Guides for Microbial Cultivation

The growth of microorganisms is not only determined by their own genetic characteristics, but also affected by many external factors, such as nutrient concentration, temperature, moisture, oxygen, pH and so on. Different types of microorganisms have different cultivation methods and conditions.

Figure 1 Microbial Cultivation

According to microorganisms that need oxygen or not during cultivation, it can be divided into two categories: aerobic culture and anaerobic culture.

Aerobic Culture

Aerobic bacteria require oxygen for survival. When aerobic microorganisms are cultivated, oxygen needs to be added, otherwise, they will not grow well. In the laboratory, agar slants can be used to culture bacterial cells for identification, providing a greater surface area for growth and obtaining sterile air from the outside.

Shake the liquid culture in the Erlenmeyer flask, so that the outside air continuously enters the flask.

Anaerobic Culture

As a general guideline, anaerobes are usually collected from a warm, moist environment that is low in oxygen. It is important to avoid shocking the anaerobes by exposing them to oxygen or allowing the sample to become dry. Such microorganisms do not need oxygen in the cultivation process. And the most important thing is to remove the oxygen in the culture medium. Generally, the following methods can be used in the effort.

a. Reduce the redox potential in the medium

Add reducing agents, such as glutathione or thioacetate, to the medium to achieve the goal. Tissues of some animals such as cattle heart and sheep brain can be added to the culture medium, which is also suitable for the growth of anaerobic bacteria.

b. Chemical deoxygenation

Deoxygenation is a chemical reaction involving the removal of oxygen atoms from a molecule. There are several methods to conduct. Use charcoal gallic acid, phosphorus, or plant tissues such as germinating seeds to absorb oxygen. Co-cultivate aerobic bacteria and anaerobic to run out of oxygen. Use the chemical method generating reducing agents to remove oxygen.

c. Block oxygen

Deep liquid culture, sealed with paraffin oil, or semi-solid puncture culture.

d. Replace oxygen

Replace oxygen with carbon dioxide, nitrogen, vacuum, hydrogen, or mixed gas.

Culture Medium

• Solid culture

Inoculate the strains into a loose and nutritious solid medium, and carry out microbial cultivation under suitable conditions.

• Liquid culture

Through liquid culture, microorganisms can multiply rapidly, and a large number of cultures can be obtained.

Strain Preservation

Preserve microorganism strains for a long time without contamination by other miscellaneous bacteria. Maintain morphological and physiological characteristics, reduce variation and prevent senescence, so that these strains can be used in the future. The strain preservation is generally to select its dormant body, such as spores, etc., and to create a low temperature, dryness, hypoxia, and lack of nutrients, so that the dormant body can stay in a rest state for a long time. For microorganisms that do not produce spores, preserve the strains when their metabolic rates decrease to a minimum level, so as to achieve the purpose of long-term preservation.

Commonly used methods for strain preservation

1. Slants or semi-solid passage preservation

The commonly used culture media for strain preservation include ordinary broth agar slants, serum slants, blood agar slants, egg slants, serum broth semi-solid medium, and so on. Sterilized liquid paraffin is often added to the surface of the slants or semi-solid culture medium to block the air.

2. Vacuum freeze-drying

Vacuum freeze-drying is the method of freezing a water-containing substance to a solid state, and then subliming the water therein from a solid state to a gaseous state to thereby remove water and preserve the substance. Add a certain protective agent (such as sterile skimmed milk), freeze the bacterial suspension and then vacuum-dry it in the frozen state to make the bacterial culture in a semi-permanent dormant state, so as to achieve the purpose of long-term preservation. Generally, strains can be preserved for several years or even decades. This method requires special drying equipment.

3. Liquid nitrogen cryopreservation

Liquid nitrogen is a non-mechanical method of cryopreserving strains. Disperse the preserved strains in a protective agent (such as glycerin, dimethyl sulphate, sucrose, Tween 80, etc.). After pre-freezing, strains stored in nitrogen can be placed above the liquid in a cold vapor phase or in the liquid nitrogen itself (-196°C). This method is currently an ideal method for the preservation of strains, which can preserve strains for a longer period of time.