Disinfection

Methods designed for the disinfection/decontamination of culture waste, work surfaces and equipment represent important means for minimizing the risk of harm.
The major disinfectants fall into four groups and their relative merits can be summarized as follows:

• Hypochlorites (e.g. Chloros, Presept) Good general purpose disinfectant
  Active against viruses,Corrosive against metals and therefore should not be used on metal surfaces e.g. centrifuges. Readily inactivated by organic matter and therefore should be made fresh daily Should be used at 1000ppm for general use surface disinfection, 2500ppm in discard waste pots for washing pipettes, and 10,000ppm for tissue culture waste and spillage
• Phenolics (e.g. Sudol, Hycolin)
  Not active against viruses
  Remains active in the presence of organic matter
• Alcohol (e.g. ethanol, isopropanol)
  Effective concentrations 70% for ethanol, 60-70% for isopropanol
  Their mode of activity is by dehydration and fixation
  Effective against bacteria. Ethanol is effective against most viruses but not nonenveloped viruses. Isopropanol is not effective against viruses.
• Aldehydes (e.g. glutaraldehyde, formaldehyde)
  Aldehydes are irritants and their use should be limited due to problems of sensitization
• Glutaraldehyde may be used in situations where the use of hypochlorites is not suitable e.g. cleaning of centrifuge bowls or materials constructed of stainless steel that may be attacked or corroded by using hypochlorite solutions.

Waste Disposal

Any employer has a ‘duty of care’ to dispose of all biological waste safely in accordance with national legislative requirements. Given below is a list of ways in which tissue culture waste can be decontaminated and disposed of safely(especially the solid waste, such as flasks, centrifuge tubes, contaminated golves etc). One of the most important aspects of the management of all laboratory-generated waste is to dispose of waste regularly and not to allow the amounts to build up. The best approach is ‘little and often’. Different forms of waste require different treatment.

Tissue culture waste (culture medium) - Inactivate overnight in a solution of hypochlorite (10,000ppm) prior to disposal to drain with an excess of water
Contaminated pipettes should be placed in hypochlorite solution (2500ppm) overnight before disposal by autoclaving and incineration
Solid waste, such as flasks, centrifuge tubes(such as 15ml Centrifuge Tube, 50ml Centrifuge Tube), contaminated gloves, tissues etc. should be placed inside heavy duty sacks for contaminated waste and autoclaved prior to incineration. These bags are available from Bibby Sterilin and Greiner.
If at all possible waste should be incinerated rather than autoclaved.

Source: Sigma-Aldrich

Aim

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.

Materials

1. Chloros / Presept solution (2.5g/l)
2. 1% formaldehyde based disinfectant e.g.Virkon,Tegador
3. 70% ethanol in water (Prod. No. R8382)
   

Equipment

1. Personal protective equipment (sterile gloves, laboratory coat, safety visor)
2. Microbiological safety cabinet at appropriate containment level
3. Cell Culture Plates(6 well plate, 24 well plate, 96 well plate)
   

Procedure

Sanitize the cabinet using 70% ethanol before commencing work.
Sanitize gloves by washing them in 70% ethanol and allowing to air dry for 30 seconds before commencing work.

Put all materials and equipment into the cabinet prior to starting work after sanitizing the exterior surfaces with 70% ethanol.
Whilst working do not contaminate gloves by touching anything outside the cabinet (especially face and hair). If gloves become contaminated re-sanitize with 70% ethanol as above before proceeding.

Discard gloves after handling contaminated cultures and at the end of all cell culture procedures.
Equipment in the cabinet or that which will be taken into the cabinet during cell culture procedures (media bottles, pipette tip boxes, pipette aids, cell culture plates) should be wiped with tissue soaked with 70% ethanol prior to use.
Movement within and immediately outside the cabinet must not be rapid. Slow movement will allow the air within the cabinet to circulate properly.
Speech, sneezing and coughing must be directed away from the cabinet so as not to disrupt the airflow.

