What Is Pure Culture

6.4B: Pure Culture

Objectives for Learning

  • Objectives of Learning

A fundamental and fundamental diagnostic procedure in molecular biology, microbial cultures are widely employed as a research tool for molecular biology researchers. It is frequently necessary to isolate a pure culture of microorganisms for several reasons. A pure (or axenic) culture is a colony of cells or multicellular creatures that is developing in the absence of any other species or types of organisms other than their own. It is possible for a pure culture to be derived from only a single cell or single organism, in which case the cells are genetic clones of each other.

Agar is a gelatinous material formed from seaweed that is used in food preparation.

  • Figure: Geomyces destructans in culture from bat tissues, grown in selective medium.
  • destructans strains develop profusely in the original culture tubes of Sabouraud agar treated with nine antibiotics and kept at 4°C for six or eight weeks; observe the abundant growth of G.
  • A close-up of a culture tube demonstrates that some fungal contamination was present on individual isolates in (B).
  • Agar-based growth media may be used to cultivate microbiological cultures in petri dishes of various sizes, which are coated with a thin coating of agar for growth.
  • Another form of bacterial culture is liquid culture, which involves suspending the desired bacteria in a liquid broth, which serves as a nutritional media.
  • The researcher would inoculate a liquid broth with bacteria and allow it to develop for an overnight period of time (they may use a shaker for uniform growth).
  • As an alternative, the microbiologist may choose to employ static liquid cultures, which are not constantly changing.

Key Points

  • A fundamental and fundamental diagnostic approach in molecular biology, microbial cultures are widely employed as a research tool for molecular biology study. A pure culture of microorganisms must be isolated on a regular basis. A pure (or axenic) culture is a population of cells or multicellular creatures that is developing in the absence of any other species or types of organisms, such as bacteria or viruses. It is possible for a pure culture to be derived from one cell or one organism, in which case the cells are genetic clones of one another. It is necessary to employ agarose gel (agar) as a medium for the microbial growth in order to gel it. Agar is a gelatinous material generated from seaweed that may be used in food preparation. When agar is not available, guar gum can be used to isolate and maintain thermophiles at a lower cost than agar. Figure: Geomyces destructans in culture from bat tissues, grown on selective media (right). Note the abundant development of G. destructans strains in the original culture tubes of Sabouraud agar treated with nine antibiotics and cultured at 4°C for six- or eight-weeks. The presence of fungal contamination on individual isolates was obvious in the close-up of a culture tube (Figure B). A culture infected with bacteria was treated with hydrochloric acid, which resulted in the enrichment and recovery of pure fungal colonies. C) It is possible to cultivate microbiological cultures in petri dishes of varying sizes that have been coated with a thin coating of an agar-based growth medium. As soon as the required bacteria have been introduced into the growth media in the petri dish, the plates are incubated at the optimal temperature for the bacteria’s development (for example, usually at 37 degrees Celsius for cultures from humans or animals or lower for environmental cultures). In addition to solid culture, liquid culture is a form of bacterial culture in which the desired bacteria are suspended in a liquid broth that contains nutrients. Antimicrobial assays may be prepared with ease using these. The researcher would inoculate a liquid broth with bacteria and allow it to develop for an overnight period of time (they may use a shaker for uniform growth). Afterwards, they would obtain aliquots of the material in order to test for the antibacterial activity of a certain medication or protein in question (antimicrobial peptides). Instead of using static liquid cultures, the microbiologist may choose to employ static liquid cultures as an alternate method. There is no shaking in these cultures, and they create an oxygen gradient for the microorganisms to thrive.

Key Terms

  • It is used as a bacterial culture medium, as a gel in electrophoresis, and as a food ingredient. Agar is made from sea algae and is gelatinous in consistency.

The Virtual Edge

Pure cultures, as previously discussed in the preceding lab, are required for microbiological research purposes. Most commonly, this is performed by spreading a pure culture of bacteria across the surface of a solid medium such that a single cell occupies an isolated part of the surface of the agar media.

