Solid medium preparation experiment

Summary

Solid culture medium can be divided into two categories: (1) a class is made of natural solid-like substances, such as potato blocks, bran, rice bran, soybean cake powder, peanut cake powder made of medium, alcohol factories, breweries and other commonly used in this medium; (2) the other class is added in the liquid coagulant and made, such as commonly used in laboratories, agar solid slant and solid plate culture medium. This medium is widely used for the separation of microorganisms, identification, preservation, counting and colony characteristics of the observation.

Operation method

Solid medium preparation experiment

Principle

Solid medium coagulant is generally not a nutrient for microorganisms, but only plays a solidifying role. Ideal coagulant should have the following conditions: will not be utilized by microbial decomposition; will not be damaged by autoclaving; in the temperature range of microbial growth to maintain a solid state; microorganisms and operators are non-toxic; good transparency, strong coagulant; inexpensive, easy to formulate. Commonly used coagulant is agar. The main component of agar is galactosaccharides sulfate, the vast majority of microorganisms can not decompose and utilize it. Agar melting point of 96 ° C, solidification point of 40 ° C, therefore, in general culture conditions are solid state, and strong transparency. It is these excellent characteristics, so that agar replaced the early use of gelatin and become a common coagulant.

Materials and Instruments

Agar Tryptone NaCl NaOH Tetracycline
Glass bottles Flasks

Move

I. Limiting medium plate

800 ml water with 15 g agar, autoclaved for 15 min. then add 200 ml sterile 5 X limiting medium and carbon source. Allow to cool to 50°C and add supplementary components. Pour 32~40 ml of medium per 10 cm plate (25~30 plates per liter of medium). Dry the plates at room temperature for 2~3 days, or slightly open the top lid of the plates and dry them at 37℃ or in a laminar flow fume hood for 30 min, then wrap the dried plates and store them at 4℃.

Enrichment Media Plates

Add water to the components of the media recipe listed below, volume to 1L, autoclave for 25 mm. Allow to cool to 50°C and add additional components. Pour 32~40 ml of medium per LB or H medium plate (25~30 plates per liter of medium) and about 45 ml of medium per λ broth medium plate (20 plates per liter of medium). Plates were dried and preserved as described above.
1. H Plates, per liter

10 g tryptone

8 g NaCl

15 g agar

2. λ-broth plate, per liter10 g Tryptone

2.5 g NaCl

10 g agar

3. LB plate, per liter

10 g tryptone

5 g yeast extract

5 g NaCl

1 ml 1 mol/L NaOH

15 g agar or agaroseIII. Additives

Other antibiotics and galactosides

1. Antibiotics (if needed)

Ampicillin 50 ug/ml Tetracycline 12 ug/ml
2. galactoside (if required):

X-gal final concentration 20 ug/ml IPTG final concentration of 0. I mmol/L

Top layer agar medium

Top layer agar medium is often used to evenly distribute phages or cells into a thin layer on the surface of a plate. Prepare top layer medium in 1 L batch, autoclave for 15 min to melt it, cool it down to 50℃, gently rotate and shake it to make it mix uniformly, and dispense it into 100 ml bottles. Autoclave again, cool, tighten the lid and store at room temperature. Before use, melt the top layer of agar in a water bath or microwave oven, cool to 45-50°C and maintain at that temperature. The agar content in the top layer agar is less than that in normal plates, and the top layer agar can remain molten for several days at this temperature.

1. LB top layer medium, 10 g tryptone per liter.

10 g tryptone

5 g yeast extract

5 g NaCl

7 g agar
2. Gamma top layer medium, per liter10 g tryptone

2.5 g NaCl

7 g agar

3. Top agarose medium, per liter

10 g tryptone

8 g NaCl

6 g agaroseV. Puncture agar medium

Puncture agar medium is used to preserve the strain. The addition of thymine results in the growth of thy- bacteria. It is believed that cysteine greatly prolongs the survival of bacteria in puncture cultures.
1. Puncture medium, per liter

10 g Nutrient broth

5 g NaCl

6 g Agar

10 mg Cysteine Salt-Cl

10 mg Thymine

Caveat

1, when the autoclave sterilization pot shows that the pot temperature reaches 121 ℃ to start timing, so that the temperature is stabilized at 121 ℃ or more (the pot pressure is strictly prohibited to exceed 0.165 M Pa), keep 15-20min.

2， cut off the power supply, let the autoclave cool naturally, must wait for the pressure to drop to below 0.05 M Pa when opening the exhaust valve.

Common Problems

The ideal coagulant should have the following conditions:

① not be utilized by the microorganisms cultured by decomposition;

② in the temperature range of microbial growth to maintain a solid state, in the cultivation of thermophilic bacteria, due to high temperatures easily caused by the liquefaction of the medium, usually in the medium to solve the problem by adding appropriate coagulants;

③ The solidifying point temperature of the coagulant should not be too low, otherwise it will be unfavorable to the growth of microorganisms;

(iv) The coagulant has no toxic effect on the cultured microorganisms;

⑤ The coagulant will not be destroyed in the sterilization process;

⑥ Good transparency and strong adhesion;

(vii) It is easy to prepare and inexpensive. The coagulants used in the experiment are agar, gelatin and silica gel, the latter is used to prepare solid culture medium for autotrophic microorganisms. For most other microorganisms, agar is the most appropriate, generally adding 1.5-2.5% can be solidified into a solid. This medium can be used for isolation, identification, viable bacteria counting, strain preservation and so on.

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