What Does A Culture Medium Provide To A Living Cell

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What does a culture medium provide to a living cel class 12 biology CBSE

A live cell’s culture medium serves as a safehouse, keeping it safe from all of the assaults and harsh circumstances of the environment, as well as from any other variables that may interfere with the cell’s ability to thrive. Answer in its entirety: The culture media is a closed medium that has been sterilized to remove germs, and then numerous nutrients in an exact known quantity have been added to the medium to provide the nutrients required for the growth and development of the live cell.

The cell contains a significant amount of plant and animal tissue extract, which may be yeast extract or even peptides.

In a typical configuration, the culture medium contains the following components: A base solution, such as agar, is used.

A carbon source, such as glucose, that provides energy.

Different types of salts are available.

Water containing a source of amino acids and nitrogen, such as meat extract or yeast extract, for example.

Note: Nutrient media are available in a variety of forms, the majority of which are semisolid materials that include a high concentration of water.

Growth medium – Wikipedia

An agar plate is an example of a bacterial growth medium*, and it looks like this: It is an astreak plate, and the orange lines and dots are generated by bacterial colonies on the surface of the plate. A growth medium, also known as a culture media, is a solid, liquid, or semi-solid that is used to promote the growth of a population of microorganisms, cells (via the process of cell proliferation), or tiny plants (such as the mossPhyscomitrella patens) in a laboratory setting. For the growth of different types of cells, different types of media are utilized.

Cell culture is a technique for growing specific cell types derived from plants or animals.

A particular habitat is required for some organisms, referred to as fastidious organisms, due to the complicated dietary requirements of these species.

Types

Nutrient broths (also known as liquid nutrient medium) and lysogeny broth medium are the most often used growth media for microorganisms. Liquid media are frequently combined with agarose and dispensed onto Petri plates using a sterile media dispenser to solidify. These agar plates serve as a solid culture medium for microorganisms to grow and multiply. They remain solid because only a few number of bacteria are capable of decomposing agar (the exception being some species in the genera:Cytophaga,Flavobacterium,Bacillus,Pseudomonas, andAlcaligenes).

It is important to understand the difference between growth media used for cell culture and growth media used for microbiological culture because cells derived from whole organisms and grown in culture frequently cannot grow without the addition of growth factors or hormones that normally occur in vivo.

  • Microorganisms, on the other hand, are frequently unicellular organisms, therefore there are no such restrictions on their growth.
  • This is another significant distinction from animal cells raised in the wild.
  • The difference between defined and undefined growth media is a significant distinction between the two forms of growth media.
  • It consists of giving trace elements and vitamins that are necessary by the bacterium, as well as specifically defined carbon and nitrogen sources.
  • Some complex compounds, such as yeast extractor casein hydrolysate, are found in an undefined medium.
  • In some cases, undefined media are chosen based on price, while in others, they are used out of necessity – for example, some bacteria have never been cultivated on defined media.
  • The wort has all of the nutrients necessary for yeast development, and when fermented in an anaerobic environment, it produces alcohol.

Beer is ready for drinking once the fermentation process is complete, and the mix of medium and dormant bacteria has been transformed into a fermentable liquid. The most common kinds are as follows:

  • Media that is cultural
  • Minimum media
  • Selective media
  • Differential media
  • Transit media
  • Indicator media

Culture media

Culture media include all of the nutrients and growth factors that most bacteria require to thrive, and because they are non-selective, they are employed for the general growing and maintenance of bacteria in laboratory cultures collections. An indefinite media (also known as a base medium or a complicated medium) has the following components:

  • The following ingredients are required: a carbon source such as glucose, water, different salts, a supply of amino acids and nitrogen (for example, beef, yeast extract)

A medium with no specified composition is created by combining amino acids from various sources, the exact composition of which is not known at the time of writing. When you work in a specified medium, you are working in an environment that has been chemically defined or synthesized.

  • Each and every one of the compounds employed is well-known
  • There is no evidence of yeast, animal, or plant tissue.

The following are examples of nutritional media:

  • Nutrient agar, plate count agar, trypticase soy agar, and other agars are used.

Minimal media

The term “minimal media” refers to a specified medium that contains just the components necessary to enable development. When growing microorganisms in a minimum medium, the quantity of components that must be added varies greatly depending on the microbe being cultivated. Minimal media are those that contain the very minimum of nutrients necessary for colony formation, and are often devoid of amino acids. They are frequently employed by microbiologists and geneticists to cultivate “wild-type” microorganisms in their laboratories.

The following are typical components of minimal medium:

  • A carbon source, which can be either a sugar such as glucose or a less energy-dense source such as succinate
  • Various salts, which can vary depending on the bacteria species and growing conditions
  • These generally provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the bacteria to synthesize protein and nucleic acids
  • Water
  • And a nitrogen source.

The term “supplemental minimum media” refers to minimal media that also include a single specified chemical, which is often an amino acid or a sugar. This supplementation enables the cultivation of specific lines of auxotrophic recombinants in the laboratory.

