Microbiological culture

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Microbial cultures on solid and liquid media Microbial cultures fridge.JPG
Microbial cultures on solid and liquid media

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.

Contents

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. [1] 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.

History

The first culture media was liquid media, designed by Louis Pasteur in 1860. [2] This was used in the laboratory until Robert Koch's development of solid media in 1881. [3] Koch's method of using a flat plate for his solid media was replaced by Julius Richard Petri's round box in 1887. [2] Since these foundational inventions, a diverse array of media and methods have evolved to help scientists grow, identify, and purify cultures of microorganisms.

Types of microbial cultures

A culture of Bacillus anthracis Anthrax culture.jpg
A culture of Bacillus anthracis

Prokaryotic culture

The culturing of prokaryotes typically involves bacteria, since archaea are difficult to culture in a laboratory setting. [4] To obtain a pure prokaryotic culture, one must start the culture from a single cell or a single colony of the organism. [5] 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.

Viral 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 culture

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.

Although macroscopic eukaryotic organisms are too large to culture in a laboratory, cells taken from these organisms can be cultured. This allows researchers to study specific parts and processes of a macroscopic eukaryote in vitro.

Culture methods

Method overview
MethodDescriptionUses and advantages
Liquid/broth culturesOrganisms are inoculated into a flask of liquid mediaGrowing up large volumes of organism, antimicrobial assays, bacterial differentiation
Agar platesOrganisms are placed or streaked onto petri dishesProvides a solid surface for stationary growth, compact and stackable
Agar based dipsticksEssentially miniature agar plates in the form of dipsticksDiagnostic purposes, can be used anywhere, cost effective, easy to use
Selective and differential mediaOrganisms are cultured in/on specific media to select for or differentiate between certain onesHelp identify unknown organisms, assist in purifying cultures
Stab culturesOrganisms are inoculated into a test tube of solid agarShort-term storage, bacterial differentiation

Liquid cultures

One method of microbiological culture is liquid culture, in which the desired organisms are suspended in a liquid nutrient medium, such as Luria broth, in an upright flask. This allows a scientist to grow up large amounts of bacteria or other microorganisms for a variety of downstream applications.

Liquid cultures are ideal for preparation of an antimicrobial assay in which the liquid broth is inoculated with bacteria and let to grow overnight (a ‘shaker’ may be used to mechanically mix the broth, to encourage uniform growth). Subsequently, aliquots of the sample are taken to test for the antimicrobial activity of a specific drug or protein (antimicrobial peptides).

Liquid cultures of the cyanobacterium Synechococcus PCC 7002 Synechococcus cyanobacteria-cultures.jpg
Liquid cultures of the cyanobacterium Synechococcus PCC 7002

Static liquid cultures may be used as an alternative. These cultures are not shaken, and they provide the microbes with an oxygen gradient. [6]

Agar plates

Example of a workup algorithm of possible bacterial infection in cases with no specifically requested targets (non-bacteria, mycobacteria etc.), with most common situations and agents seen in a New England setting. The grey box near top left shows a Venn diagram of what culture media are routinely used for various sources or purposes. Diagnostic algorithm of possible bacterial infection.png
Example of a workup algorithm of possible bacterial infection in cases with no specifically requested targets (non-bacteria, mycobacteria etc.), with most common situations and agents seen in a New England setting. The grey box near top left shows a Venn diagram of what culture media are routinely used for various sources or purposes.

Microbiological cultures can be grown in petri dishes of differing sizes that have a thin layer of agar-based growth medium. Once the growth medium in the petri dish is inoculated with the desired bacteria, the plates are incubated at the optimal temperature for the growing of the selected bacteria (for example, usually at 37 degrees Celsius, or the human body temperature, for cultures from humans or animals, or lower for environmental cultures). After the desired level of growth is achieved, agar plates can be stored upside down in a refrigerator for an extended period of time to keep bacteria for future experiments.

There are a variety of additives that can be added to agar before it is poured into a plate and allowed to solidify. Some types of bacteria can only grow in the presence of certain additives. This can also be used when creating engineered strains of bacteria that contain an antibiotic-resistance gene. When the selected antibiotic is added to the agar, only bacterial cells containing the gene insert conferring resistance will be able to grow. This allows the researcher to select only the colonies that were successfully transformed.

