Beef paste peptone medium preparation experiment

Summary

Beef Paste Peptone Medium is one of the most widely used and common bacterial bases for the growth and propagation of microorganisms.

Operation method

Preparation of Beef Paste Peptone Medium

Principle

Beef Paste Peptone Medium is one of the most widely used and common bacterial basal media, sometimes called general medium. Because this medium contains the most basic nutrients needed for general bacterial growth and reproduction, it can be used for microbial growth and reproduction. The base medium contains beef paste, peptone and NaCI, of which beef paste provides carbon, energy, phosphate and vitamins for microorganisms, peptone mainly provides nitrogen and vitamins, and NaCI provides inorganic salts. In the preparation of solid culture medium should also add a certain amount of agar as a coagulant, agar in the common concentration of 96 ℃ when dissolved, the actual application, generally in a boiling water bath or the following pad to the asbestos mesh boiling melting, so as not to agar burnt. Agar solidifies at 40℃ and is usually not utilized by microbial decomposition. The amount of agar in the solid medium varies according to the quality of the agar and the temperature. Since this medium is mostly used for the cultivation of bacteria, its pH should be adjusted to neutral or slightly alkaline with dilute acid or alkali to facilitate the growth and multiplication of bacteria. Beef paste peptone medium is formulated as follows: beef paste: 3.0 g peptone: 10.0 g NaCI: 5.0 g water: 1 000 mlpH: 7.4-7.6

Materials and Instruments

Beef paste Peptone NaCI Water Agar Sodium hydroxide Hydrochloric acid
Test tubes Triangular flasks Beakers Measuring cylinders Glass rods Medium dispensers Scales Bullhorn spoons Autoclave pH test paper Cotton Kraft paper Markers Sisal twine Gauze?

Move

I. Weighing
According to the proportion of the medium formula in order to accurately weigh the beef paste, peptone, NaCl in a beaker, beef paste commonly used glass rod pick, placed in a small beaker or surface dish weighing, with hot water to dissolve and pour the beaker. Can also be placed on the weighing paper, weighing directly into the water, such as a little heat, beef paste will be separated from the weighing paper, and then immediately remove the paper.

Peptones are very hygroscopic and should be weighed quickly. In addition, when weighing drugs to prevent mixing drugs, a horn spoon for a drug, or weighing a drug, wash, dry, and then weigh another drug. Don't put the wrong cap on the bottle.
II. Dissolution
Add less than the required amount of water in the above beaker, stir well with a glass rod, and then heat it on an asbestos net to dissolve it, or heat it on a magnetic stirrer to dissolve it (Fig. 1) After the drug has been completely dissolved, replenish the water to the total volume required, if the preparation of solid culture medium, the weighed agar will be placed in the dissolved drug, and then be heated to dissolve it, and then finally replenish the loss of water, and the agar will be added directly into each triangle bottle without heating. In the preparation of solid culture medium in triangular flasks, generally can also be a certain amount of liquid culture medium divided into triangular bottles, and then according to the amount of 1.5% -2.0% of the agar directly added to the triangular bottles, do not have to heat to dissolve, but sterilization and heating and dissolution of the synchronization, saving time.


During agar melting, the fire should be controlled so that the culture gene does not boil and overflow the container. At the same time, constant stirring is required to prevent the agar from burning at the bottom of the paste. When preparing culture medium, do not use copper or iron pot to heat and dissolve, so as to avoid ions entering into the culture medium and affecting bacterial growth.
III. Tuning pH
Before adjusting the pH, measure the original pH of the medium with pH paper, if it is acidic, use a dropper to add 1 mol/L NaOH drop by drop into the medium while stirring, and use pH paper to measure the pH at any time until the pH reaches 7.6. Instead, use lmol/L HCl to adjust.
For some microorganisms that require a more precise pH, their pH adjustment can be carried out with an acidimeter (refer to the relevant instruction manual for use).
The pH should not be over-adjusted to avoid back-adjustment which affects the concentration of each ion in the medium. When preparing agar medium with low pH, if the pH is adjusted in advance and sterilized under autoclave, the agar cannot solidify due to hydrolysis. Therefore, the components of the culture medium and agar should be separated and sterilized before mixing, or sterilized under neutral pH conditions before adjusting the pH.
IV. Filtration
Filter with filter paper or multi-layer gauze while hot, in order to facilitate the observation of certain experimental results, generally no special requirements, this step can be omitted (this experiment do not need to filter).

V. Packing
1. Depending on the requirements of the experiment, the prepared medium may be dispensed into test tubes or triangular flasks. The dispensing device is shown in Figure 2.

Figure 2 Medium dispensing unitA. funnel dispensing device; B. automatic dispenser 1. irons; 2. funnels; 3. latex tubes; 4. spring clamps; 5. glass tubes; 6. flow rate adjustments; 7. volume adjustments; 8. switches2. Liquid dispensing

The dispensing height should be about 1/4 of the height of the test tube. The amount of the triangular flask should be determined according to the need, generally not more than half of the volume of the triangular flask is appropriate, if it is used for oscillation culture, it is appropriate to reduce the amount of aeration according to the requirements of the liquid medium; some liquid medium should be supplemented with a certain amount of other sterile components, such as antibiotics, etc., after sterilizing the liquid medium, the amount of loading must be accurate.3. Solids Dispensing

Dispense test tubes in a volume not exceeding 1/5 of the height of the tube, sterilized to make a slant. Dispensing of triangular flasks in an amount not exceeding half the volume of the triangular flask is appropriate.
4. Semi-solid fractionation

Test tubes are usually 1/3 of the height of the test tube, sterilized vertically for condensation.
In the process of dispensing, be careful not to make the culture even on the tube (bottle), so as not to stain the cotton plug and cause contamination.
VI. Congestion
After the medium is dispensed, plug the mouth of the test tube or triangular flask with cotton plugs (or foam plugs and test tube caps, etc.) to prevent outside microorganisms from entering the medium and causing contamination, and to ensure that there is good aeration.
VII. Dressing
After stuffing, all test tubes tied with twine, and then a layer of kraft paper wrapped around the cotton plug to prevent condensation when sterilizing the cotton plug, and then tied with a twine outside, with a marker to indicate the name of the medium, the group, the date of preparation. Triangular flasks plus plugs, kraft paper outside, tied with twine in the form of a live knot, easy to unravel when in use, the same with a marker to indicate the name of the medium, group, date of preparation (the conditions of the laboratory, commercially available aluminum foil instead of kraft paper, eliminating the need to use the rope tie, and the effect is good.)
VIII. Sterilization
The above medium was autoclaved at 0.103 MPa, 121°C, 20 min.IX. Shelving ramps
Chill the sterilized test tube medium to about 50°C (to prevent too much condensation on the slant) and rest the mouth end of the test tube on a glass rod or other utensil of suitable height, with the length of the slant of the rest not exceeding half of the total length of the test tube (Fig. 3).

Figure 3 Setting up the inclined plane

X. Sterility check
The sterilized medium was incubated in a greenhouse at 37 ℃ for 24-48 h to check whether the sterilization was complete.

Caveat

Wipe the key promptly when weighing reagents to avoid reagent contamination.

Common Problems

Beef paste is often difficult to weigh and has a high viscosity, so final weighing is recommended.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/