A microbiological culture is a foundational method used to multiply microbial organisms by allowing them to reproduce in a predetermined medium under controlled laboratory conditions. The goal is to grow the microorganisms to a large population size, enabling scientists to study their characteristics, genetic material, and behavior. The technique of growing bacteria in a liquid medium, known as a liquid culture or broth culture, facilitates the growth of uniform, high-density bacterial populations. Culturing bacteria is a primary diagnostic method in microbiology, often used to determine the type of organism present or its abundance in a sample.
Defining Liquid Culture
Liquid culture involves suspending the desired organisms in a liquid nutrient medium, often referred to as a broth. This liquid state allows for the uniform distribution of nutrients and the microbes themselves, which is a major benefit over solid media. Unlike solid media, where bacteria form fixed colonies on a surface, a liquid culture results in a turbid, or cloudy, suspension of cells throughout the entire volume.
The composition of the liquid medium is designed to mimic the nutritional requirements the bacteria encounter in their natural habitat. A complete broth must contain a source of carbon and energy, a source of nitrogen, inorganic salts, and water, with the pH adjusted to be optimal for the target species. Common ingredients include peptone, which provides both carbon and nitrogen, yeast extract for vitamins and trace elements, and sugars like glucose for energy.
Steps for Preparation and Inoculation
Preparation begins with the formulation and sterilization of the growth medium. The dry components of the nutrient broth are dissolved in water, and the pH is checked and adjusted before the solution is dispensed into a flask or tube. Sterilization is achieved using an autoclave, which applies high-pressure steam at 121°C for 15 to 20 minutes. This heat eliminates all pre-existing microorganisms, ensuring the culture remains pure and contamination-free.
Once the medium is prepared and cooled, inoculation, or adding the starter bacteria, takes place. This step requires strict adherence to aseptic technique to minimize the risk of introducing unwanted microbes. A small sample of the target bacteria, often taken from a pure colony on a solid plate, is transferred into the sterile liquid broth using a sterile loop or pipette. The flask is then incubated at the organism’s optimal temperature, sometimes with gentle agitation to enhance aeration and nutrient distribution.
How Bacteria Grow in Liquid Media
When bacteria are introduced into a closed liquid system, their population growth follows a predictable pattern known as the bacterial growth curve. This curve is divided into four distinct phases, each reflecting a change in the microbes’ physiological state in response to the environment. The increase in cell number throughout these phases is monitored in a liquid culture by measuring its turbidity, or cloudiness, using a spectrophotometer.
Lag Phase
The first stage is the Lag Phase, where cells are metabolically active, synthesizing proteins and molecules necessary for replication, but are not yet dividing. The cells are adapting to the new medium and increasing in size during this initial period.
Exponential or Log Phase
Following this adaptation, the population enters the Log Phase, characterized by rapid and predictable cell division via binary fission. During this phase, the population doubles at a constant rate, and the cells are considered the healthiest and most uniform for experimental use. The Log Phase continues until resources become depleted or toxic waste products accumulate.
Stationary Phase
In the Stationary Phase, the rate of new cell division equals the rate of cell death, resulting in no net increase in the overall population. Competition for the remaining nutrients increases, and cells often become less metabolically active or produce specialized compounds to survive the stress.
Decline or Death Phase
The Decline or Death Phase occurs when the number of dying cells exponentially exceeds the number of new cells, leading to a steady decrease in the number of viable organisms.
Primary Uses in Science and Industry
Liquid culture is a standard technique in laboratories for generating large, uniform quantities of bacteria necessary for various downstream applications. One major application is genetic analysis, where researchers require a high biomass of cells to extract and purify DNA, RNA, or proteins for molecular studies. The suspended nature of the culture ensures that all cells are exposed to the same conditions, yielding a homogeneous sample.
In the biotechnology and pharmaceutical industries, liquid culture is scaled up in large fermenters to produce valuable compounds. Bacteria like E. coli can be genetically engineered and grown in massive liquid batches for the large-scale production of human proteins, such as insulin and growth hormones. Similarly, liquid fermentation processes are the foundation for manufacturing antibiotics, enzymes, and other industrial products.
The technique is also regularly used in clinical and research settings for standardized testing and diagnostics. For instance, liquid media allows for accurate, standardized testing of antibiotic sensitivity by exposing the microbes to varying concentrations of drugs to determine the minimum inhibitory concentration. Liquid enrichment broths are also employed in clinical diagnostics to increase the concentration of rare pathogens from a patient sample before plating, making them easier to isolate and identify.