Selective media

Campylobacter isolates were obtained using a blood-free, charcoal-based selective medium agar (CSM). Blood agar plates are frequently used in the diagnosis of infectious diseases. A positiveStaphylococcusculture is shown on the right, while a positiveStreptococcusculture is shown on the left. Selective media are used to promote the development of just specific microorganisms in a laboratory setting. Suppose a microbe develops resistance to a certain antibiotic, such as ampicillin or tetracycline.

  • The use of media missing an amino acid, such as proline, in combination with E.
  • Selection media are also utilized in the cell culture process in order to guarantee the survival or multiplication of cells that possess certain characteristics, such as antibiotic resistance or the ability to produce a specific metabolite.
  • In such instances, the gene is referred to as a marker.
  • Gancycloviris an exception to the norm, since it is used to precisely destroy cells that have the Herpes simplex virus thymidine kinase, which is a marker for the virus in question.

Four distinct types of agar plates were used to demonstrate how bacterial metabolism might influence growth. Examples of selective media include the following:

  • To isolate Campylobacter bacteria, blood-free, charcoal-based selective medium agar (CSM) was utilized. Infections are frequently diagnosed using blood agar plates. Those on the right have a positiveStaphylococcusculture, and those on the left have a positiveStreptococcusculture, respectively. In order to promote the development of certain bacteria, selective media must be utilized. A microbe that is resistant to a certain antibiotic, such as ampicillin or tetracycline, may be grown in the presence of that antibiotic, which prevents other cells from developing that do not have the resistance from multiplying. Before the advent of genomics, geneticists often utilized media missing an amino acid, such as proline, in combination with E. coli strains that were unable to manufacture the amino acid. Certain growth media are also employed to ensure the survival or multiplication of cells with specific characteristics, such as antibiotic resistance or the ability to produce a specific metabolite, in cell culture. A cell’s capacity to develop in a selective medium is normally conferred upon it by the presence of a specificgeneor an alleleof a gene. It is referred to be a marker gene when this occurs. Neomycin is often used in selective growth medium for eukaryotic cells to identify cells that have been successfully transfected with a plasmid containing the neomycin resistance gene as a signal for successful transfections. As an exception to the rule, Gancycloviris used to target destroy cells that have the Herpes simplex virus thymidine kinase, which is a flag for the virus. Based on bacterial metabolic activity, four distinct types of agar plates were used to demonstrate differential growth. Selective media examples include:

Differential media

Media that identify one species of microbe from another that is growing on the same medium are referred to as differential or indicator media. To visually identify the defining characteristics of a microorganism, this type of media employs the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, ormethylene blue) that have been added to the medium. These media are used by microbiologists to detect microorganisms, and by molecular biologists to identify recombinant strains of bacteria in a variety of settings.

  • It is utilized in strep tests because it includes bovine heart blood that turns translucent in the presence of -hemolytic organisms such as Streptococcus pyogenes andStaphylococcus aureus. Lactose fermentation is aided by the use of eosin methylene blue
  • Nevertheless, This medium is selective and differential for Streptococcus agalactiae (group B streptococcus), which develops as characteristic red colonies on the surface of the culture media. Lactose fermentation is facilitated by MacConkey agaris differential
  • Agar containing mannitol salt has a difference effect on mannitol fermentation, and X-galplates have a differential effect on lac operonmutants.

Transport media

The following requirements should be met by transportation media:

  • The temporary storage of specimens while they are being transferred to the laboratory for culture
  • Maintain the viability of all organisms present in the specimen without affecting their concentration
  • And. Only buffers and salt are used in this formulation. Lack of carbon, nitrogen, and organic growth factors, which prevents microbial reproduction
  • Lack of organic growth nutrients. In order to isolate anaerobes, the transport media utilized in the isolation process must be devoid of molecular oxygen.
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Examples of transportation media include:

  • In the case of strictanaerobes, thioglycolate broth is used. Stuart transfer medium is a non-nutritional soft agar gel that contains a reducing chemical to avoid oxidation as well as charcoal to neutralize the environment. Gonococci are treated with specific bacterial inhibitors, while enteric bacilli are treated with buffered glycerol saline. For V. cholerae, the Venkataraman Ramakrishna (VR) medium is employed.

Enriched media

Increased nutrient availability provides the nutrients necessary to sustain the growth of a wide range of species, including some of the more finicky organisms. They are typically used to collect as many distinct types of bacteria from a specimen as there are germs present in it. Blood agar is an enhanced media in which nutritionally dense whole blood replenishes the basic nutrients in the presence of other nutrients. It is made up of dark chocolate agaris that have been heat-treated to 40–45 degrees Celsius (104–113 degrees Fahrenheit), which turns brown and provides the medium its name-giving hue.

Physiological relevance

In tissue culture investigations, the choice of culture media can have an impact on the physiological significance of the findings, which is especially true for metabolic research. Furthermore, it was shown that the kind of media used can influence the dependency of a cell line on a metabolic gene. When doing a study with several cell lines, using a homogeneous culture medium for all of the cell lines may help to limit the amount of bias in the datasets that are created. Increased physiological relevance of in vitro research can be achieved by using a growth medium that more accurately replicates physiological amounts of nutrients.