This single cell will undergo recurrent multiplication, resulting in the formation of a visible colony of similar cells, often known as clones. When it comes to creating a pure culture, there are three ways that are widely used:

  1. Pure cultures are required for microbiological experiments, as was discussed in the last lab. Most commonly, this is performed by spreading a pure culture of bacteria across the surface of a solid medium so that a single cell occupies an isolated part of the surface of the agar media. In order to form a visible colony of similar cells (clones), this single cell will go through serial multiplication. When it comes to creating a pure culture, there are three typical methods:
All of these methods dilute or “thin out” a heavy population of bacteria across an agar surface.The spread plate technique was used in lab5 to obtain isolated colonies.In this lab we will learn to streak a plate with a mixed culture containing more than one bacterial species.Since most bacterial samples encountered by a microbiologist (in the clinic, environment, industry, etc.) are mixed cultures, this is a very important microbiological technique.If this procedure is performed correctly, a number of isolated colonies will grow that will be a source of pure bacterial cultures.Streak plate – Triplet Streak.There are many variations of the streaking technique, but in this lab we will use the triplet streak as described here.Be sure to note the diagrams and description of the technique on the following page before starting with your streak plate.

Pure Culture

The bacteria that live in our surroundings are a diverse group of organisms. Each of these categories of species makes a positive contribution to the ecology of the area. Having said that, have you ever pondered how research on such bacteria are carried out? Is it possible to investigate and experiment with all of these at the same time? No, not at all. It isn’t the case. In order to do research on these bacteria, it is necessary to separate the mixed culture into a pure culture before proceeding.

What is the function of this item?

What exactly are pure culture methods, or pure culture approaches, in this context?

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Pure Culture Definition

Put another way, a pure culture in microbiology is described as a laboratory culture that includes just one species of organisms. This definition comes from the American Society for Microbiology. The majority of the time, microbes are found in mixed cultures. However, by transferring a little amount of its sample onto a fresh and sterile growing media, it is possible to obtain a pure culture of the organism. The dispersion of cells across the surface medium is typically used in the process of pure culture isolation to achieve this result.

  • Pure culture techniques are used by scientists to carry out the process of creating discrete colonies of pure microorganisms.
  • During the mid-19th century, Robert Koch was principally responsible for the development of procedures for the separation of pure cultures.
  • The introduction of such pure culture techniques resulted in the identification of bacteria that were responsible for the transmission of anthrax, TB, and other serious illnesses.
  • Do you want to know if pure culture organisms may be used for any kind of commercial purposes?
  • For many years, it has been primarily utilized for commercial fermentation purposes, including the production of yoghurt, alcohol, citric acid, lactic acid, and a variety of other drinks.

Pure Culture Collections:

For the uninitiated, pure culture is described as a laboratory culture that includes just one species of organisms in microbiology, and it is a straightforward definition. The majority of the time, microbes are found in mixtures. However, by transferring a little amount of its sample into a fresh and sterile growing medium, it is possible to obtain a pure culture. The dispersion of cells across the surface medium is a common method of achieving pure culture separation in most cases. Preparation for inoculation into a new media mostly entails thinned samples being introduced into the old medium.

  • During the mid-19th century, Robert Koch was principally responsible for the development of procedures for isolating pure cultures.
  • The introduction of pure culture methods led to the identification of bacteria that were responsible for the transmission of illnesses such as anthrax and TB.
  • Interested in knowing whether organisms cultivated in pure culture may be used for a variety of commercial applications?
  • Over the last several decades, it has mostly been utilized for commercial fermentation purposes, such as the production of yoghurt, alcohol, citric acid, lactic acid, and a variety of other drinks.

It has also aided in the creation of various vaccines and medicines through the use of pure culture techniques.

How is the Purity of Culture assessed?

There are a number of various approaches that may be used to determine the purity of culture. These are the ones to look out for:

  • The same cultural features are demonstrated on the media derived from isolated colonies of pure culture organisms
  • The same culture organisms seem to be the same when seen under the microscope under the microscope. Essentially, this means that they both get the same dye and have the same morphology. Similarly to similar biochemical results, isolated colonies of pure culture perform equally, i.e., independently of one another.

Biochemical Characteristics of Pure Culture

  • In the case of tests, the mixed culture organisms provide a variety of varied effects. In the event of tests, the pure culture organisms produce findings that are identical to those obtained from the wild.

What is the Importance of Pure Culture in Microbiology?

  • If you do tests using mixed culture organisms, you will get various findings. Even in the case of tests, the pure culture organisms provide identical findings.

Pure Culture Techniques Microbiology

The following are the many ways that may be used to obtain pure cultures in microbiology:

  • Preparation of media and glassware for sterilization
  • Individual cells disperse over the media as a result of this process. Before the inoculation of fresh media, the samples are thinned many times before they are inoculated with the new media itself.

