Sources of microbial contamination in tissue culture. A cell culture contaminant can be defined as some elements in the culture system thats undesirable because of its possible adverse effects on either the system or its use. Understanding and managing cell culture contamination. Preventing, detecting, and eliminating mycoplasma contamination once mycoplasma contaminates a culture, it can quickly spread to other areas of the lab. The cultured tissue may consist of a single cell, a population of cells, or a whole or part of an. Cross contamination can occur when working with multiple cell lines at the same time. Although contamination remains a serious challenge for in vitro plant cell, tissue or organ culture researchers, over the past 25 years a large body of evidence has accumulated that the presence of microorganisms in plant in vitro cultures, while often harmful, may also, depending on the specific organism, have no effect on cultures or even be.
In this issue, we also present invivogens highly cited reagents and frequently asked questions to help you keep your valuable cells safe from any contamination. Pdf contaminants of planttissue and cell cultures carlo leifert. Epitheliallike cells are polygonal in shape with more regular dimensions, and grow. Yeast is used to ferment the sugars of various grains to produce continued. Animal cell culture protocol aseptic technique and good cell culture practice to ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines. Waites is with the department of applied biochemistry and food science, university of nottingham, sutton bonington le12 5rd, uk. Different experimental procedures including chemical sterilization and antibiotics have been used at various levels of success to minimize or eliminate such. Some of the advantages of in vitro cultures are their. Laboratories must assess their situation, detennine contamination sources, and change their laboratory operalions to avoid or eliminate most of the contaminants. The study overviews results of bacterial incidence in in vitro plant tissue cultures obtained. Faulty aseptic technique and overconfidence in antibiotics are the main causes of contamination in tissue cultures contamination may be confined to one laboratory or be connected with the individual technician, or may be more widespread. Biological contamination is the dread of every person working with cell culture.
Pdf regeneration of multiple individual plantlets from a single cell or explant is one of the main aims of plant tissue culture. Plant tissue culture terminology differentiatedcells that maintain, in culture, all or much of the specialized structure and function typical of the cell type in vivo. This chapter discusses contamination of tissue culture and its cure. The biochemical engineer can grow plant cells in liquid culture on a large scalebioreactor 2. Cell culture basics handbook thermo fisher scientific. No cell culture problem is as universal as that of culture loss due to contamination. For this method, explants tissue from the plant are obtained from an in vitro derived plant and introduced in to an artificial environment, where they get to proliferate.
Research paper on plant tissue culture biology discussion. Tissue culture the maintenance or growth of tissue, in vitro, in a way that may allow differentiation and preservation of their function. A method for eliminating bacterial contamination from in. Culture contaminants may be biological or chemical, seen or unseen, destructive or seemingly benign, but in all cases they adversely affect both the use of your cell cultures and the quality of your research. Contamination is not always seen at the culture establishment stage.
Contamination in tissue culture can originate from two sources, either through carry over of microorganisms on the surface or in the tissues of explants. Serious problemswhen contamination frequency increases or entire experiments or cell culture are. Totipotencya cell characteristic in which the potential for forming all the cell types in the adult organism are retained. Thereafter, nonattached epithelial cells still present were removed and a purified stromal preparation was obtained on the surface of the culture dishes. Fibroblastic or fibroblastlike cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate. Contamination contamination is a staple problem in cell culture all cell culture laboratories and workers have experienced contamination affects both the use of cultures and research quality most apparent consequences of contamination. Purchase contamination in tissue culture 1st edition. Introductionwe can define to contamination as the impurity in a cell culture produced by chemicals, microbes, other cells called contamination. In particular, prompt removal of contaminated cultures is needed to protect neighboring cultures, as well as the sterile tissue culture environment including culture incubators, baths, and the biosafety cabinet. I need procedures to control fungal contamination during tissue culture. Endophytic bacterial contamination is an important problem in plan tissue culture kneifel and leonhardt. Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. Endometriotic cell culture contamination and authenticity. The inside tissue of a fruiting body is exposed by pulling apart the mushroom cap to expose the inside uncontaminated tissues figure 3.