See also

  • Cell culture, impedance microbiology, and modified Chee’s medium are among topics covered in this course.

References

  1. Madigan, M., and Martinko, J. (eds). (2005). Brock’s Microorganism Biology is a textbook that teaches microorganism biology (11th ed.). It is published by Prentice Hall with the ISBN number 0-13-144329-1. Birgit Hadeler, Sirkka Scholz, and Ralf Reski are three of the most talented people in the world (1995). A moss’ cytokinin-sensitivity is influenced differentially by gelrite and agar, according to the researchers. Journal of Plant Physiology, vol. 146, no. 3, pp. 369–371. maintainer of CS1: makes use of the authors parameter (link)
  2. K.J. Ryan and C.G. Ray, eds (2004). Sherris Medical Microbiology is a medical microbiology company (4th ed.). Hans Günter Schlegel’s book is published by McGraw Hill and has the ISBN number 0-8385-8529-9. (1993). Cambridge University Press, p. 459. ISBN 978-0-521-43980-0. General Microbiology. Cambridge University Press. retrieved on August 6, 2013
  3. Parija and Shubhash Chandra are two of the most talented musicians in the world (1 January 2009). Textbook of Microbiology and Immunology, Elsevier India, p. 45, ISBN 978-81-312-2163-1. Textbook of Microbiology and Immunology, Elsevier India, p. 45, ISBN 978-81-312-2163-1 Cooper, GM (retrieved on August 6, 2013)
  4. (2000). “Cell Biology Instruments” is an abbreviation for “Cell Biology Tools.” The Cell: A Molecular Approach to Understanding It ASM Press, Washington, D.C., ISBN 0-87893-106-6
  5. Catherine A. Ingraham and John L. Ingraham are the authors (2000). G. D. W. Curtis and Rosamund Baird are the editors of the textbook Introduction to Microbiology (Corry et al., ed). (1995-01-01). An agar containing dichloran rose bengal chloramphenicol (DRBC) was developed. Progress in Industrial Microbiology, vol. 34, no. 3, pp. 303–305, published by Elsevier, doi: 10.1016/s0079-6352(05)80036-0, ISBN 9780444814982. Retrieved2020-04-20
  6. s^ John A. Washington, Jr. (1996). “Principles of Diagnosis” is an acronym for “Principles of Diagnosis.” S. Baron and colleagues (eds.). Baron’s Medical Microbiology is a textbook that teaches medical microbiology (4th ed.). Univ of Texas Medical Branch.ISBN0-9631172-1-1
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  9. Lagziel S, Lee WD, Shlomi T. Nature Metabolism.2(12): 1369–1372.doi: 10.1038/s42255-020-00299-y.PMID33046912.PMID33046912.S2CID222319735
  10. Lagziel S, Lee WD, Shlomi T. Lagziel S, Lee WD, Shlomi T. (2019). “Inferring cancer dependencies on metabolic genes from large-scale genetic screens” is the title of this article. Vande Voorde J, Ackermann T, Pfetzer N, Sumpton D, Mackay G, Kalna G
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  12. Vande Voorde J, Ackermann T, Pfetzer N, Sumpton D, Mackay G, Kal (2019). Improved metabolic fidelity of cancer models using a physiological cell culture medium is the subject of this study. Science Advances.5(1): eaau7314 (early access). The Bibcode for this paper is 2019SciA.5.7314V.doi: 10.1126/sciadv.aau7314.PMC6314821.PMID30613774.CS1 maint: multiple names: authors list (link)
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  15. And others (2017). A study published in the journal Physiologic Medium revealed that Uric Acid is an endogenous inhibitor of UMP Synthase and that it rewired cellular metabolism. Cell.169(2): 258–272.e17.doi: 10.1016/j.cell.2017.03.023.PMC5421364.PMID28388410.CS1 maint: multiple names: authors list (link)
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The creation of adequate circumstances for the survival and/or multiplication of live cells is essential while doing laboratory research in the biological sciences. Cell cultures need the presence of specific environmental conditions. Cells require the following nutrients for proper development and proliferation:

  • Temperature regulation
  • PH and osmolality levels that are appropriate
  • A surface on which cells can adhere themselves
  • It refers to a specific type of culture media
  • It is necessary to have an incubator to keep the conditions steady.

One of these crucial components, cell culture medium, will be discussed in detail in this article. What cell culture media is, what components make it up, what varieties there are, and what to look for when selecting an appropriate cell culture medium will all be covered in this section.

What is cell culture media?

One of these crucial components, cell culture medium, will be discussed in depth in this article.

What cell culture media is, what components make it up, what varieties there are, and what to look for when choosing an appropriate cell culture medium will all be covered in this section.

What are the components of cell culture media?