Microbiological culture – Wikipedia

Cultures of microorganisms on solid and liquid medium A microbiological culture, also known as a microbial culture, is a process of multiplying microorganisms by allowing them to reproduce in a specified culture medium under controlled laboratory circumstances, also known as a microbiological culture. The utilization of microbial cultures as a research tool in molecular biology is based on the foundational and fundamental diagnostic approaches. The term culture can also apply to the microorganisms that are being raised in a laboratory.

  1. It is one of the basic diagnostic tools in microbiology, and it is used as a tool to pinpoint the source of an infectious illness by allowing the agent to proliferate in a specific medium.
  2. This procedure is called a throat culture.
  3. It is frequently necessary to isolate a pure culture of microorganisms for several reasons.
  4. An isolated cell or single organism may be responsible for the formation of the pure culture; in this instance, all of the cells are genetic clones of one another.
  5. Agar is a gelatinous material formed from seaweed that is used in food preparation.

Bacterial culture

There are various different types of bacterial culture methods, and the method that is used is determined by the agent that is being grown and the downstream use.

Broth cultures

Bacterial culture can be accomplished by the use of liquid culture, in which the desired bacteria are suspended in an upright flask of liquid nutritional media (such as Luria Broth) and allowed to grow. Using this method, a scientist may cultivate huge quantities of bacteria for use in a number of downstream applications. When doing an antimicrobial assay, liquid cultures are useful because they allow the researcher to inoculate liquid broth with bacteria and allow it to develop overnight in the laboratory (they may use a shaker for uniform growth).

As an alternative, the microbiologist may choose to employ static liquid cultures, which are not constantly changing. These cultures are not disturbed, and they offer a gradient of oxygen to the bacteria in the culture.

Agar plates

It is possible to cultivate microbiological cultures in petri dishesof various sizes that contain a thin coating of agar-based growth media on the bottom. After the chosen bacteria have been introduced into the growth media in the petri dish, the plates are incubated at a temperature that is appropriate for the development of the selected bacteria (for example, usually at 37 degrees Celsius, or thehuman body temperature, for cultures from humans or animals, or lower for environmental cultures).

In addition to the ingredients listed above, agar can also be mixed with other ingredients before being put into a plate and allowed to set.

When producing engineered strains of bacteria that have an antibiotic-resistance gene, this method can also be applied.

In this way, only the colonies that were successfully changed may be chosen by the researcher for further study.

Agar based dipsticks

Agar plates that have been miniaturized and integrated into dipstick forms, such as Dip Slide and Digital Dipstick, have the potential to be employed at the point of care for diagnostic purposes. They offer benefits over agar plates in that they are less expensive and do not need the use of specialized knowledge or a laboratory setting, allowing them to be employed at the point of care.

Stab cultures

Along the stab lines, bacteria that are motile and bacteria that are not motile may be distinguished. Unlike non-motile bacteria, motile bacteria will expand out from the stab line, but non-motile bacteria are only present along the stab line. The formation of stab cultures is identical to that of agar plates, except that solid agar is used in a test tube. Bacteria are injected into the agar with the use of an innoculation needle or a pipette tip that is inserted into the middle of the plate.

For short-term culture storage or transport, Stab cultures are the most widely employed culture media type.

Culture collections

Cell lines and other materials for research in microbiological systematics are collected and catalogued in microbial culture collections. Microbial culture collections are concerned with the acquisition of standard reference microorganisms, cell lines, and other materials for research in microbiological systematics. Culture collections serve as both storage facilities and repositories for type strains.

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Major national culture collections.

Collection Acronym Name Location
ATCC American Type Culture Collection Manassas,Virginia
BCCM Belgian Co-ordinated Collections of Micro-organisms Decentralized, Coordination Cell inBrussels,Belgium
CCUG Culture Collection University of Gothenburg Gothenburg, Sweden
CECT Colección Española de Cultivos Tipo Valencia, Spain
CIP Collectiond’Institut Pasteur Paris,France
DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen Braunschweig,Germany
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

Solid plate culture of thermophilic microorganisms

Cell lines and other materials for research in microbiological systematics are collected and catalogued in microbial culture collections.

Microbial culture collections are concerned with the acquisition of standard reference microorganisms, cell lines, and other materials for research in microbiology systematics. It is also possible to find strains of interest in culture collections.

