biology Biology is the scientific study of life. It is a natural science with a broad scope but has several unifying themes that tie it together as a single, coherent field. For instance, all organisms are made up of cells that process hereditary i ...

, a subculture is either a new

cell culture Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This te ...

or a microbiological culture made by transferring some or all

cell Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery ...

s from a previous culture to fresh

growth medium A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss ''Physcomitrella patens''. Differen ...

. This action is called subculturing or passaging the cells. Subculturing is used to prolong the lifespan and/or increase the number of cells or microorganisms in the culture.

Role

Cell lines and microorganisms cannot be held in culture indefinitely due to the gradual rise in

toxic Toxicity is the degree to which a chemical substance or a particular mixture of substances can damage an organism. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a subst ...

metabolites In biochemistry, a metabolite is an intermediate or end product of metabolism. The term is usually used for small molecules. Metabolites have various functions, including fuel, structure, signaling, stimulatory and inhibitory effects on enzymes, ...

, use of

nutrients A nutrient is a substance used by an organism to survive, grow, and reproduce. The requirement for dietary nutrient intake applies to animals, plants, fungi, and protists. Nutrients can be incorporated into cells for metabolic purposes or excre ...

and increase in cell number due to growth. Once nutrients are depleted and levels of toxic byproducts increase, the bacteria in the overnight culture enter the stationary phase, where proliferation is greatly reduced or ceased (the cell density value plateaus). When microorganisms from this overnight culture are transferred into the fresh media, nutrients trigger the growth of the microorganism and it goes through the lag phase, a period of slow growth and adaptation to the new environment, and then the log phase, a period where the cells grow exponentially. Subculture is therefore used to produce a new culture with a lower density of cells than the originating culture, fresh nutrients and no toxic metabolites allowing continued growth of the cells without risk of cell death. Subculture is important for both proliferating (e.g. a microorganism like '' E. coli'') and non-proliferating (e.g. terminally differentiated

white blood cells White blood cells, also called leukocytes or leucocytes, are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and derived from mult ...

) cells. Subculturing can also be used for growth curve calculations (ex. generation time) and obtaining log-phase microorganisms for experiments (ex. Bacterial transformation)."Bacterial Culture Techniques." Cold Spring Harbor Laboratory. 331-32. ''DNA Learning Center Lab Protocols''. Web. 15 Mar. 2021. http://labprotocols.dnalc.org/files/017_bacteria_culture_techniques.pdf Typically, subculture is from a culture of a certain volume into fresh growth medium of equal volume, this allows long-term maintenance of the cell line. Subculture into a larger volume of growth medium is used when wanting to increase the number of cells for, for example, use in an industrial process or scientific experiment.

Passage number

It is often important to record the approximate number of divisions cells have had in culture by recording the number of passages or subcultures. In the case of

plant Plants are predominantly photosynthetic eukaryotes of the kingdom Plantae. Historically, the plant kingdom encompassed all living things that were not animals, and included algae and fungi; however, all current definitions of Plantae exclud ...

tissue cells somaclonal variation may arise over long periods in culture. Similarly in

mammal Mammals () are a group of vertebrate animals constituting the class Mammalia (), characterized by the presence of mammary glands which in females produce milk for feeding (nursing) their young, a neocortex (a region of the brain), fur or ...

ian cell lines

chromosomal aberrations A chromosomal abnormality, chromosomal anomaly, chromosomal aberration, chromosomal mutation, or chromosomal disorder, is a missing, extra, or irregular portion of chromosomal DNA. These can occur in the form of numerical abnormalities, where ther ...

have a tendency to increase over time. For microorganisms there is a tendency to adapt to culture conditions, which is rarely precisely like the microorganism's natural environment, which can alter their biology.

Protocols for passaging

The protocol for subculturing cells depends heavily on the properties of the cells involved.

Non-adherent cells

Many cell types, in particular, many microorganisms, grow in solution and not attached to a surface. These cell types can be subcultured by simply taking a small volume of the parent culture and diluting it in fresh growth medium. Cell density in these cultures is normally measured in cells per

milliliter The litre (international spelling) or liter (American English spelling) (SI symbols L and l, other symbol used: ℓ) is a metric unit of volume. It is equal to 1 cubic decimetre (dm3), 1000 cubic centimetres (cm3) or 0.001 cubic metre (m3). ...

for large eukaryotic cells, or as optical density for 600nm light for smaller cells like bacteria. The cells will often have a preferred range of densities for optimal growth and subculture will normally try to keep the cells in this range.

Adherent cells

Adherent cells, for example many mammalian cell lines, grow attached to a surface such as the bottom of the culture flask. These cell types have to be detached from the surface before they can be subcultured. For adherent cells cell density is normally measured in terms of

confluency In cell culture biology, confluence refers to the percentage of the surface of a culture dish that is covered by adherent cells. For example, 50 percent confluence means roughly half of the surface is covered, while 100 percent confluence means ...

, the percentage of the growth surface covered by cells. The cells will often have a preferred range of confluencies for optimal growth, for example a mammalian cell line like

HeLa HeLa (; also Hela or hela) is an immortalized cell line used in scientific research. It is the oldest and most commonly used human cell line. The line is derived from cervical cancer cells taken on February 8, 1951, named after Henrietta ...

or Raw 264.7 generally prefer confluencies over 10% but under 100%, and subculture will normally try to keep the cells in this range. For subculture cells may be detached by one of several methods including

trypsin Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting these long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the dig ...

treatment to break down the proteins responsible for surface adherence, chelating calcium ions with

EDTA Ethylenediaminetetraacetic acid (EDTA) is an aminopolycarboxylic acid with the formula H2N(CH2CO2H)2sub>2. This white, water-soluble solid is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-soluble complexes eve ...

which disrupts some protein adherence mechanisms, or mechanical methods like repeated washing or use of a cell scraper. The detached cells are then resuspended in fresh growth medium and allowed to settle back onto their growth surface.

References