Agar based dipsticks

Miniaturized version of agar plates implemented to dipstick formats, e.g. Dip Slide, Digital Dipstick [7] show potential to be used at the point-of-care for diagnosis purposes. They have advantages over agar plates since they are cost effective and their operation does not require expertise or laboratory environment, which enable them to be used at the point-of-care.

Selective and differential media

Selective and differential media reveal characteristics about the microorganisms being cultured on them. This kind of media can be selective, differential, or both selective and differential. Growing a culture on multiple kinds of selective and differential media can purify mixed cultures and reveal to scientists the characteristics needed to identify unknown cultures.

Selective media

Selective media is used to distinguish organisms by allowing for a specific kind of organism to grow on it while inhibiting the growth of others. For example, eosin methylene blue (EMB) may be used to select against Gram-positive bacteria, most of which have hindered growth on EMB, and select for Gram-negative bacteria, whose growth is not inhibited on EMB [8] .

Differential media

Scientists use differential media when culturing microorganisms to reveal certain biochemical characteristics about the organisms. These revealed traits can then be compared to attributes of known microorganisms in an effort to identify unknown cultures. An example of this is MacConkey agar (MAC), which reveals lactose-fermenting bacteria through a pH indicator that changes color when acids are produced from fermentation [9] .

Multitarget panels

On multitarget panels, bacteria isolated from a previously grown colony are distributed into each well, each of which contains growth medium as well as the ingredients for a biochemical test, which will change the absorbance of the well depending on the bacterial property for the tested target. The panel will be incubated in a machine, which subsequently analyses each well with a light-based method such as colorimetry, turbidimetry, or fluorometry. [10] The combined results will be automatically compared to a database of known results for various bacterial species, in order to generate a diagnosis of what bacterial species is present in the current panel. Simultaneously, it performs antibiotic susceptibility testing.

Stab cultures

Motile and non-motile bacteria can be differentiated along the stab lines. Motile bacteria (left) will grow out from the stab line while non-motile bacteria (right) are present only along the stab line. Stab culture.png
Motile and non-motile bacteria can be differentiated along the stab lines. Motile bacteria (left) will grow out from the stab line while non-motile bacteria (right) are present only along the stab line.

Stab cultures are similar to agar plates, but are formed by solid agar in a test tube. Bacteria is introduced via an inoculation needle or a pipette tip being stabbed into the center of the agar. Bacteria grow in the punctured area. [11] Stab cultures are most commonly used for short-term storage or shipment of cultures. Additionally, stab cultures can reveal characteristics about cultured microorganisms such as motility or oxygen requirements.

Solid plate culture of thermophilic microorganisms

For solid plate cultures of thermophilic microorganisms such as Bacillus acidocaldarius, Bacillus stearothermophilus, Thermus aquaticus and Thermus thermophilus etc. growing at temperatures of 50 to 70 degrees C, low acyl clarified gellan gum has been proven to be the preferred gelling agent comparing to agar for the counting or isolation or both of the above thermophilic bacteria. [12]

Cell Culture Collections

Microbial culture collections focus on the acquisition, authentication, production, preservation, cataloguing and distribution of viable cultures of standard reference microorganisms, cell lines and other materials for research in microbial systematics. [13] [14] Culture collection are also repositories of type strains.

Major national culture collections. [13] [14]
Collection AcronymNameLocation
ATCC American Type Culture Collection Manassas, Virginia
BCCM Belgian Co-ordinated Collections of Micro-organisms Decentralized, Coordination Cell in Brussels, Belgium
CCUG Culture Collection University of Gothenburg Gothenburg, Sweden
CECTColección Española de Cultivos TipoValencia, Spain
CIPCollection d'Institut Pasteur Paris, France
DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen Braunschweig, Germany
NCPPB National Collection of Plant Pathogenic Bacteria York, UK
ICMP International Collection of Microorganisms from Plants Auckland, New Zealand
JCM Japan Collection of Microorganisms Tsukuba, Ibaraki, Japan
NCTC National Collection of Type Cultures Public Health England, London, United Kingdom
NCIMB National Collection of Industrial, Food and Marine Bacteria Aberdeen, Scotland
NCPPB National Collection of Plant Pathogenic Bacteria York, England

See also

Related Research Articles

<span class="mw-page-title-main">Agar</span> Thickening agent used in microbiology and food

Agar, or agar-agar, is a jelly-like substance consisting of polysaccharides obtained from the cell walls of some species of red algae, primarily from "ogonori" (Gracilaria) and "tengusa" (Gelidiaceae). As found in nature, agar is a mixture of two components, the linear polysaccharide agarose and a heterogeneous mixture of smaller molecules called agaropectin. It forms the supporting structure in the cell walls of certain species of algae and is released on boiling. These algae are known as agarophytes, belonging to the Rhodophyta phylum. The processing of food-grade agar removes the agaropectin, and the commercial product is essentially pure agarose.

