negative, because we also need to consider the influence of Vi antigen. The Vi antigen belongs to the K antigen group, which can block the combination of the 0 antigen and the corresponding antibody, so when the Vi antigen exists, the 0 polyvalent serum will not agglutinate. The GB4789.4-2016 Salmonella inspection standard mentions that the bacteria types known to have Vi antigens are: Salmonella typhi, Salmonella paratyphi C and Salmonella Dublin. Therefore, we had to remove the effect of Vi antigen. The specific method is to make the bacteria to be tested into a concentrated bacterial solution, put it into boiling water at 100°C and boil for 15-60 minutes, the purpose is to destroy the Vi antigen, and then pick the bacterial solution to make 0 polyvalent serum. If it still does not agglutinate, it can be judged as negative.
Observation of fluorescence experiment results
In daily experiments, it is often encountered that Escherichia coli needs to observe the fluorescence. National standard GB 4789.38-2012 Escherichia coli enumeration second method (VRBA-MUG enumeration), GB/T 5750.12-2006 Escherichia coli test in water and source water, all need to observe fluorescence, it is important to observe fluorescence results sex.
MUG is the abbreviation of 4-methylumbelliferone-β-D-galactoside, which is the substrate of β-galactosidase, and β-galactosidase binds to MUG and cuts β-galactosidase The glycosidic bond releases the fluorescent chromogenic group 4-methylumbelliferone, and when observed under ultraviolet light (In = 366nm), the culture emits blue fluorescence.
Conditions for observing fluorescence:
1) Ultraviolet light has a wavelength of 366nm;
2) Observing in a dark environment helps to better observe fluorescence;
3) It is better to directly irradiate the ultraviolet light tube on the plate or test tube to be tested, without glass and other items to absorb ultraviolet light.
4) Use blank tubes, positive bacteria, and negative bacteria as controls.
It is recommended to use the "simple portable fluorescent detection lamp", which not only meets the above three requirements, but also avoids the direct exposure of ultraviolet light to people, which protects the experimenters.
Cause analysis of blank fluorescence:
1) The test tube or flat blood is not cleaned cleanly. Residual 4-methylumbelliferone in the test tube or flat blood will cause the blank to have fluorescence.
2) The ultraviolet lamp for observing fluorescence is not suitable. Although the wavelength of the ultraviolet lamp is 366nm, there is a glass lampshade on the outside, or the power is too high (too high wattage, too bright lamp) will affect the fluorescent effect.
3) The reason of the test tube or flat blood itself. Some test tubes or flat blood itself have light under ultraviolet light due to quality reasons. Before use, you can take several test tubes or flat blood and fill them with distilled water, first observe them under the ultraviolet light, and then pick the containers with darker brightness for use.
suggestion:
It is recommended to use positive control bacteria to observe at the same time when doing the fluorescence test, which can be simpler and clearer.
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