Contamination in tissue culture covers the sources, prevention, detection, and elimination of contamination in tissue culture. Agar is generally used at a concentration of 612 gliter. 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. This in turn will be contaminated by the tissue culture users of that incubator. Determination of the sources of microbial contaminants of. Sources of microbial contamination in tissue culture laboratories in. Cell culture contamination thermo fisher scientific us. Tissue cultures can become contaminated at any stage of the tissue culturing process leifert, 2000. 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. Cauliflower florets in particular give excellent results since they can be grown into a complete plant in the basic tissue culture media, without the need for additional growth or root hormones. Contaminants transferred in or on the plant material include plant pathogens and environmental microorganisms.
Chemical contamination is often overlooked as a source of cell growth problems. Applications of plant tissue culture in crop improvement 5. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including. Dec 22, 2011 the microbiological culture method is a gold standard to detect every kind of mycoplasma contamination in cell culture without considering the origin and species of mycoplasma. The cultured tissue may consist of a single cell, a population of cells, or a whole or part of an. Tissue culturethe maintenance or growth of tissue, in vitro, in a way that may allow differentiation and preservation of their function. Plant tissue cultures may be contaminated by a wide variety of bacteria, many of which are species specific. Good quality reagents and materials are available from numerous manufacturers of tissue culture media and supplements.
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. Isolation of mother cultures and preparation of spawn for oyster mushroom cultivation 4 the sterilized seeds, which are the substrate for the oyster mushroom culture to grow on to produce spawn, should be inoculated with mycelium from a pure mother culture under aseptic conditions to prevent contamination and produce clean, healthy spawn. Ritchie was formerly with the department of physiology and environmental science, university of nottingham, sutton bonington le12 5rd, uk. 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. Potential sources of contamination are reagents and materials, in particular bovine serum which has been identified as a source of bovine viral diarrhea virus bvdv. Green shoots are generally observable within three weeks, and roots develop within six weeks. Tissue culture involves the in vitro maintenance and propagation of cells in optimal conditions. Most mycoplasmas are typical extracellular microorganisms able to adhere to the surface of tissue culture cells. Micropropagation is the art and science of plant multiplication in vitro. Importance of controlling contamination the medium contains many different bacterialnutrients, both original constituents of the medium and exudates from the plant cells. Introduction no cell culture problem is as universal as that of culture loss due to contamination. The most obvious consequence of cell culture contamination is the loss of your time, money for cells, culture vessels, media and sera and effort spent develop. Growth of potato shoot cultures on media with antibiotics. Cell culture materials, used to grow the vaccine antigens.
The culture medium, especially when it contains sugar, will also support the growth of microorganisms like bacteria, fungi etc. In my experience the fungus will be from the co2 incubator. Here, any organ plant can be used as an explant for the culture process shoot, root, leaf, and flower. 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. Waites is with the department of applied biochemistry and food science, university of nottingham, sutton bonington le12 5rd, uk. Contaminants of planttissue and cell cultures springerlink.
Project report on the meaning of plant tissue culture project. Microbial contamination of cell cultures is a serious and relentless threat to your research. Plant cultures may be contaminated by a wide variety of. Chapters 9 and 10 examine the occurrence of tissue culture contaminants by. Cell culture is a fussy discipline in the tissue culture laboratory. Agrobacterium, biotization, in vitro culture, meristem culture, pathogen elimination.
Some strains of mycoplasma hyorhinis cannot cultivate well in this method. 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. Pdf curbing the menace of contamination in plant tissue culture. The nature and origin of cell culture contamination. Keep incubators free from contamination, change the water onceweek and spray all flasks in and out of the incubator. Cell culture contamination thermo fisher scientific id. Pdf contamination of tissue cultures by mycoplasmas.
A wide variety of luminoldependent chemiluminescence and bioluminescent methods were described 5, 63. Contamination of tissue cultures by mycoplasmas 49. Prevention and detection of mycoplasma contamination in. Contamination in tissue culture 1st edition elsevier. The adherence of mycoplasmasto host cells is an initial and essential step in tissue colonization.
With organ culture, or as is with their various tissue components, the method is used for preserve their structure or functions, which allows the. Many mycoplasmas exhibit the typical polymorphism of mycoplasmas, with the most common filamentous, flask shapes or ovoid structures figure 1, ref. Project report on plant tissue culture biology discussion. Whereas lung, kidney, or liver tend to be mycoplasmaf ree, the foreskin, the low er femaleurogenital tract, or tumor ti ssues, are subject. Types and sources of potential chemical contaminants. Isolation of mother cultures and preparation of spawn for. Plant tissue culture, cell culture or micropropagation is the technique of producing selected plants of known desirable agriculture qualities, in large numbers of plants from small pieces of plant in relatively short period times. Ideal antibiotics should be soluble, stable, unaff by ph, unaffectai by media. Contamination in tissue culture can originate from two sources, either through carry over of microorganisms on the surface or in the tissues of explants, or through faulty procedures in the laboratory. Organ culture is a type of tissue culture that involves isolating an organ for in vitro growth. The microbiological culture method is a gold standard to detect every kind of mycoplasma contamination in cell culture without considering the origin and species of mycoplasma. An exclusive project report on plant tissue culture.
Biological contamination in plant cell cultures originates from two sources, namely, from the tissue used to initiate the culture, and from the laboratory environment. Modification of the micro tissue culture technique, including incubation under increased co2, resulted in prolongation of the viability of the cells. Inactivating ingredients, used to kill viruses or inactivate toxins. Modern applications of plant biotechnology in pharmaceutical sciences, 2015.