<span class="mw-page-title-main">Petri dish</span> Shallow dish used to hold cell cultures

A Petri dish is a shallow transparent lidded dish that biologists use to hold growth medium in which cells can be cultured, originally, cells of bacteria, fungi and small mosses. The container is named after its inventor, German bacteriologist Julius Richard Petri. It is the most common type of culture plate. The Petri dish is one of the most common items in biology laboratories and has entered popular culture. The term is sometimes written in lower case, especially in non-technical literature.

<span class="mw-page-title-main">Bacteriology</span> Subdiscipline of microbiology that studies bacteria

Bacteriology is the branch and specialty of biology that studies the morphology, ecology, genetics and biochemistry of bacteria as well as many other aspects related to them. This subdivision of microbiology involves the identification, classification, and characterization of bacterial species. Because of the similarity of thinking and working with microorganisms other than bacteria, such as protozoa, fungi, and viruses, there has been a tendency for the field of bacteriology to extend as microbiology. The terms were formerly often used interchangeably. However, bacteriology can be classified as a distinct science.

<span class="mw-page-title-main">Agar plate</span> Petri dish with agar used to culture microbes

An agar plate is a Petri dish that contains a growth medium solidified with agar, used to culture microorganisms. Sometimes selective compounds are added to influence growth, such as antibiotics.

<span class="mw-page-title-main">Bacteriological water analysis</span>

Bacteriological water analysis is a method of analysing water to estimate the numbers of bacteria present and, if needed, to find out what sort of bacteria they are. It represents one aspect of water quality. It is a microbiological analytical procedure which uses samples of water and from these samples determines the concentration of bacteria. It is then possible to draw inferences about the suitability of the water for use from these concentrations. This process is used, for example, to routinely confirm that water is safe for human consumption or that bathing and recreational waters are safe to use.

<span class="mw-page-title-main">Blood culture</span> Test to detect bloodstream infections

A blood culture is a medical laboratory test used to detect bacteria or fungi in a person's blood. Under normal conditions, the blood does not contain microorganisms: their presence can indicate a bloodstream infection such as bacteremia or fungemia, which in severe cases may result in sepsis. By culturing the blood, microbes can be identified and tested for resistance to antimicrobial drugs, which allows clinicians to provide an effective treatment.

<span class="mw-page-title-main">Growth medium</span> Solid, liquid or gel used to grow microorganisms or cells

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. Different types of media are used for growing different types of cells.

In microbiology, colony-forming unit is a unit which estimates the number of microbial cells in a sample that are viable, able to multiply via binary fission under the controlled conditions. Counting with colony-forming units requires culturing the microbes and counts only viable cells, in contrast with microscopic examination which counts all cells, living or dead. The visual appearance of a colony in a cell culture requires significant growth, and when counting colonies, it is uncertain if the colony arose from one cell or a group of cells. Expressing results as colony-forming units reflects this uncertainty.

<span class="mw-page-title-main">Simmons' citrate agar</span> Differential culture medium

Simmons' citrate agar is used for differentiating gram-negative bacteria on the basis of citrate utilization, especially for distinguishing Gammaproteobacteria of the family Enterobacteriaceae or even between species of the same genus. For example, Salmonella enteritidis would yield a positive (blue) result on Simmons’ agar and thus be distinguished from other Salmonella species like Salmonella typhi, Salmonella pullorum, and Salmonella gallinarum, which would yield a negative (green) result.

<span class="mw-page-title-main">Disk diffusion test</span> Microbiology assay used in diagnostic and drug discovery laboratories

The disk diffusion test is a culture-based microbiology assay used in diagnostic and drug discovery laboratories. In diagnostic labs, the assay is used to determine the susceptibility of bacteria isolated from a patient's infection to clinically approved antibiotics. This allows physicians to prescribe the most appropriate antibiotic treatment. In drug discovery labs, especially bioprospecting labs, the assay is used to screen biological material and drug candidates for antibacterial activity. When bioprospecting, the assay can be performed with paired strains of bacteria to achieve dereplication and provisionally identify antibacterial mechanism of action.

