Microbial culture is a fundamental technique that enables microorganisms to grow, be isolated, or produce specific metabolites in vitro by controlling nutrient composition, pH, osmotic pressure, oxygen supply, temperature, and other conditions. Culture-medium composition directly affects strain recovery, colony formation, metabolic characteristics, and fermentation yield.

Keywords: microbiological culture media; peptone; tissue infusion powder; lactic acid bacteria; yeast; mold; anaerobic bacteria; bile salts; lactose; industrial fermentation

1 Major Functions of Culture-Medium Raw Materials

1.1 Peptones and Protein Hydrolysates

Peptones and protein hydrolysates mainly provide polypeptides, oligopeptides, free amino acids, and organic nitrogen. Casein peptone, tryptone, and bacteriological peptone are suitable for routine bacterial culture. Peptic peptone, blood powder peptone, and gelatin peptone can be used in nutrient-rich or complex culture systems. Pea peptone, potato peptone, and papain-digested soybean meal are suitable for plant-derived or animal-origin-free culture media.

1.2 Animal-Tissue Extracts

Beef powder and bacteriological beef extract powder mainly supplement vitamins, minerals, nucleotides, and tissue-derived growth factors and are commonly used in nutrient broth and nutrient agar. Bovine brain infusion powder, bovine heart infusion powder, and bovine liver infusion powder have relatively complex nutrient compositions and are suitable for fastidious bacteria, anaerobic bacteria, and highly nutritious culture systems.

1.3 Plant Extracts

Malt extract powder and potato infusion powder are mainly used for culturing yeasts, molds, and certain lactic acid bacteria. Corn steep powder, soybean cake powder, and soybean meal extract can provide amino acids, small peptides, carbohydrates, vitamins, and minerals and are mainly used in industrial fermentation media.

1.4 Carbohydrates and Selective Components

Lactose can serve as a carbon source or as a differential substrate for lactose fermentation. Bovine bile salts, porcine bile salts, and bile salts No. 3 are mainly used to inhibit bile-sensitive microorganisms and are suitable for selective isolation of intestinal bacteria and coliforms. They are not nutrient components required for routine expansion culture.

2 Rapid Selection for Different Microbial Cultures

Table 1 Culture-Medium and Raw-Material Formulation for Different Microorganisms and Culture Purposes

3 Selection of Culture Media for Common Bacteria

3.1 Escherichia coli Culture

(1) LB-Type Media

LB-type media are suitable for E. coli expansion, plasmid preparation, and routine molecular biology experiments. Tryptone provides peptides and amino acids, yeast extract supplements vitamins, nucleotides, and growth factors, and sodium chloride regulates osmotic pressure. Beef powder and beef extract powder are not equivalent substitutes for yeast extract.

(2) Tryptic Soy Media

Tryptic soy broth and tryptic soy agar are suitable for routine bacterial culture, strain expansion, and sterility testing. Their formulations use tryptone and papain-digested soybean meal as combined nitrogen sources, together with carbohydrates, sodium chloride, and buffer salts.

3.2 Nutrient Broth and Nutrient Agar

Nutrient broth and nutrient agar are suitable for most nonfastidious bacteria. Peptone provides organic nitrogen, beef powder or beef extract powder supplements vitamins and growth factors, and sodium chloride maintains osmotic pressure. Agar is added when a solid medium is required.

3.3 Strain Recovery and Pre-Enrichment

Strain recovery should use a culture system with low selective pressure. Bacteriological peptone, casein peptone, or tryptone can serve as basic nutrients and should be combined with a phosphate-buffering system and sodium chloride. After injured cells have recovered, they can be transferred to selective media.

3.4 Staphylococci and Bacillus

Nonselective culture of staphylococci and Bacillus species can use nutrient broth or tryptic soy medium. Selective isolation of salt-tolerant staphylococci generally depends on a high concentration of sodium chloride rather than bile salts.

4 Selection of Culture Media for Lactic Acid Bacteria and Bifidobacteria

4.1 Lactobacilli and Lactococci

(1) Basal Medium

Lactobacilli are generally cultured in MRS-type media. Casein peptone, tryptone, and papain-digested soybean meal provide small peptides and amino acids, while lactic acid bacteria nutrients supplement vitamins and other growth-promoting components.

(2) Carbon-Source Formulation

Most lactic acid bacteria culture systems use glucose. Lactose is suitable only for strains capable of lactose transport and catabolism and should not be used as a universal carbon source for lactic acid bacteria.

(3) Auxiliary Components

A complete MRS-type medium also requires acetate, citrate, phosphate, magnesium salts, manganese salts, and surfactants. Malt extract powder or corn steep powder may be used as complex nutritional supplements but cannot replace the complete formulation.

4.2 Bifidobacteria

Bifidobacteria can be cultured in modified MRS or specialized bifidobacterial media. In addition to peptones, lactic acid bacteria nutrients, and fermentable carbohydrates, reducing agents such as cysteine should be added, and culture should be performed under anaerobic or low-oxygen conditions.

5 Selection of Culture Media for Yeasts and Molds

5.1 Yeast Culture

(1) YPD-Type Media

YPD-type media are suitable for yeast biomass expansion and fermentation studies. Peptone provides organic nitrogen, yeast extract supplies vitamins and growth factors, and glucose serves as the main carbon source.

