Agar-Application in Culture Medium

Agar-Application in Culture Medium

Agar plays a key and irreplaceable role in microbial culture medium. Its main value lies in providing a solid, stable support surface for microbial growth, greatly expanding the possibilities of microbiological research and application. The following is a detailed description of its core applications and advantages:

​Creating solid surfaces for separation and pure culture:

​Key functions: This is the core application of agar. Agar (usually at a concentration of 1.0%-1.5%) is heated and dissolved in a nutrient solution, and a semi-solid gel is formed after cooling.

​Microbial separation: When different microorganisms are mixed together (such as from environmental samples, clinical specimens), single microbial cells or spores can grow and reproduce in fixed positions on the surface of the agar by streaking, coating or pouring plates on the surface of the solid culture medium, forming visible, isolated colonies. The morphology, size, color, texture, etc. of the colonies are important bases for identifying and identifying microorganisms.

​Pure culture acquisition: The isolated single colony theoretically originates from a microbial cell and can be picked and transferred to a new culture medium to obtain a pure culture, which is the basis for subsequent research (physiological and biochemical, genetic, drug sensitivity, etc.).

​As an inert support for physical and chemical properties:

​Chemical inertness: Agar itself is not decomposed and utilized by most microorganisms. This ensures that:

The nutrients required for microbial growth are completely provided by various added nutrients (peptone, beef extract, sugars, inorganic salts, growth factors, etc.).

Agar does not react significantly with the components in the culture medium and does not interfere with the experimental results. This is a huge advantage of agar over the gelatin used in the early days (which can be liquefied by many microorganisms).

The morphological characteristics of the colonies grown on the surface of agar can truly reflect the characteristics of the microorganisms themselves.

​Special culture applications:

​Deep Agar: A culture medium containing agar that is melted and cooled to about 45°C and solidified in a test tube. Commonly used for:

Anaerobic culture: puncture inoculation, forming an anaerobic environment inside.

Observe the motility of bacteria: motile bacteria can grow around the puncture line.

Determination of oxygen demand (such as in thioglycollate fluid culture medium).

​Semi-solid agar culture medium: ​ The agar concentration is low (about 0.3%-0.7%), forming a soft and flowable state.

Mainly used to detect the motility of bacteria: motile bacteria can grow from the puncture line to the surrounding areas in a cloud-like manner; unmotile bacteria only grow on the puncture line.

​Special research: ​

​Plaque assay: ​ Phages lyse cells on a plate covered with host bacterial lawns (growing on the agar surface) to form transparent empty spots (plaques), which are used for phage isolation, counting and titer determination.

​Antimicrobial drug sensitivity test: ​ Such as K-B paper diffusion method (agar diffusion method), the agar plate containing bacteria is used as the basis, and the size of the inhibition zone around the drug paper reflects the sensitivity of the microorganism to the drug.

​Colony hybridization:​​ The colonies on the agar plate are transferred in situ to the membrane for nucleic acid hybridization analysis for clone screening.

​Gradient plate (such as antibiotic gradient, pH gradient):​​ The solid state of agar allows it to form a stable physical and chemical gradient.

​Inverted culture:​​ Solid agar plates are convenient for inverted culture to prevent condensed water from dripping onto the colonies.

​Promote characteristic growth of colonies:​​

The solid surface restricts the spread of microorganisms, allowing them to multiply in large numbers in a limited space, forming colonies with specific morphological characteristics. These characteristics are important indicators for classification and identification (such as flat, raised, convex, round, filamentous, root-like, wrinkled, moist, dry, etc.).

​Counting microorganisms:​​

​Pour plate method:​​ Mix the diluted microbial suspension with the melted and cooled agar medium, pour it into a plate and culture it after solidification. The number of colonies multiplied by the dilution is the number of live bacteria in the original solution.

​Spread plate method: Spread the dilution on the solidified agar plate, count the colonies after incubation.

​Advantages of agar:

​Ideal melting-freezing point: Typical commercial agar melts above 85-90℃ and freezes at around 32-40℃ (freezing point is usually <40℃). This makes it:

Easy to heat and melt and then mix and sterilize in the hot solution of the culture medium (such as high-pressure steam sterilization at 121℃).

Frozen (cooled) under conditions higher than the growth temperature of most microorganisms, convenient for pouring plates or filling test tubes without scalding the microorganisms (cooling to the culture temperature after solidification can be inoculated).

Stay stable in a solid state at the culture temperature.

​Thermoreversibility: It can be repeatedly heated, melted, cooled and solidified without significant changes in physical properties (such as the formation of breakpoints), which is convenient for reuse or re-pour.

​Transparency: Pure agar gel is transparent, which is convenient for observing changes in colony morphology and culture medium color (such as indicator color change circle). ​High water holding capacity: It can lock the water in the culture medium (more than 95% is water), providing the necessary water environment for the growth of microorganisms.

​Physiological inertness: It is not decomposed and utilized by most microorganisms (only a very small number of special microorganisms can degrade it), and does not affect the composition of the culture medium.

​Appropriate gel strength: Agar gel of appropriate concentration has sufficient hardness to support the colony, but it is not too brittle. The concentration of 1.5% is the general standard for solid culture.

​Relative stability: It is stable under normal culture conditions and during storage (but it should be kept away from light and dry).

​Controllable purity: Modern refined agar has few impurities (such as inorganic salts and residual compounds) and has little interference with microbial growth.

​Summary: Agar has become the absolute core coagulant for the preparation of microbial solid and semi-solid culture media due to its unique physical and chemical properties (especially ideal solidification/melting temperature, thermal reversibility, high inertness, good gel strength and transparency). Without agar, there would be no convenient microbial separation and purification technology, colony morphology observation, quantitative counting methods, and a variety of important experimental techniques (such as drug sensitivity, plaque assay, motility detection, clone screening, etc.). It is the key substance that promotes microbiology from liquid culture to solid culture era, and it is still an indispensable basic material for microbiology laboratories.