Quality control considerations in cell culture sigmaaldrich. In the course of plant growth and development many surface and rhizosphereinhabiting. Leifert is with the department of cell and structural biology, manchester university, manchester m 9pt, uk. Live imaging movie produced in the leib laboratory at dartmouth college to help diagnose different contaminants of eukaryotic cell cultures. The minimal infectious dose of mycoplasma for tissue cultures was measured, and it was determined that one organism was capable of initiating an infection in a tissue culture.
Pdf regeneration of multiple individual plantlets from a single cell or explant is one of the main aims of plant tissue culture. Microbial contaminants in plant tissue culture propagation. Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Rooting rate is the percentage of the number of rooted seedlings divided by the total. Jun 01, 2012 importance of controlling contamination the medium contains many different bacterialnutrients, both original constituents of the medium and exudates from the plant cells. Contamination problems can be divided into 3 classes. Microbial contamination is a constant problem, which often compromise development of all in vitro techniques. I have proven this over the years by settle plates. Considerations for contamination cleanup in plant tissue. Contamination of tissue cultures by mycoplasmas intechopen. Waites is with the department of applied biochemistry and food science, university of nottingham, sutton. Totipotencya cell characteristic in which the potential for forming all the cell types in the adult organism are retained. Endophytic bacterial contamination is an important problem in plan tissue culture kneifel and leonhardt.
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. So if they come in contact with medium either in cellular form or in spore form, the microorganisms grow faster than the higher plant tissue due to their brief life cycle and will kill the tissue. Plant tissue culture terminology adventitiousdeveloping from unusual points of origin, such as shoot or root tissues, from callus or embryos, from sources other than zygotes. Bacterial contamination remains a continuing thrt to plant tissue culture, but techniques for reducing con,taminatton are available. It is widely used to produce clones of a plant in a method known as micropropagation. Plant surfaces are habitats for microorganisms campbell, 1985.
Antibiotics, used to prevent contamination by bacteria. An intracellular organism that looks like moving black dots is growing inside cultured cells. Agara polysaccharide powder derived from algae used to gel a medium. Understanding and managing cell culture contamination technical. Two basic culture systems are used for growing cells. The black dots will grow to sufficient number to break out of the cell and migrate onto other cells. Tissue culture contamination frequently originates with the introduction into culture of explants contaminated with endophytic microorganisms or surface sterilisationresistant microorganisms e. Contamination in tissue culture can originate from two sources, either through carry over of microorganisms on. Minor annoyanceswhen up to several plates or flasks are occasionally to contamination. Some of the advantages of in vitro cultures are their. Invasive mycoplasma, bacteria, and fungi can kill or drastically alter. Laboratories must assess their situation, detennine contamination sources, and change their laboratory operalions to avoid or eliminate most of the contaminants.
How can one reduce contamination in the tissue culture lab. Examples of ingredients added in complex media beef extract. Cells, differentiated and undifferentiated calli and suspended cells tissues, and organs can be maintained in vitro. Bacterial contamination can reduce growth rate, retard rooting, and even cause plant death leifert and waites, 1992. Understanding and managing cell culture contamination. Life sciences corning incorporated 4 consequences of contamination personal embarrassment loss of time, money and effort adverse effects on the cultures loss of valuable cells andor products bad dataerroneous publications. Tissue culture the maintenance or growth of tissue, in vitro, in a way that may allow differentiation and preservation of their function. Yeast is used to ferment the sugars of various grains to produce alcoholic beverages and in the baking industry to expand dough. Porcine trypsin is also a potential source of mycoplasma. The process includes many stepsstock plant care, explant selection and sterilization, media manipulation to obtain proliferation, rooting, acclimation, and growing on of liners.
Bradbury 1988when microbes come in contact with plant tissue or. The principle of meristem culture is that most endophytes are absent from apical tissue. No cell culture problem is as universal as that of culture loss due to contamination. Contamination issue in cell culture tissue and cell culture. Composed of 12 chapters, the book describes the frequency of occurrence of contamination and the many different effects of contamination on cultured cells. The pattern of contamination and the small dose required for infection indicated that mycoplasma contamination was spread from one tissue culture to another via aerosols. Most epiphytes are eliminated by surface sterilants. Mar 10, 2011 an intracellular organism that looks like moving black dots is growing inside cultured cells. This study aimed at investigating the sources of microbial contamination in tissue culture laboratories in southwestern nigeria. Nineteen microbial contaminants consisting of eleven bacteria and eight fungi were found associated with the tissue culture plants and the laboratory.
Contamination detection and elimination in plant cell culture. Plant tissue culture an overview sciencedirect topics. Contamination in tissue culture can originate from two sources, either through carry over of microorganisms on the surface or in the tissues of explants, or through. Introduction to animal tissue culture science book. Plant tissue culture is defined as culturing plant seeds, organs, explants, tissues, cells, or protoplasts on a chemically defined synthetic nutrient media under sterile and controlled conditions of light, temperature, and humidity. Culturing animal cells, tissue or organs in a controlled artificial environment is called animal tissue culture. Effects of different light qualities on root growth characteristics of c. The file will be downloaded and can be opened with adobe acrobat reader. In vitro plant cell cultures are defined as a culture in aseptic conditions of any part of the plant body in a nutritive media. Techniques used in plant tissue culture with diagram. Pdf plant tissue culture contaminants identification and its. Bacteria can be isolated from nearly any surface including inanimate objects and human skin. Detection and control of bacterial contaminants of plant.