A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture medium under controlled laboratory conditions. Microbial cultures are foundational and basic diagnostic methods used as research tools in molecular biology.

The term culture can also refer to the microorganisms being grown.

Microbial cultures are used to determine the type of organism, its abundance in the sample being tested, or both. It is one of the primary diagnostic methods of microbiology and used as a tool to determine the cause of infectious disease by letting the agent multiply in a predetermined medium. For example, a throat culture is taken by scraping the lining of tissue in the back of the throat and blotting the sample into a medium to be able to screen for harmful microorganisms, such as Streptococcus pyogenes, the causative agent of strep throat. Furthermore, the term culture is more generally used informally to refer to "selectively growing" a specific kind of microorganism in the lab.

It is often essential to isolate a pure culture of microorganisms. A pure (or axenic) culture is a population of cells or multicellular organisms growing in the absence of other species or types. A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another. For the purpose of gelling the microbial culture, the medium of agarose gel (agar) is used. Agar is a gelatinous substance derived from seaweed. A cheap substitute for agar is guar gum, which can be used for the isolation and maintenance of thermophiles.

The first culture media was liquid media, designed by Louis Pasteur in 1860. This was used in the laboratory until Robert Koch's development of solid media in 1881. Koch's method of using a flat plate for his solid media was replaced by Julius Richard Petri's round box in 1887. Since these foundational inventions, a diverse array of media and methods have evolved to help scientists grow, identify, and purify cultures of microorganisms.

The culturing of prokaryotes typically involves bacteria, since archaea are difficult to culture in a laboratory setting. To obtain a pure prokaryotic culture, one must start the culture from a single cell or a single colony of the organism. Since a prokaryotic colony is the asexual offspring of a single cell, all of the cells are genetically identical and will result in a pure culture.

Virus and phage cultures require host cells in which the virus or phage multiply. For bacteriophages, cultures are grown by infecting bacterial cells. The phage can then be isolated from the resulting plaques in a lawn of bacteria on a plate. Viral cultures are obtained from their appropriate eukaryotic host cells. The streak plate method is a way to physically separate the microbial population, and is done by spreading the inoculate back and forth with an inoculating loop over the solid agar plate. Upon incubation, colonies will arise and single cells will have been isolated from the biomass. Once a microorganism has been isolated in pure culture, it is necessary to preserve it in a viable state for further study and use in cultures called stock cultures. These cultures have to be maintained, such that there is no loss of their biological, immunological and cultural characters.

Eukaryotic cell cultures provide a controlled environment for studying eukaryotic organisms. Single-celled eukaryotes - such as yeast, algae, and protozoans - can be cultured in similar ways to prokaryotic cultures. The same is true for multicellular microscopic eukaryotes, such as C. elegans.