Cell culture medium are made up of a combination of chemicals and nutrients that are specifically intended to promote cellular proliferation. The following are common components of cell culture media:

  • Amino acids are the building blocks of protein, and every cell culture medium contains a combination of amino acids. It is possible to employ both essential and non-essential amino acids to increase cell viability and proliferation. Vitamins: Vitamins are added in order to aid in the formation and multiplication of cellular structures. A large number of vitamins are obtained from serum when using serum-containing media
  • However, vitamins must be added to serum-free media. Energy-producing carbohydrates: Carbohydrates are the primary source of energy for living cells. Although glucose is the most often utilized carbohydrate, other sugars such as galactose, fructose, and maltose are also accessible. Inorganic salts are required for the regulation of membrane potential and osmolality
  • However, inorganic salts are not required for the regulation of membrane potential. Basic and trace elements: For cells to develop, they require elements such as iron, potassium, magnesium, and zinc. Proteins in serum include growth factors and inhibitors, hormones, protease inhibitors, chelators, amino acids, carbohydrate and lipid metabolites, vitamins, trace elements, minerals, and other substances that are essential for cellular development and growth. The most widely used serum is bovine serum. In order to alter cell activity, growth, and proliferation, certain hormones may be added to the mixture. pH buffering systems: pH buffering systems are used to adjust the pH of a solution. Supplements: Supplements such as hormones, enzyme inhibitors, and trace elements are occasionally added to cell culture medium to make them more suitable for the cell type and study purpose in question. Drugs that suppress fungal and bacterial development are added to cell culture conditions to prevent the growth of these organisms. Thanks to proteins that bind portion of the antibiotic load, serum media are the optimum medium for antibiotics to be used in. Cells cultured in serum-free medium, on the other hand, are more susceptible to antibiotic toxicity.

What are the different types of cell culture media?

Natural media and synthetic media are the two basic types of cell culture media that may be used in cell culture.

Natural media

Natural media are those that are obtained from tissue extraction or animal bodily fluids, such as plasma, lymph, and serum, among other sources of information. Generally speaking, natural media may be divided into three categories:

  • Biological fluids include serum, plasma, lymph, and amniotic fluid, to name a few examples. Tissue extracts: Examples of tissue extracts include embryo, bone marrow, tumor, and liver extracts, among others. The presence of clots or coagulants is an example. For example, plasma clots and coagulants are examples.

Natural mediums such as serum are the most popular. It is even employed as a supplement in synthetic media cultures, according to the manufacturer. The popularity of bovine serum can be attributed to a variety of factors, including abundant resources and a lengthy application time.

Synthetic media

Synthetic media (also known as artificial media) are those that have been manufactured by combining different organic and inorganic substances. The following are examples of synthetic media:

  • The term “serum-containing medium” refers to synthetic media that contain supplementary serum. Serum-free media (SFM) are more consistent than serum-containing media, allowing researchers to sustain cellular growth and proliferation without the need of serum. Inorganic and organic components are purified to ensure that chemically defined media are contamination-free
  • Chemically defined media are also known as chemically defined solutions. Protein-free media: Protein-free media do not include any protein in their composition. Depending on the situation, protein supplements may be administered. Balanced salt solutions (BSS): BSS are used to maintain the viability of cells on their own. A variety of nutrients and other substances are added to BSS in order to generate culture medium that is capable of sustaining cellular development and proliferation. As a result, BSS serve as the foundation for many other sorts of sophisticated media.

Serum-free media (SFM) are frequently divided into two categories: basic/basal and complicated. Basal media are ones that do not include any supplements. As soon as growth supplements are introduced to a basal medium, the media is transformed into a complex medium. The following is a list of some popular forms of culture media.

Media Name Media Type Description
MEM (Minimum Essential Medium) Basal media Also known as Eagle’s minimal essential medium (EMEM), this medium is used for primary and diploid cultures. MEMs contain only vitamins, non-essential amino acids, inorganic salts, and glutamine.
DMEM (Dulbecco’s Modified Eagle’s Medium) Basal media Similar to MEM, but with additional amino acids and vitamins. Contains no growth promoters or proteins.
IMDM (Iscove’s Modified Dulbecco’s Medium) Complex media A modified version of DMEM with more amino acids and vitamins. It contains no iron but does have selenium and ferric nitrate. Suitable for high-density cultures.
RPMI-1640 Complex media A medium that works for most types of mammalian cells. One of the most used mediums.
Ham’s F-10 and F-12 Serum-free media Ham’s F-10 was the original Ham nutrient mixture, designed for use with Chinese hamster ovary (CHO) cells. It’s suitable for use with human diploid cells. Ham’s F-12 is more complex, suited for rat prostate epithelial and rat hepatocyte cells.

What to look for in a cell culture media

The most critical stage is deciding which type of cell culture media will be used in the experiment. When there are so many commercial channels to choose from, it may be difficult to choose the best decision. Begin by conducting research on the cell line you intend to use in order to determine which different growth media are advised for it. You may then choose a handful and experiment with them to find which ones work best for you. Here are some considerations to keep in mind.