(2) Malt Extract Media

Malt extract powder provides carbohydrates, vitamins, and plant-derived nutrients and is suitable for culturing yeasts and certain molds. When a higher organic-nitrogen level is required, casein peptone, pea peptone, or potato peptone may be added.

5.2 Mold Culture

(1) Potato Dextrose Medium

Potato dextrose medium is suitable for mold isolation, colony observation, and sporulation culture. Potato infusion powder provides plant-derived extract nutrients, glucose provides the carbon source, and agar forms the solid medium.

(2) Malt Extract Medium

Malt extract medium is suitable for various molds and yeasts. Malt extract powder provides complex plant nutrients, peptone supplements organic nitrogen, and carbohydrates and agar serve as the carbon source and solidifying agent, respectively.

(3) Potato Peptone and Potato Infusion Powder

Potato peptone mainly provides peptides and amino acids generated by hydrolysis of potato proteins, whereas potato infusion powder mainly provides plant-derived extract components. These materials have different functions and can be combined according to the requirements for nitrogen sources and complex nutrients.

6 Selection of Culture Media for Fastidious and Anaerobic Bacteria

6.1 Fastidious Bacteria Such as Streptococci

Brain-heart infusion medium is suitable for streptococci and other nutritionally demanding bacteria. Bovine brain infusion powder and bovine heart infusion powder supplement tissue-derived growth factors, whereas peptic peptone, casein peptone, or gelatin peptone provides organic nitrogen. The medium should also contain glucose, sodium chloride, and phosphates. Some strains additionally require blood, serum, hemin, or NAD.

6.2 Blood- and Serum-Derived Nutrients

Blood meal peptone provides peptides, amino acids, and other nitrogenous nutrients derived from blood proteins and may also supply growth-promoting components, whereas Digested Serum Powder is a dried animal-blood-derived nutrient material that can be used in carbohydrate-fermentation media and differential plates; it should not be defined directly as a hydrolysate of serum proteins. Neither material is an equivalent substitute for intact blood or serum. When a standard formulation requires blood, hemin, or another specific factor, it must still be added separately.

6.3 Anaerobic Bacteria

Anaerobic culture can use peptic peptone, blood powder peptone, casein peptone, or polypeptone as nitrogen sources, with bovine liver infusion powder added to supply complex growth factors. Reducing agents such as cysteine or thioglycollate are also required. Some strains require hemin, vitamin K, or blood and must be cultured under strictly anaerobic conditions.

7 Selective Culture of Intestinal Bacteria and Differentiation of Lactose Fermentation

7.1 Composition of Selective Differential Media

(1) Basal Nutrients

Bacteriological peptone, peptic peptone, or casein peptone provides basic organic nitrogen, while beef extract powder supplies vitamins and minerals.

(2) Selective Effects of Bile Salts

Bovine bile salts, porcine bile salts, and bile salts No. 3 can inhibit certain Gram-positive and other bile-sensitive microorganisms, increasing the selectivity of the medium for intestinal Gram-negative bacteria.

(3) Differential Effects of Lactose

Lactose serves as the differential substrate. Acids produced during lactose fermentation cause indicators such as neutral red or phenol red to change color, thereby distinguishing lactose-fermenting from non-lactose-fermenting bacteria.

(4) Other Components

Solid selective differential media also require sodium chloride, buffer salts, and agar. The concentration of bile salts should balance inhibition of nontarget organisms with recovery of the target bacteria.

7.2 Scope of Bile Salt Use

Bile salts are suitable for selective isolation of intestinal bacteria, bile-tolerance studies, and lactose-containing selective differential media. They are generally not used for routine bacterial expansion, recovery of injured cells, or culture of bile-sensitive microorganisms.

8 Selection of Nitrogen Sources for Industrial Fermentation Media

8.1 Rapidly Available Nitrogen Sources

Casein peptone, tryptone, bacteriological peptone, and polypeptone contain relatively high levels of readily utilizable peptides and amino acids and are suitable for seed culture and rapid growth during the early stages of fermentation.

8.2 Complex Fermentation Nutrients

Corn steep powder provides amino acids, small peptides, organic acids, carbohydrates, minerals, and vitamins. Defatted soy flour can provide soybean-derived protein and complex nutrients, whereas soybean meal extract provides a relatively dispersible or soluble plant-derived nitrogen source. These materials are suitable for the fermentation production of amino acids, organic acids, enzymes, and other industrial products.

8.3 Plant-Derived and Nontraditional Nitrogen Sources

Pea peptone, potato peptone, and papain-digested soybean meal are suitable for animal-origin-free culture systems. Poultry peptone and silkworm pupae peptone can be used as nontraditional animal-protein hydrolysates in fermentation nitrogen-source screening.

8.4 Raw-Material Combinations

Rapidly available peptones can be combined with corn steep powder to support both early-stage growth and complex nutrient supply. Plant peptones can be combined with soybean meal extract or corn steep powder to reduce the proportion of animal-derived raw materials. Specific ratios should be determined through fermentation trials.

9 Nutrient Raw Materials and Selective Components for Microbiological Culture Media

Culture media should be formulated according to the target microorganism and culture objective and should be validated through actual culture experiments.

For more related articles, please see below:

[1] Culture and identification of microorganisms

[2] Microbial Morphological Identification: Observation Systems, Staining Methods, and Result Interpretation

[3] Elimination of microbial contamination

[4] Reagents Suitable for Microbiology

[5] Classification, Experimental Positioning, and Selection Logic of Culture Media Systems