Microbial contaminants in plant tissue culture propagation. Three different surveys in japan showed an incidence of mycoplasma contamination of 6080%, and an incidence of 65% was reported in argentina 5. Abstract biological contamination in plant cell cultures originates from two sources, namely, from the tissue used to initiate the culture, and from. Composed of 12 chapters, the book describes the frequency of occurrence of contamination and the many different effects of contamination on cultured. Originally, the term is not limited to animal cells, but includes the in vitro cultivation of plant cells. Contamination in tissue culture covers the sources, prevention, detection, and elimination of contamination in tissue culture. Common fungi contamination affecting tissuecultured. Biological contamination in plant cell cultures originates from two sources, namely, from the tissue used to initiate the culture, and from the. However, some cell cultures are so precious that they cannot be lost i. Isolation of mother cultures and preparation of spawn for. The file will be downloaded and can be opened with adobe acrobat reader.
Contamination problems can be divided into 3 classes. Sources of microbial contamination in tissue culture laboratories in. Approximately 180 different species exist the most common method of transmission is from other contaminated cultures a. Detection of ftingi and yeasts most fungi and yeasts grow well on planttissue culture media and can be detected by visual assessment of cultures cassells 1990. All cell culture laboratories and cell culture workers have experienced it. Pdf using hydrogen peroxide for reducing bacterial. Pdf plant tissue culture contaminants identification and its. The best strategy to control tissue culture contamination is to establish aseptic cultures and to maintain good laboratory practice, including routine testing for contamination by cultivable microorganisms and microarthropods.
Yeasts, such as candida and rhodotortila, found as contaminants in planttissue cultures. Us5750402a compositions and methods to prevent microbial. Microbial contamination is a constant problem, which often compromise development of all in vitro techniques. Cultures, that do not show good growth or infected, are discarded. How to control fungal infection in plant tissue culture. During micropropagation, bacterial contamination can remain undetected because the salt concentration, sucrose concentration, ph. Considerations for contamination cleanup in plant tissue culture.
Tissue cultures can become contaminated at any stage of the tissue culturing process. Additionally, specific testing for bacteria and fungi should be used as part of a routine and regular quality control screening procedure. Yeast is used to ferment the sugars of various grains to produce alcoholic beverages and in the baking industry to expand dough. Need for certification of tissue culture plants 10. Dec 15, 2009 biological contamination in plant cell cultures originates from two sources, namely, from the tissue used to initiate the culture, and from the laboratory environment. Plant tissue cultures can become contaminated with bacteria either because of poor aseptic technique while handling the cultures or because of animal vectors leifert et al. When cultures become infected with microorganisms, or crosscontaminated by foreign cells, these cultures usually must be destroyed. Continuous use of antibiotics is unnecessary and can lead to the development of resistant strains that are difficult to eradicate and may require the use of more exotic antibiotics that may be toxic to the cell cultures. Higher incidences of contamination have also been reported.
The information available on the contamination of cultures of differentiated cell lines is limited, and more data are needed before a proper assessment can be made. Introduction to animal cell culture university of western. Ritchie was formerly with the department of physiology and environmental science, university of nottingham, sutton bonington le12 5rd, uk. Leifert is with the department of cell and structural biology, manchester university, manchester m 9pt, uk. This is the first in a threepart series on co 2 incubation. Fundamental techniques in cell culture sigmaaldrich. Biological contamination in plant cell cultures originates from two sources, namely, from the tissue used to initiate the culture, and from the laboratory environment. In vitro plant cell cultures are defined as a culture in aseptic conditions of any part of the plant body in a nutritive media. Basic characteristics of tissue culture tissue culture tissue culture is used as a generic term to include the in vitro cultivation of organs, tissues and cells. Strict adherence to good laboratory practices is key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination. This has changed the view on bacteria in plant tissue culture and led to the differentiation between artificially introduced contaminations and naturally occurring endophytes with neutral, negative, or positive impact on the plant propagation process. If youve ever enjoyed really good bread and a cold beer good only in this combination when nothing else is in the fridge you would agree that, in general, yeast is mans friend.
Nineteen microbial contaminants consisting of eleven bacteria and eight fungi were found associated with the tissue culture plants and the laboratory. Cell culture mixups contamination of one cell line by another was first reported in the late1950s hela cells are the most famouscell culture contaminant can you tell what cell henrietta lacks line this is. The present invention encompasses compositions and methods to reduce or prevent microbial growth in plant tissue culture media, comprising adding a chemical agent comprising methylchloroisothiazolinone, methylisothiazolinone, magnesium chloride and magnesium nitrate to a plant culture medium at a concentration that reduces or prevents microbial contamination of the plant tissue culture medium. Tissue culture isolation for mother culture production pure cultures of oyster mushroom can be obtained by tissue culture from desired mushroom fruiting bodies, the basidiocarps. What to do in the event of cell culture contamination. Endophytic bacteria have been increasingly in the focus of research projects during the last decade. Jan 05, 2015 bacterial contamination is a recurrent problem in moss tissue culture. Considerations for contamination cleanup in plant tissue culture david s.
