Preparation of microbiological test medium details 1

First,


The medicine required for the medium was weighed on an electronic balance of 1/100 degree. According to the formula, the amount of various ingredients needed to make a certain amount of medium is calculated first, and then it is accurately weighed by the balance. When weighing, fold the weighing paper into a dustpan to hold the medicine. Weighing paper folding method is shown in the figure.



Second, 



Place the medium in a beaker or enamel jar and slowly add a small amount of the required water, stirring with a glass stick as you add it. If the medium does not contain AGAR, the medium does not need to be heated; If AGAR is present, it should be boiled with a Bunsen burner or induction cooker, completely dissolved, then filled with the required water and stirred evenly (as shown in FIG. 3). If the preparation of the medium amount is large, can be used stainless steel pot heating melting, can first use warm water heating and stir at any time, to prevent coking, such as coking phenomenon, the preparation of the medium can not be used, to reformulate.



It is not allowed to dissolve in containers of copper or iron in the preparation of culture medium, because containers of copper or iron may cause the content of copper and iron in the culture medium to exceed the standard, affecting the experiment (the content of copper in the culture medium is greater than 0.3mg/L, the growth of bacteria is not suitable, and the content of iron is greater than 0.14mg/L, hinding the production of toxins by bacteria). For drugs that are prone to reaction and precipitate, dissolve them separately and then add medium, such as dipotassium phosphate and magnesium sulfate.



Third, 



Although the medium contains buffering elements that keep the pH of the medium within the required range as far as possible, adjustments are necessary if the medium does not meet the requirements. If you have a calibrated pH meter, use a pH meter; if not, use a precise pH test paper, and then use 1mol/L sodium hydroxide or 1mol/L hydrochloric acid as needed (fine tuning with 0.1 sodium hydroxide or 0.1mol/L hydrochloric acid) to modulate the required pH. The pH of the base is generally 7.4 ~ 7.6, but also acidic or alkaline. When adjusting the medium with sodium hydroxide, the pH value of the medium should be adjusted to 0.1~0.2 units higher than the required value. When adjusting with sodium hydroxide, the pH value of the medium should be reduced by 0.1~0.2 after autoclaved. If the medium contains calcium carbonate, not pH.



Four, filtering,



If there are no special requirements, this step can be omitted. If there is precipitation or turbidity. Need to clarify. Liquid medium can be filtered by oil paper. The solid medium can be filtered by a double layer of gauze sandwiched with a thin layer of absorbent cotton. If the filtration method cannot meet the requirements for clarification, the egg white clarification method can be used, namely, the medium is heated and cooled to 50~60℃, and then put into a triangular bottle (no more than half of the capacity). The egg white of 1 ~ 2 eggs is added by 1000mL, shaken vigorously for 3-5min, sterilized by high-pressure steam at 121℃ for 20min, and then taken out and filtered by hot.



Five, the partial shipments



Prepared culture media according to different purposes in triangular bottles, test tubes and other containers, in the test tube, if the tube is large, can be used for automatic separators, if the tube is small, can be used funnel. The volume shall not exceed two thirds of the volume of the container. Triangle bottles should not exceed one half of the volume. The AGAR slant should not exceed one fifth of the length of the test tube. After sterilization, the slant formed should be one third of the medium volume and two thirds of the substrate volume. The amount of semi-solid AGAR is one third of the tube length. The upper layer AGAR used to inoculate or protect bacteria should be divided into one fourth or one third of the tube length and two thirds of the anaerobe used to inoculate. AGAR plates with an inner diameter of 90mm are suitable for 13mL~ 15ml, while those with an inner diameter of 70mm are suitable for 8mL~ 10ml. If the surface moisture of AGAR plates is too high, the plates can be inverted and placed in the incubator at 37℃ for 30min, and then used after drying. Each batch of medium should be separately packed with a small glass bottle (about 20mL) and sterilized at the same time with the batch of medium to determine the final pH of the batch of medium.