Preparation of "microorganism" detection medium details
Preparation of microbiological testing medium details
1. Weighing
Use an electronic balance with an accuracy of 1/100 to weigh the medicines required for the culture medium. First calculate the amount of each component medicine required to prepare a certain amount of medium according to the formula, and then accurately weigh it with a balance. When weighing, use weighing paper to fold into a dustpan to hold the medicine. The weighing paper folding method is shown in the figure.
Second, melting
Put the culture medium into a beaker or enamel jar, slowly add a small amount of required water, and stir with a glass rod while adding. If the medium does not contain agar, the medium does not need to be heated; if it contains agar, it needs to be heated and boiled with a Bunsen burner or an electromagnetic oven. After it is completely dissolved, add the required water and stir evenly (as shown in Figure 3) . If the amount of the prepared medium is large, it can be heated and melted in a stainless steel pot. It can be heated with warm water and stirred at any time to prevent coking. If there is a phenomenon of coking, the prepared medium cannot be used and must be reconstituted.
When preparing the medium, do not use copper or iron containers to dissolve, because the copper or iron containers may make the contents of copper and iron in the medium exceed the standard, affecting the experiment (the copper content in the medium is greater than 0.3mg / L, bacteria should not grow , Iron content exceeds 0.14mg / L, which hinders bacterial toxin production). For drugs that easily react and produce precipitation, dissolve separately, and finally add culture medium, such as dipotassium hydrogen phosphate and magnesium sulfate.
3. Adjust the pH
Although the medium contains buffer substances, the pH of the medium can be kept within the required range as much as possible, but if the prepared medium does not meet the requirements, it must be adjusted as necessary. If a calibrated pH meter is available, a pH meter can be used. If not, a precision pH test paper can be used, and then 1 mol / L sodium hydroxide or 1 mol / L hydrochloric acid can be used as needed (for fine adjustment, 0.1 sodium hydroxide or 0.1 mol / L hydrochloric acid can be used) Adjust the desired pH. The pH of the base is generally 7.4 ~ 7.6, there are also acidic or alkaline. The medium that needs to be autoclaved adjusted with sodium hydroxide should be adjusted to 0.1 to 0.2 units higher than the required pH when adjusting the pH, because when adjusted with sodium hydroxide, the pH of the medium should be reduced after autoclaving 0.1 ~ 0.2. If the medium contains calcium carbonate, it may not be pH.
Four, filtering
If there is no special requirement for the prepared medium, this step can be omitted. If there is precipitation or turbidity. Need to be clarified. Liquid medium can be filtered with oil paper. The solid medium can be filtered with a double layer of gauze with a thin layer of absorbent cotton in between. If the filtration method can not meet the requirements of clarification, you can use egg white clarification method, that is, the medium is heated and cooled to 50 ~ 60 ℃, filled into a triangular flask (not exceeding one-half the capacity), add 1 per 1000mL The egg whites of 2 eggs are shaken vigorously for 3-5 minutes, autoclaved at 121 ° C for 20 minutes, and then taken out and filtered while hot.
5. Packing
The prepared culture medium is divided into containers such as flasks and test tubes according to different purposes. The test tubes are divided into tubes. If the amount of test tubes is large, an automatic dispenser can be used. If the amount of test tubes is small, the funnel can be used. The filling volume does not exceed two thirds of the container volume. The flask should not exceed one-half of the volume; the agar slope should not exceed one-fifth of the length of the test tube. After sterilization, the slope should be one-third of the amount of medium, and the bottom layer should be three The amount of two-half is appropriate; the volume of semi-solid agar is one-third of the length of the test tube; the high-level agar used for inoculation or bacteria preservation is the volume of one-quarter or one-third of the length of the test tube. It is necessary to inoculate anaerobic bacteria by two thirds; for agar plates, the inner diameter of 90mm is preferably 13mL ~ 15mL, and the inner diameter of 70mm is 8mL ~ 10mL. If the surface of the agar plate is made of more water, it can be Invert the plate and place it in a 37 ° C incubator for 30 min. Dry it before use. Each batch of medium should be aliquoted in a small glass bottle (about 20 mL) and sterilized simultaneously with the batch of medium, in order to determine the final pH of the batch of medium.
6. Sterilization
The aliquoted medium should be sterilized immediately. The types of sterilization methods are as follows:
1. High-pressure steam sterilization
This method can be used for most heat-resistant media. When small amounts are packed, use 121 ℃, 15min; when the amount of sterilization is large, use 121 ℃, 30min; the medium containing sugar can only be 113 ~ 115 ℃, 15min Sterilize to avoid damage to sugar.
2. Boiling sterilization method
This method can be used for media containing substances that do not tolerate high temperatures.
3. Filter sterilization
It is sterilized by filtration, and the medium is added quantitatively using aseptic technique. Blood and antibiotics can be extracted by aseptic technique and added to the medium cooled to about 50 ℃. This method can be used when the medium contains heat-resistant substances.
When sterilizing LST medium, there may be air bubbles in the fermentation tube. In order to prevent bubbles in the fermentation tube, the following measures can be taken:
1. Fill the small inverted tube with the medium (without air bubbles) before adding it to the test tube containing LST.
2. Before closing the bleed valve in the sterilization pot, drain the gas in the pot.
3. Do not plug the test tube plug too tightly (when using a silicone plug), do not use a rubber plug.
4 Do not open the sterilization pot prematurely. Wait until the air pressure and temperature in the sterilization pot are reduced to the same temperature as the room temperature or the temperature is not too different before opening the sterilization pot.
If there are bubbles in the above situation, water can be used as a control test for the medium group. If the medium group has bubbles and the control group has no bubbles, it can be determined that the medium itself is the cause.
Seven, inverted tablet
After the sterilized and melted medium is cooled to 50 ° C, it is poured into a sterile dry petri dish. The temperature of the culture medium should not be too high, otherwise it will easily form too much condensed water on the inner cover of the petri dish; if the temperature is too low, the culture medium will easily solidify into a block and cannot be made into a flat plate. When pouring the plate, it should be carried out near the flame of the alcohol lamp to prevent external bacteria from falling into the plate. Hold the Petri dish in the left hand and the bottom of the triangular bottle in the right hand. Use the little finger and palm of the left hand to pull out the tampon of the triangular bottle and burn At the mouth of the flask, use your thumb and index finger to open the slit of the Petri dish lid until the mouth of the flask just reaches into, pour the culture medium to the bottom of the bottom, not more than one third of the height of the Petri dish, quickly close the lid , Place it on the table, and gently turn the petri dish to make the medium evenly distributed, just after condensation.
8. Pendulum
After the sterilization is completed, the agar culture medium in the test tube is placed on a wooden rod (or glass rod) while it is hot, and at an appropriate slope. After cooling, the agar solidifies and becomes a slope. The length of the bevel need not exceed half of the test tube.
Nine, quality inspection
After sterilizing the culture medium, double check it and find that there are cracks, water intrusion, abnormal color, and tampon contaminated with the culture medium. They must be discarded and cannot be used again. And determine its final pH. Sterility inspection and effect inspection are also required. Sterility check is to take 1 tube (bottle) ~ 2 tubes (bottle) of the sterilized culture medium and incubate at 37 ℃ for 1d ~ 2d to confirm the growth of no bacteria; the effect check is to inoculate the relevant culture medium with standard strains The upper inspection checks the growth, morphology and biochemical conditions of the bacteria, which are consistent with the known conditions. Both cases are qualified, the prepared medium can be used.
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