After completing work disinfect all equipment and material before removing from the cabinet. Spray the work surfaces inside the cabinet with 70% ethanol and wipe dry with tissue. Dispose of tissue by autoclaving.
Cell culture discard in chloros (10,000) ppm must be kept in the cabinet for a minimum of two hours (preferably overnight) prior to discarding down the sink with copious amounts of water.
Periodically clean the cabinet surfaces with a disinfectant such as Presept,Tegador or Virkon or fumigate the cabinet according to the manufacturers instructions. However you must ensure that it is safe to fumigate your own laboratory environment due to the generation of gaseous formaldehyde, consult your on-site Health and Safety Advisor.

Source: Sigma-Aldrich

Given below are a few of the essential "do’s and don'ts" of cell culture. Some of these are mandatory e.g. use of personal protective equipment (PPE). Many of them are common sense and apply to all laboratory areas. However some of them are specific to tissue culture.

The Do’s

1. Use personal protective equipment, (laboratory coat/gown, gloves and eye protection) at all times. In addition, thermally insulated gloves, full-face visor and splash-proof apron should be worn when handling liquid nitrogen.
2. Always use disposable caps to cover hair.
3. Wear dedicated PPE for tissue culture facility and keep separate from PPE worn in the general laboratory environment. The use of different colored gowns or laboratory coats makes this easier to enforce.
4. Keep all work surfaces free of clutter.
5. Correctly label reagents including flasks, medium and ampules with contents and date of preparation.
6. Only handle one cell line at a time. This common-sense point will reduce the possibility of cross contamination by mislabeling etc. It will also reduce the spread of bacteria and mycoplasma by the generation of aerosols across numerous opened media bottles and flasks in the cabinet.
7. Clean the work surfaces with a suitable disinfectant (e.g. 70% ethanol) between operations and allow a minimum of 15 minutes between handling different cell lines.
8. Wherever possible maintain separate bottles of media for each cell line in cultivation.
9. Examine cultures and media daily for evidence of gross bacterial or fungal contamination. This includes medium that has been purchased commercially.
10. Quality Control all media and reagents prior to use.
11. Keep cardboard packaging to a minimum in all cell culture areas.
12. Ensure that incubators, cabinet, centrifuges and microscopes are cleaned and serviced at regular intervals.
13. Test cells for mycoplasma on a regular basis.
    

The Don’ts

1. Do not continuously use antibiotics in culture medium as this will inevitably lead to the appearance of antibiotic resistant strains and may render a cell line useless for mmercial purposes.
2. Don’t allow waste to accumulate particularly within the microbiological safety cabinet or in the incubators.
3. Don't have too many people in the lab at any one time.
4. Don't handle cells from unauthenticated sources in the main cell culture suite. They should be handled in quarantine until quality control checks are complete.
5. Avoid keeping cell lines continually in culture without returning to frozen stock.
6. Avoid cell culture becoming fully confluent. Always sub-culture at 70-80% confluency or as advised on ECACC's cell culture data sheet.
7. Do not allow media to go out of date. Shelf life is only 6 weeks at +4ºC once glutamine and serum is added.
8. Avoid water baths from becoming dirty by using Sigma Clean (Prod. No. S5525).
9. Don’t allow essential equipment to become out of calibration. Ensure microbiological safety cabinets are tested regularly.