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1. Media preparation

Cell culture mediums are available in three different forms: 1.Powdered medium: Powdered media are the least costly type of medium, however they must be disinfected and produced by the researcher themselves. 2.Concentrated medium: Concentrated media are those that just require the researcher to dilute them. Three-dimensional working solution: Working solutions are the simplest sort of media to use. Because they are meant to be utilized without any modification by the researcher, they are often employed in clinical trials.

2. Type of media to use: natural vs synthetic media

Generally speaking, all cell culture medium may be divided into two categories: natural media and synthetic media. Each has its own set of advantages and disadvantages, which help investigators decide which media to use. Hemaleucin is removed from plasma in order to produce serum medium. The resultant mixture has a unique blend of components that are beneficial for the development and proliferation of cell cultures. The following are some of the advantages of serum media:

  • Consist of an extensive array of cellular elements, including amino acids, vitamin B1, vitamin D3, minerals, and fatty acids Provide components that aid in cell adhesion and adhesion
  • Growth factors and hormones in a variety of forms are found in them.

However, there are several disadvantages to using serum media. When it comes to its disadvantages, serum media has the following:

  • The lack of compositional consistency makes the material unsuitable for large-scale investigations
  • And Contained inside them is a complex variety of substances, some of which might be detrimental to or hinder the growth of specific cell types
  • Comparing natural media versus manufactured media, there is a larger danger of contamination
  • It is possible that the isolation of cell culture products will be hampered. It can be prohibitively pricey.

The constancy of synthetic media is a notable advantage over natural media. It is possible for researchers to be confident that they are utilizing the same culture media, even when their study is scaled up.

Artificial media are also often less expensive than serum-based media, which is another advantage. However, there are several drawbacks to adopting a serum-free media. These cultures are more susceptible to extremes in terms of temperature, pH, and osmolality than other types of cultures.

3. Which specific medium to use

Once you’ve decided between natural and artificial media, you’ll need to choose a specific medium to work with. Many laboratories make use of commercial media, which ensures high quality and uniformity with little effort. In order to pick the greatest one, you must first ask yourself the following questions:

  • What kinds of cells will you be employing, and what are the specifications of those cells
  • In what ways does your laboratory excel and where it falls short
  • Was the experiment carried out for a specific reason?

For example, stem cells require medium that can allow their preservation, differentiation, growth, and use in subsequent procedures. In order to avoid producing anything other than their intended product, stem cells must be kept in carefully controlled medium conditions until they are fully matured. There are various commercial stem cell media, such as RPMI-1640, that are specifically intended for use with stem cells and that are effective with specific kinds of stem cells. If you are unclear about which medium is suitable for your needs, media businesses may assist you in narrowing down your choices.

What does a cultural medium provide to a living cell? – Easierwithpractice.com

“Culture Medium” is just a word used to describe the local environment in which to live. It might be in the form of a gel (agar) or a solution (water). It “cultures” the cell, which means that it is meant to provide a favorable environment for the cell’s development. Nutrients should be provided to live cells by the medium in order to boost their growth circumstances.

What do you mean by culture media?

A culture medium, also known as a growth medium, is a liquid or gel that is used to promote the development of microorganisms in culture. There are several distinct types of media available for the cultivation of various cell types. We shall cover microbiological cultures in this section, which are used to cultivate microorganisms such as bacteria or yeast.

What are the types of culture media?

Each of the six types is divided into the following categories: (1) Basal medium; (2) Enriched medium; (3) Selective; (4) Indicator; (5) Transport medium; and (6) Storage medium. 1. BASAL MEDIA ENTERTAINMENT. A basal medium is one that may be utilized for the growth (culture) of bacteria without the requirement for further enrichment of the media.

What type of media is blood agar?

Blood agar is a bacterial growth media that has been supplemented with nutrients. Organisms that are picky about their development, such as streptococci, do not fare well on standard growth media, but do well on blood agar. Blood agar is a form of growth media that has a trypticase soy agar foundation that has been enhanced with 5% sheep blood.

What is a culture media and its examples?

Nutrient broth, nutrient agar, and peptone water are all examples of this. These media support the growth of Staphylococcus and Enterobacteraceae. The media are often supplemented by the addition of blood, serum, or eggs. Blood agar and Lowenstein-Jensen media are two examples of enriched media that can be used.

What does mixed culture mean?

Mixed-culture fermentations are those in which the inoculum is always composed of two or more organisms, as opposed to single-culture fermentations.

Mixed cultures can be formed of recognized species that are isolated from all others, or they can be composed of mixes of unknown species that are isolated from each other.

What are the pure culture techniques?

It is referred to as the Enrichment Culture Method.

  • The Streak Plate Method is the most often used method for isolating pure bacteria cultures
  • It is also known as the agar plate method. Pour Plate Method
  • Spread Plate Method
  • Serial Dilution Method
  • Pour Plate Method
  • Pour Plate Method Single Cell Isolation Methods:
  • Enrichment Culture Methods:
  • Single Cell Isolation Methods:

Is microbial culture dangerous?