Virus and phage culture

A viral culture or phage culture requires the presence of host cells in which the virus or phage may reproduce. Bacteriophage cultures are created by infecting and multiplying within bacterial cells. The phage may then be separated from the plaques that form in a lawn of bacteria on a plate by using a phage isolation kit. Viruscultures are produced from eukaryotic host cells that have been selected for their virus. The streak plate method is a technique for physically separating the microbial population.

Upon incubation, colonies will form and single cells will be extracted from the biomass, indicating that the procedure was successful.

It is necessary to preserve stock cultures in such a way that their biological, immunological, and cultural characteristics are not compromised.

Eukaryotic cell culture

The separation of pure cultures from single-celled eukaryotes, such as yeast, is accomplished using the same procedures as those used for bacterial cultures. Pure cultures of multicellular organisms are frequently more easily isolated by simply selecting a single individual from which to start a culture than by using a culture medium. Among other things, this approach is beneficial for the pure cultivation of fungi, multicellularalgae, and smallmetazoa, among others. To properly observe the specimen in issue, it is critical to develop pure culture procedures that are specific to the specimen in question.

Streak plate technique is a physical separation of the microbiological population that is accomplished by distributing the inoculate back and forth over the solidagar plate using an inoculating loop over the solidagar plate.

In order to research and utilize a microbe once it has been isolated in pure culture, it is important to keep it alive until it can be studied and used.

See also

  • Cellular colony-forming unit
  • Blood culture
  • Microbial dark matter
  • Microbial food cultures
  • Screening cultures
  • Sputum culture
  • Synchronized culture
  • Gellan gum

References

  1. Healthwise, Inc. is a for-profit corporation (2010-06-28). “Culture of the Throat.” WebMD. The original version of this article was archived on 2013-03-17. Old, D.C., and Duguid, J.P. (2013). Retrieved 2013-03-10
  2. Old, D.C., and Duguid, J.P. (1970). “Selective Outgrowth of Fimbriate Bacteria in Static Liquid Medium” is a scientific paper that describes the selective outgrowth of fimbriate bacteria in a static liquid medium. In 1970, the Journal of Bacteriology, published by the American Society for Microbiology, was published in two parts: 447–456. doi: 10.1128/JB.00447–456.1970.PMC248102.PMID4914569 and Iseri, Emre, Biggel, Michael, Goossens, Herman, Moons, Pieter, and van der Wijngaart, Wouter (2020). In the paper “Digital dipstick: miniaturized bacteria detection and digital quantification for the point-of-care,” the authors describe a dipstick that may be used to detect bacteria. The article, “Addgene: Streaking a Plate from an Addgene Stab Culture”, appears in Lab on a Chip, volume 20, number 23, pages 4349–4356. doi:10.1039/D0LC00793E.ISSN1473-0197.PMID33169747. The original version of this article was published on April 8, 2018. retrieved on the 21st of March, 2018
  3. Michael T. Madigan, abMadigan, Michael T. (2012). Microorganisms and their Biology by Brock (13th ed.). abUruburu, F. (2003). “History and services of cultural collections.” San Francisco: Benjamin Cummings, ISBN 9780321649638
  4. AbUruburu, F. (2003). “History and services of culture collections” (PDF). 6.(2): 101–103
  5. Doi: 10.1007/s10123-003-0115-2
  6. Hdl:10550/12955
  7. PMID12811589
  8. S2CID19711069
  9. A gelling agent in media for the growth of thermophilic microorganisms was developed by Chi Chung Lin and L. E. Casida in 1984, and the results were published in the journal GELRITE. 427-429 in Applied and Environmental Microbiology, vol. 47.

External links

  • EFFCA is an acronym for the European Food and Feed Cultutes Association. Information about the development and applications of microbial cultures, as well as legislative considerations

