Sterile Technique 2009

Sterile Technique 2009 - Sterile (Aseptic) Technique...

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Sterile (Aseptic) Technique Aseptic technique is absolutely necessary for the successful establishment and maintenance of plant cell, tissue and organ cultures. The in vitro environment in which the plant material is grown is also ideal for the proliferation of microorganisms. In most cases the microorganisms outgrow the plant tissues, resulting in their death. Contamination can also spread from culture to culture. The purpose of aseptic technique is minimize the possibility that microorganisms remain in or enter the cultures. The environmental control of air is also of concern because room air may be highly contaminated. Example: Sneezing produces 100,000 - 200,000 aerosol droplets which can then attach to dust particles. These contaminated particles may be present in the air for weeks. (Have you ever viewed the air around you when you open the curtains on a sunny day?). Air may also contain bacterial and fungal spores, as do we. I. Contaminants A. Bacteria, fungi, and insects 1. Bacteria Bacteria are the most frequent contaminants. They are usually introduced with the explant and may survive surface sterilization of the explant because they are in interior tissues. So, bacterial contamination can first become apparent long after a culture has been initiated (see below). Some bacterial spores can also survive the sterilization procedure even if they are on the tissue surface. Many kinds of bacteria have been found in plant tissue cultures including Agrobacterium , Bacillus , Corynebacterium , Enterobacter , Lactobacillus , Pseudomanas , Staphylococcus , and Xanthomonas . Bacteria can be recognized by a characteristic "ooze"; the ooze can be many colors including white, cream, pink, and yellow. There is also often a distinctive odor. 2. Fungi Fungi may enter cultures on explants or spores may be airborne. Fungi are frequently present as plant pathogens and in soil. They may be recognized by their "fuzzy" appearance, and occur in a multitude of colors. 3. Yeast Yeast is a common contaminant of plant cultures. Yeasts live on the external surfaces of plants and are often present in the air.
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4. Viruses, etc. Viruses, mycoplasma-like organisms, spiroplasmas, and rickettsias are extremely small organisms that are not easily detected. Thus, plant culture is not necessarily pathogen-free even if microorganisms are not detected, and this can influence culture success. Special measures such as meristem culture are often necessary to eradicate such contaminants. 5. Insects The insects that are most troublesome in plant cultures include ants, thrips, and mites. Thrips often enter cultures as eggs present on the explants. Ants and mites, however, usually infest already established cultures. Mites feed on fungus and mite infestations are often first detected by observing lines of fungal infection that lead from the edge of the culture vessel to the plant tissue, having been introduced by the insect. It is very difficult to eradicate insect infestations. Careful lab practices and
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Sterile Technique 2009 - Sterile (Aseptic) Technique...

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