<span class="mw-page-title-main">Streaking (microbiology)</span> Method for isolation of bacterial strains

In microbiology, streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria. Samples can then be taken from the resulting colonies and a microbiological culture can be grown on a new plate so that the organism can be identified, studied, or tested.

<span class="mw-page-title-main">Medical microbiology</span> Branch of medical science

Medical microbiology, the large subset of microbiology that is applied to medicine, is a branch of medical science concerned with the prevention, diagnosis and treatment of infectious diseases. In addition, this field of science studies various clinical applications of microbes for the improvement of health. There are four kinds of microorganisms that cause infectious disease: bacteria, fungi, parasites and viruses, and one type of infectious protein called prion.

<span class="mw-page-title-main">Etest</span>

Etest is a way of determining antimicrobial sensitivity by placing a strip impregnated with antimicrobials onto an agar plate. A strain of bacterium or fungus will not grow near a concentration of antibiotic or antifungal if it is sensitive. For some microbial and antimicrobial combinations, the results can be used to determine a minimum inhibitory concentration (MIC). Etest is a proprietary system manufactured by bioMérieux. It is a laboratory test used in healthcare settings to help guide physicians by indicating what concentration of antimicrobial could successfully be used to treat patients' infections.

Plate count agar (PCA), also called standard methods agar (SMA), is a microbiological growth medium commonly used to assess or to monitor "total" or viable bacterial growth of a sample. PCA is not a selective medium.

A dip slide is a test for the presence of microorganisms in liquids. The use of dip slides is the method most frequently used to measure and observe microbial activity in liquid-based systems. It is often used in testing cooling systems. Dip slides are often used to determine the presence of slime forming bacteria in cooling & industrial water systems. The Health and Safety Executive's (HSE) recommends the use of dipslides to monitor the general activity of aerobic bacteria. The dip slide test consists of a sterile culture medium on a plastic carrier that is dipped into the liquid to be sampled. The culture is then incubated, allowing for microbial growth. Most Dip slides consist of 1 - 2 agars attached to a flexible plastic paddle, this allows full contact of the agar onto the desired area for testing. Most Dipslides come in a circular clear shatterproof tube that can be inserted into a dip-slide incubator.

<span class="mw-page-title-main">Fanny Hesse</span> German biologist (1850–1934)

Fanny Hesse is best known for her work in microbiology alongside her husband, Walther Hesse. Following her initial suggestion of using agar as an alternative to gelatin, they were instrumental in pioneering agar's usage as a common gelling agent for producing media capable of culturing microorganisms at high temperatures.

In microbiology, the term isolation refers to the separation of a strain from a natural, mixed population of living microbes, as present in the environment, for example in water or soil, or from living beings with skin flora, oral flora or gut flora, in order to identify the microbe(s) of interest. Historically, the laboratory techniques of isolation first developed in the field of bacteriology and parasitology, before those in virology during the 20th century.

<span class="mw-page-title-main">Inoculation needle</span>

An inoculation needle is a laboratory equipment used in the field of microbiology to transfer and inoculate living microorganisms. It is one of the most commonly implicated biological laboratory tools and can be disposable or re-usable. A standard reusable inoculation needle is made from nichrome or platinum wire affixed to a metallic handle. A disposable inoculation needle is often made from plastic resin. The base of the needle is dulled, resulting in a blunted end.

Diagnostic microbiology is the study of microbial identification. Since the discovery of the germ theory of disease, scientists have been finding ways to harvest specific organisms. Using methods such as differential media or genome sequencing, physicians and scientists can observe novel functions in organisms for more effective and accurate diagnosis of organisms. Methods used in diagnostic microbiology are often used to take advantage of a particular difference in organisms and attain information about what species it can be identified as, which is often through a reference of previous studies. New studies provide information that others can reference so that scientists can attain a basic understanding of the organism they are examining.

<span class="mw-page-title-main">Colonial morphology</span> Examination of microbial colonies

In microbiology, colonial morphology refers to the visual appearance of bacterial or fungal colonies on an agar plate. Examining colonial morphology is the first step in the identification of an unknown microbe. The systematic assessment of the colonies' appearance, focusing on aspects like size, shape, colour, opacity, and consistency, provides clues to the identity of the organism, allowing microbiologists to select appropriate tests to provide a definitive identification.

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