However, while the vast majority of microbes are not harmful to people and have never been demonstrated to cause sickness, a small number of germs, which are not ordinarily dangerous, can become pathogens under rare circumstances. Treatment of any microorganisms, especially unfamiliar cultures, should be done as if they were potentially harmful.

Why bacteria is used in laboratory culture?

Under general, bacteria and viruses are not harmful to people and have never been demonstrated to cause sickness, but in rare conditions, a few bacteria and viruses that are not ordinarily pathogenic can become pathogens and cause illness. Treatment of any microorganisms, especially unknown cultures, should be performed as if they were harmful.

What does it mean to grow a culture?

Culture is the process of propagating microorganisms in a growth media, which is used in microbiology. It is possible to assess any human tissue or fluid in the laboratory using culture methods to discover and diagnose infectious processes in any body tissue or fluid. Cells can also be cultivated in a laboratory setting.

How long does it take for bacteria culture to grow?

The sample is placed in a container containing a material (referred to as growth media or culture medium) that aids in the development of bacteria, fungi, or viruses after it has been collected. Bacteria typically take 1 to 2 days to develop from start to finish.

What are the 4 conditions that bacteria need to grow?

It is possible to have an influence on the proliferation of bacteria in four ways. Temperatures, moisture, oxygen, and a certain pH are all factors to consider.

How do you identify bacterial culture?

Traditional bacteria identification include doing a Gram stain, followed by bacterial identification and antibiotic susceptibility testing, as well as other procedures. Beginning with the first step and progressing through the entire procedure can take up to five days, delaying the time required for antibiotic de-escalation.

What indicates growth in a broth culture?

In broth, different organisms will develop in a variety of ways with differing growth properties. A few organisms will disperse equally throughout the broth, while others will sink to the bottom and produce a sediment; others will develop in clumps, resulting in flocculent growth; and yet others will float on the surface of the soup, generating pellicle growth (see figure).

Introduction to Cell Culture – NL

Cell culture is the process of removing cells from an animal or plant and allowing them to develop in a suitable artificial environment once they have been removed. Before culture, cells can be isolated directly from the tissue and disaggregated using enzymes and/or mechanical methods. Alternatively, cells can be isolated from an existing cell line or cell strain and cultivated in the same way that they were originally isolated. Primary culture refers to the stage of the culture after the cells have been separated from the tissue and have multiplied under the suitable circumstances until they have taken up the whole accessible surface area of the substrate (i.e., reachconfluence).

Upon the completion of the first subculture, the primary culture is known as a cell line or a subclone.

It is possible to transform a cell line into a cell strain by selectively cloning or using other methods to isolate a subpopulation of the cell line from the rest of the culture.

Additional genetic alterations are frequently acquired by a cell strain after it has been established as a part of the parent line.

Finite vs Continuous Cell Line

A limited number of times in normal cells before they lose their capacity to multiply, which is a genetically regulated phenomenon known as senescence; these cell lines are referred to as finite. Although some cell lines become immortal naturally, some cell lines become immortal by a process known as transformation, which can occur spontaneously or be driven chemically or virally. The transformation of a finite cell line into a continuous cell line occurs when the cell line gains the ability to divide endlessly as a result of the transformation.

Culture Conditions

The culture conditions for each cell type differ significantly, but the artificial environment in which the cells are cultivated always consists of an appropriate vessel that contains the following components:

  • A substrate or medium that provides the required nutrients (amino acids, carbohydrates, vitamins, and minerals)
  • Growth factors
  • Hormones
  • Gases (oxygen and carbon dioxide)
  • And other substances (such as oxygen and carbon dioxide). It is necessary to have a controlled physico-chemical environment (pH, osmotic pressure, temperature).

Adherent or monolayer culture is required for the majority of cells, which must be attached to a solid or semi-solid substrate (adherent or monolayer culture), while some can be grown floating in the solution (floating culture) (suspension culture).

Cryopreservation

If there is an excess of cells available after subculturing, they should be treated with a suitable protective agent (e.g., DMSO or glycerol) and kept at temperatures below –130°C (cryopreservation) until they are required again. Refer to theGuidelines for Maintaining Cultured Cells for further information on subculturing and cryopreservation of cells.

Morphology of Cells in Culture

It is possible to classify cells in culture into three fundamental types depending on the shape and appearance of the cells (i.e.,morphology). Bipolar or multipolar fibroblastic (or fibroblast-like) cells have elongated forms and grow connected to a substrate, whereas fibroblastic cells do not. A polygonal structure with more regular proportions characterizes epithelial-like cells, which develop in distinct patches linked to a substrate as they expand. Lymphoblast-like cells are spherical in form and are often cultivated in suspension, without adhering to a surface, in order to maximize their growth potential.