Contamination and browning in tissue culture of curculigo. Contaminants isolated from planttissue cultures and their effect on plants. Cell culture contaminants can be divided into two main categories, chemical contaminants such as impurities in media, sera, and water, endotoxins, plasticizers, and detergents, and biological contaminants such as bacteria, molds, yeasts, viruses, mycoplasma, as well as cross contamination by other cell lines. Instead, these guidelines describe the aatbs current thinking on this topic. Ideal antibiotics should be soluble, stable, unaff by ph, unaffectai by media. Minor annoyanceswhen up to several plates or flasks are occasionally to contamination. Plant surfaces are habitats for microorganisms campbell, 1985. Pdf on nov 18, 2015, gaurav kumar sharma and others published general techniques of plant tissue culture find, read and cite all the research you need on researchgate. In tissue culture, by using optimum concentration of cytokinin or combination of cytokinin and auxin the dormancy of the axillary buds can be broken. A very useful list of antibiotics, the organisms they are effective against, and recommended working concentrations compiled by the sigmaaldrich company can be found as a pdf document 129kb, 3 pages by clicking on the following link. Pdf contaminants of planttissue and cell cultures carlo. Modifications of new cells to form tissues or organs with a specific function.
Seed culture is the type of tissue culture that is primarily used for plants such as orchids. Different concentrations of hydrogen peroxide at different time. Bacterial contamination remains a continuing thrt to plant tissue culture, but techniques for reducing con,taminatton are available. Cell culture contamination this comprehensive guide highlights the different categories of cell culture contaminants and how they can impact your research. Explant tissue taken from its original site and transferred to an artificial medium. Bacterial contamination in tissue culture is well documented, and the failure of surface sterilization procedures to produce aseptic cultures is a major problem with woody plants. Composed of 12 chapters, the book describes the frequency of occurrence of contamination and the many different effects of contamination on cultured cells. Tissue culture, a method of biological research in which fragments of tissue from an animal or plant are transferred to an artificial environment in which they can continue to survive and function. Contamination of tissue cultures by mycoplasmas intechopen. Contaminants of planttissue and cell cultures springerlink. Nineteen microbial contaminants consisting of eleven bacteria and eight fungi were found associated with.
Contaminants transferred in or on the plant material include plant pathogens and environmental microorganisms. Ritchie was formerly with the during in vitro culture of plants cassells 1986. Culture contaminants may be biological or chemical. Here we describe an agarembedding method that is reliable, rapid, and relatively inexpensive, using materials that are readily available in most laboratories that conduct sterile tissue culture. Th d f dh l d l f h l d lhe production of dihaploid plants from haploid cultures sh h hortens the time taken to achieve uniform homozygous lines and varieties 3.
One hugely underestimated problem in tissue culture is the routine use of antibiotics. Cell culture cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells e. The use of hydrogen peroxide as a disinfectant material was evaluated for its effect relevant to bacterial contamination which usually occurs in the micropropagation of date palm. Contamination detection and elimination in plant cell culture. Mitigating risks of commercial plant tissue culture 10 7. Mycoplasmas are spread by using laboratory equipment, media, or reagents that have been contaminated by previous use in processing mycoplasmainfected cell cultures. Culture media, bovine sera, reagents, and plasticware can also be major sources of biological contaminants.
Bacterial contamination is a recurrent problem in moss tissue culture. The cultured tissue may consist of a single cell, a population of cells, or a whole or part of an organ. Cells, differentiated and undifferentiated calli and suspended cells tissues, and organs can be maintained in vitro. Pdf curbing the menace of contamination in plant tissue culture. New cell culture acquisitions should be quarantined, tested and guaranteed mycoplasmafree before introduction into the tissue culture laboratory. Quality control considerations in cell culture sigmaaldrich. Detection and control of bacterial contaminants of plant. Identification and elimination of bacterial contamination. Feb 27, 2020 the stromalenriched fraction was washed twice in complete medium and allowed to adhere selectively to 25cm 2 tissue culture dishes for 15 min at 37c in 5% co 2.
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