ex 12 -​ Pure culture technique

  1. Obtain three empty plates (Petri dishes) for this project. Label them with the numbers 1, 2, and 3 as well as their names, lab days and times, and the organism
  2. Acquire the broth containing the mixed bacterial culture as well as a loop, and then ignite the loop. To begin, remove from the waterbath one TSA tube (TSA tube 1) that has melted and quickly add one loopful of mixed culture broth to the tube of melted TSA (TSA tube1)
  3. Make use of a loop to mix for 5 seconds
  4. Have your partner remove another melted TSA tube (TSA tube 2) from the water bath and quickly transfer one loopful of the mixture from the first melted TSA tube (TSA tube 1) to the second melted TSA tube (TSA tube 2)
  5. Repeat the process with the third melted TSA tube (TSA tube 3). 5 seconds should be mixed using the loop. Pour the contents of TSA tube 1 into the petri dish labeled 1 as soon as it is possible. Have your partner remove a third melted TSA tube from the water bath (TSA tube 3) and immediately transfer one loopful of the mixture from the second melted TSA tube (TSA tube 2) to the third melted TSA tube (TSA tube 3)
  6. Immediately pour the contents of TSA tube 2 into the petri plate labeled 2
  7. Immediately pour the contents of TSA tube 3 into the petri plate labeled 3
  8. Immediately pour the contents of TSA tube 2 into the petri plate labeled Allow for gentle rotation of the plate to assist the media in spreading out before it solidifies. To minimize condensation, incubate your plates upside down for a few hours after they have solidified

Why are Petri dishes stored upside-down?Petri platesare incubatedupside-downto lessen the risk of contamination from airborne particles settling on them and to prevent the accumulation of any water condensation that may otherwise disturb or compromise a culture.

When it comes to isolating your colonies, you’ll need your streak plates and pour plates along with three TSA slants.

  • You should notice distinct independent colonies that are different shades of red, purple, and white
  • This is the ideal situation. Take one of each colour colony and place it in its own dedicated slant tube, which should be kept at 25 degrees C.

Following incubation, prepare a smear prep of the contents of each of your slants and do a Gram-stain on each of the samples you collected.

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Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment

The vast majority (ninety-nine percent) of microorganisms from the environment are resistant to laboratory culture. It appears that uncultivated organisms may be discovered in virtually every bacterial group, and numerous divisions have no known cultivable members, according to ribosomal RNA research. Our team developed a diffusion chamber that permitted the development of microorganisms that had never been before cultured in a mimicked natural environment. In pure culture, colonies of representative marine creatures were extracted and studied.

It was discovered that these isolates did not grow on artificial media alone, but instead formed colonies when exposed to other microbes. This insight may be useful in understanding the nature of microbial uncultivability in general.

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An impure “pure culture”

It was previously discovered that a culture of the electrical current generating bacteria Geobacter sulfurreducansstr. DL1 may be developed in order to select an isolate with the ability to create greater amounts of electricity. A DNA sequence analysis of the isolate (designated KN400) revealed that it included more than 27,000 SNPs and 139 distinct ORFs, indicating that it could not have developed fromG. sulfurreducansstr. DL1. As a result, it was thought to be an external contaminant that had been introduced accidently during the experiment.

sulfurreducansstr.

This was the case despite the DL1 strain having undergone the standard approach of serial dilution and repeated re-streaking of isolated colonies on solid agar to ensure its purity.

Because of these findings, it is clear that deep sequencing might miss exceedingly uncommon variations, and that the repeated-streaking strategy is insufficient for obtaining a pure culture.

References

  1. It was previously discovered that a culture of the electrical current generating bacteria Geobacter sulfurreducansstr. DL1 may be developed in order to select an isolate with the ability to create greater amounts of electrical current. A DNA sequence analysis of the isolate (designated KN400) revealed that it included more than 27,000 SNPs and 139 unique ORFs, indicating that it could not have developed from G. sulfurreducansstr. DL1. Because of this, it was considered to be an external contaminant that had been introduced accidently throughout the trial. The KN400 strain was present in very low abundance in the initialG. sulfurreducansstr. DL1 culture, according to Shresthaet al., who discovered this by sequencing a gene that differed by 14 bp between the DL1 and KN400 strains. This was discovered despite the DL1 strain having undergone the standard approach of serial dilution and repeated re-streaking of isolated colonies on solid agar to ensure its purity. A prior deep sequencing experiment using purified DL1 strain to 80-fold coverage failed to find any sequences peculiar to the KN400 strain. Because of these findings, it is clear that deep sequencing can miss exceedingly uncommon variations, and that the repeated-streaking method is insufficient for obtaining a pure culture. A single cell culture method is recommended by the authors in order to eliminate undetectable contamination, which may be difficult to implement for many environmental bacteria because of their complex growth patterns.

References can be downloaded.

About this article

C. Khrström’s impure “pure culture” is discussed in detail in this paper. Nature Reviews Microbiology11,301 (2013). citation

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