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Applications of Cell Culture

When it comes to cell culture, it is one of the most important tools in the field of cellular and molecular biology. It provides excellent model systems for studying the normal physiology and biochemistry of cells (for example, metabolic studies, aging), the effects of drugs and toxic compounds on the cells, as well as the mutagenesis and carcinogenesis of cells, among other things. It is also employed in the screening and development of pharmaceuticals, as well as the large-scale production of biological substances (e.g., vaccines, therapeutic proteins).

Related Cell Culture Basics Videos

An overview of the fundamental equipment needed in cell culture as well as the suitable laboratory set-up is provided in this video. Instructions and demonstrations on how to operate safely and aseptically in a cell culture hood are provided.

It is the objective of this video to discuss the precautions you should take to avoid contamination of your cell culture. There is a demonstration of all of the fundamental procedures necessary to accomplish cell culture utilizing best-practice sterile methods.

Cell Culture Contamination Troubleshooting

Cell culture contamination is often associated with bacteria amid cells and hazy media, although unwelcome intruders in the culture flask can take on a variety of shapes and forms depending on their origin. Cell culture health can be adversely affected by viral and chemical contaminants, and ensuring that cell lines are not cross-contaminated is crucial to guaranteeing repeatability of results in the laboratory. How widespread is the problem of cell contamination? According to research conducted by the FDA, the American Type Culture Collection, and others, it is estimated that 5 – 30 percent of all cell cultures are contaminated with mycoplasma species alone today.

Figure 1.

Microbial contaminants such as bacteria, fungal, and yeast pollutants are generally apparent to the untrained eye and can quickly kill cells in culture; however, modest morphology can make substantial microbial intrusions such as mycoplasma more difficult to detect.

Contamination of research cell lines, or even simple misidentification of research cell lines, has emerged as one of the most critical issues facing the biology community in recent years.

Types of Culture Contamination

In most cases, unaided eyes can detect bacterial, fungal (including mold), and yeast contamination as a rapid-onset turbidity and color change in the culture media (provided that themedium is supplemented with phenol red, the most common non-toxic pH indicator). As bacterial cells and fungal structures may be seen under standard light microscopy, daily microscopic monitoring of cultures will allow for the early diagnosis of microbial contamination and the prompt implementation of corrective measures when the first symptoms of contamination become apparent.

As an additional measure of quality control, specialized testing for bacteria and fungus should be utilized as part of a normal and regular quality control screening program.

Common Causes and Prevention of Microbial Contamination

Ignored by antibiotics, Mycoplasma are a genus of bacteria that does not have cell walls and is thus untouched by antibiotics that prevent bacteria from growing by blocking cell wall development. As a result of their flexible architecture and small cell size (in the 0.15 to 0.3 m range), they are unlikely to be captured by normal cell culture filtration methods, which typically employ filters with 0.22 m holes. For example, unlike most other bacterial contaminants, mycoplasma can go undetected on the surface of a sample and are difficult to identify under a light microscope due to their unique form and relatively tiny size.

In most cases, they do not kill the mammalian cells that they infect, but they have a substantial influence on cultures by affecting cellular metabolism, inducing chromosomal abnormalities, decreasing cell development, and interfering with cell attachment, among other effects.

When used in conjunction with fluorescence microscopy (right), common DNA detection methods such as DAPI or Hoechst indicate the presence of mycoplasma in contaminated samples (Figure 2).

Because certain mycoplasma species may be found on human skin, they can be introduced into the body through inadequate aseptic procedures, as well as through contaminated supplements such as fetal bovine serum, among other means.

Filtering media and buffers with membranes with holes of 0.1 micron or less is required for mycoplasma treatment, since ordinary media filtration systems with pores of 0.22 or 0.45 micron will fail to eliminate these microscopic organisms from the treatment process.

Preventing, Detecting, and Eliminating Mycoplasma Contamination

As a result, Mycoplasma bacteria do not have cell walls and are therefore unaffected by antibiotics that prevent the production of cell walls, which are commonly used to control bacterial growth. As a result of their flexible architecture and small cell size (in the 0.15 to 0.3 m range), they are likely to evade normal cell culture filtration methods, which frequently employ filters with 0.22 m holes. Mycoplasma, in contrast to the majority of other bacterial pollutants, will not be obvious upon casual examination and will be difficult to identify under a microscope due to their shape and relatively tiny size.

  • They normally do not kill the mammalian cells that they infect, but they have a substantial influence on cultures by modifying cellular metabolism, inducing chromosomal abnormalities, decreasing cell development, and interfering with cell attachment.
  • For the most part, they are likely to have a significant impact on the outcomes of most tests conducted on the cell lines in question.
  • Mycoplasma contamination can occur from a variety of sources in the laboratory, which can be difficult to manage and control.
  • Because mycoplasma is very contagious, it is easy to spread it between cell cultures in close proximity.

Viral Contamination

Viruses are among the most difficult cell culture contaminants to detect in culture, necessitating the use of microscopy procedures that are impracticable for the majority of research labs to use. Endogenous retroviruses can come from either the patient or the host animal cell source, and endogenous retroviruses have been found in a number of cell lines of biotechnological importance. Cells are more frequently infected by viruses that are present in the animal-derived materials that are used to cultivate them.