Source: Sigma-Aldrich ECACC Handbook

Presenter: Lydiane (Ann) Kyte

Host: Kathy Liu

Discussion

Did you ever have a plant that was so unique or so beautiful that you wished you had hundreds or thousands of them to enjoy or to sell? Plant tissue culture (micropropagation) is a technique which will do just that for us. We are going to discuss this tool which is used so extensively in the nursery business and in plant biotechnology. It is a fascinating and useful tool which allows the rapid production of many genetically identical plants using relatively small amounts of space, supplies and time. Basically the technique consists of taking a piece of a plant (such as a stem tip, node, meristem, embryo, or even a seed) and placing it in a sterile, (usually gel-based) nutrient medium where it multiplies. The formulation of the growth medium is changed depending upon whether you are trying to get the plant to produce undifferentiated callus tissue, multiply the number of plantlets, grow roots, or multiply embryos for "artificial seed".For many who become superficially aware of the technique it seems shrouded in mystery and is shrugged off as too technical to be of concern. Actually, it is no more of a mystery than taking a cutting of your favorite house plant and growing it to share with a friend. As for being technical, you can begin plant tissue culture with as little as a cookbook approach and a feeling for sterile technique.Some people have visions of scientists doing plant tissue cultures in white gowns and masks in hospital-clean environments. Such conditions are excessive. While it is true that mold spores, bacteria, and other contaminants will grow and overrun a culture, air that is not moving has a minimum of contaminants. In addition, disinfection of implements, work surface and nearby areas helps eliminate contaminants.The guidelines for preparation and the laboratory protocol provided here are given as a place to begin. Included with is a limited discussion of some of the many options you have as you explore micropropagation. We can discuss these in more depth if you have questions, concerns or related experiences to share. I would be particularly interested in success and challenges you may have had or are currently having in your classroom.Some suggestions are given for the following(a) Selecting plant sources. Some species, or even clones are easier to grow in culture than others. Some respond reluctantly to culture, some do not respond at all, and many plants have never been tried.(b) Choosing a growth medium (price, convenience, type of plant and purpose of the micropropagation all enter into this decision.) How important are the kinds of hormones used? On limited scale, media ingredients are available at the grocery and health food stores. (c) Suggestions for media preparation and sterilization. There are alternatives to sterilization in a pressure cooker or an autoclave.(d) Methods for cleaning, storing and manipulating explants (plant pieces to be cultured).Given certain basics there are many options for procedure, equipment and supplies for plant tissue culture. Some of your decisions will be based upon the amount of time, money and space you have. Other decisions will be based upon why you are doing plant tissue culture and what you expect as a result (more plants?) . Catalogs, such as Sigma, Carolina Biological, or Edmund Scientific are good reference and they are for purchasing needed materials..I look forward to sharing tissue culture experiences with you.

References:

Debergh, P.C. and R.H. Zimmerman, eds. 1991. Micropropagation, Technology and Application. Kluwer Academic Publishers. $61.50. Lab design, info on labs worldwide, in depth discussions of problems. Not for the beginner.

Donnelly, D.J., and W.E.Vidaver, 1988. Glossary of Plant Tissue Culture, Portland, OR. Timber Press, $22.95. Good definitions of tissue culture terms.

Kyte, Lydiane and J. Kleyn, 1996. Plants from Test Tubes: An Introduction to Micropropagation, 3rd ed., Timber Press, 1996 $29.95. Good basics for the beginning amateur or grower.

Smith, Roberta H., 1992. Plant Tissue Culture-Techniques and Experiments. Academic Press. $35.00. Good introduction and broad base for college course.

Trigiano, Robert N, and Dennis J. Gray, eds.1996,Plant Tissue Culture Concepts and Laboratory Exercises. CRC Press. $65.00. For the advanced student.

From Wikipedia, the free encyclopedia

1 Historical usage
2 Modern usage
3 References
4 See also
5 External links

Tissue culture is the growth of tissues and/or cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid, or solid growth media, such as broth or agar. Tissue culture commonly refers to the culture of animal cells and tissues, while the more specific term plant tissue culture is used for plants.

Historical usage
In 1885 Wilhelm Roux removed a portion of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the basic principle of tissue culture.[1]
In 1907 the zoologist Ross Granville Harrison demonstrated the growth of frog nerve cell processes in a medium of clotted lymph.

Modern usage
Main article: cell culture
In modern usage, "tissue culture" generally refers to the growth of eukaryotic cells in vitro. It is often used interchangeably with cell culture to specifically describe the in vitro culturing of sperm donor cells.
However, "tissue culture" can also be used to refer to the culturing of tissue pieces, i.e. explant culture or whole organs, i.e. organ culture

About Plant Cell, Tissue and Organ Culture publishes articles of scientific and practical importance, including research articles, brief research notes, reviews, and book reviews, covering all areas of research in which plant tissue culture is a major aspect.

Coverage extends to biotechnological breeding including genetic modification, somatic hybridisation and embryo rescue; propagation in vitro, including micropropagation, somatic embryogenesis and freeing plants from diseases; the use of tissue culture in physiological or molecular researches; production of secondary metabolites; storage of valuable genetic material including slow growth in vitro and cryopreservation.

The Editor-in-Chief of Plants and Tissue Culture is Ir. Edhi Sandra MSi, ESHA FLORA Tissue Culture Research, and Lecturer of Forestry Faculty Institut Pertanian Bogor (IPB) - Bogor Agricultural University, Indonesia.