  • Nevertheless, because most viruses are host- and even tissue-specific, their ability to infect other species or tissues may be constrained.
  • Using virally contaminated cell cultures is fraught with danger, not only because of the possible impact on cultured cells, but also because of the potential health hazard they provide to laboratory employees.
  • The environmental safety officials at the institution should be advised about the protocols to be followed while working with potentially hazardous tissues, cultures, or viruses.
  • Guidelines for limiting the likelihood of viral contamination of cell cultures include the following: Cells should be obtained from repositories that perform virus testing and provide certification of virus-free cell lines.

*Biological sources (suppliers, animals) from which cells should be obtained should be limited. *Animals and cells should be obtained from sources that are less virus-susceptible.

Chemical Contamination

When it comes to cell culture contamination, viruses are among the most difficult to detect in culture, necessitating microscopy approaches that are unfeasible for most research institutions. Retroviruses can be derived from either the patient’s or the host animal’s cell source, and endogenous retroviruses have been found in a number of cell lines of biotechnological value. Most of the time, viruses contained in the animal-derived materials used to cultivate the cells cause infection. Viruses are extremely difficult to remove from media, sera, and other biological solutions due to the tiny size of their particles.

In spite of the fact that viruses are more abundant in cell cultures than many researchers know, they may or may not be a significant confounder in cell culture experiments if they do not cause cytotoxicity or other harmful effects on cells.

When working with tissues or cells from humans or other primates, special safety precautions should always be taken to avoid the possibility of viral infection (including HIV, hepatitis B, Epstein-Barr, and simian herpes B virus, among others) being transmitted from the cell cultures to laboratory staff.

Please see this link for further information on risk assessment for laboratory employees who work with cell culture.

*Biological sources (suppliers, animals) from which cells should be obtained should be limited.

General Tips and Techniques for Preventing and Eliminating Contamination

Keep in mind that air movement is critical when working in a biosafety cabinet to ensure that the atmosphere remains as sterile as possible during your time inside. To ensure efficient ventilation, both the back and front vents should be kept free of obstructions at all times. Before beginning any practical activity, make sure that the cabinet is fully supplied with all of the necessary ingredients. This will reduce the likelihood of pollutants being transferred from the operator’s sleeves and hands.

All things that enter the cabinet must be sprayed with 70 percent (v/v) sterile alcohol and then wiped off using lint-free wipes to prevent dust and particles from entering the cabinet during the cleaning process.

Before opening any tops or containers, be sure that the airflow has been set thoroughly. This will allow the airflow to cleanse the work area of any particles that may have been introduced. Drawing of a person working in a safety cabinet.

Pipetting and Prevention of Aerosols

Disposable plastic pipettes, sometimes known as serological pipettes, and available in sizes ranging from 1-100 mL, are critical instruments in the cell culture laboratory. To guarantee sterility, each of them must be separately wrapped. Following the recommendations in this document can help to reduce the hazards of contamination and safety related with pipetting.

  • Make use of automated pipette aids, with each pipette assist being confined to a particular cabinet of storage. Disassemble and sanitize the pipette aid components on a regular basis to prevent infection. Maintain a sufficient supply of pipette aid filters and replace them on a regular basis. Plugged pipettes (pipettes with cotton inserted into the top) should be used whenever feasible, especially while transferring medium. It is important not to pull liquid into the pipette plug. If a plug is accidently wetted, replace the pipette assistance filter as soon as possible. Avoid contacting the serological pipette entirely by unwrapping only a portion of it, fitting the end to the pipette assist, and then removing the paper sleeve. Do not produce bubbles in the medium or pipette in order to avoid the generation of contaminated aerosols.

Disinfection

In addition to minimizing contamination risk, methods intended for the disinfection/decontamination of culture debris, work surfaces and equipment must be safe for lab staff. When working with intense disinfectants, always sure to use proper personal protective equipment (PPE) such as gloves and eye protection. The gloves that are chosen should provide protection against the material that is being handled. Manufacturers’ charts will assist you in determining which gloves are most appropriate for a certain work.

Sodium Hypochlorite or Bleach

  • This product is a good general purpose disinfectant. It has antiviral properties. Corrosive to metals—should not be used on metal surfaces such as centrifuges because it will corrode them. Because it is easily inactivated by organic debris, it should be replaced on a regular basis. Make a 10 percent (v/v) solution of commercial bleach in waste liquid or use it to clean surfaces by soaking them in it.

Alcohol (e.g. Ethanol, Isopropanol)

  • Concentrations that are effective include: 70% for ethanol and 60-70 percent for isopropanol
  • And Bacterial resistance is demonstrated. Ethanol is effective against the majority of viruses, but not against viruses that are not enclosed. Isopropanol does not have any antiviral properties. Aldehydes are irritants, and their usage should be restricted to prevent irritation.

Phenolic-based disinfectants should be avoided since they are not supported by the EU’s Biocidal Products Directive review program, which is now in progress.

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