Maintenance of Aseptic Environment

It is very important to maintain aseptic environment during the in vitro culture of plant cells and tissues. Following are some of the methods adopted for sterilization:

(a) Sterilization of Glassware- The glassware can be sterilized in a hot air oven at 160-1800C for 2-4 hours.

(b) Sterilization of instruments- The metallic instruments are incinerated by dipping them in 75% ethanol followed by flaming and cooling.

(c) Sterilization of nutrient media- The culture media are transferred into glass container, plugged with cotton or sealed with plastic closures and sterilized by autoclaving at 15 psi for 30 min. The autoclaving denatures the vitamins, plant extracts, amino acids and hormones therefore the solution of these compounds are sterilized by using Millipore filter paper with pore size of 0.2 micrometer diameter.

(d) Sterilization of plant materials- The surface of the plant material is made sterile by using disinfectants e.g. sodium hypochlorite, hydrogen peroxide, mercuric chloride, or ethanol. The transfer of sterile plant material on to the nutrient medium is done under the cabinet of laminar airflow.

(e) Sterilization of Culture room and transfer area- the floor and walls of the culture room should be washed with detergent followed by 2% sodium hypochlorite or 95% ethanol. The sterilization can also be done by exposure to UV light. The cabinet of laminar air flow is sterilized by exposing to UV light for 30 min. and 95% ethanol 15 minutes before starting the work.

Progress of work:

Preparation of ware and media for sterilisation.

Ware before sterilisation carefully wash, dry and paper for preservation of sterility after warming up. Each pipette wrap separately in long strips of a paper, in the width 4-5 cm. In the ends of pipettes which take in a mouth, preliminary insert wadded tampons. A winding begin with the opposite end gradual movement of a paper on a spiral and end at the end with a tampon. The paper should cover a pipette densely. Wrapped up pipettes for protection of a paper from pollution and ruptures turn on some pieces together or place in special metal or cardboard cases.

Spreading rod wrap up separately to similarly pipettes, using for a winding of a strip of a bigger paper width. Petri dishes wrap together on 2А pieces.

Flasks, etc. vessels close test tubes wadded stoppers, and from above paper napkins.

Nutrient media preparation

Potato dextrose agar. Potato dextrose agar and potato dextrose broth are common microbiological growth media made from potato infusion, and dextrose. Potato dextrose agar (abbreviated "PDA") is the most widely used medium for growing fungi and bacteria which attack living plants or decaying dead plant matter. Potato infusion can be made by boiling 300 grams of sliced (washed but unpeeled) potatoes in ~ 1 litre (0.22 imp gal; 0.26 US gal) water for 30 minutes and then decanting or straining the broth through cheesecloth. Distilled water is added such that the total volume of the suspension is 1 litre (0.22 imp gal; 0.26 US gal). 20 grams (0.71 oz) dextrose and 20 grams (0.71 oz) agar powder is then added and the medium is sterilized by autoclaving at 15 pounds per square inch (100 kPa) for 15 minutes.

A similar growth medium, Potato dextrose broth (abbreviated "PDB") is formulated identically to PDA, omitting the agar. Common organisms that can be cultured on PDB are yeasts such as Candida albicans and Saccharomyces cerevisiae and molds such as Aspergillus niger.

Wort agar medium. Well I've taken a try at making plates and slants. I've tried a couple different amounts of agar for setting my wort. All the different sources I'm learning from tell me many different things.

One source told me 1.5-2 grams agar per 400ml. These plates ended up a bit watery, and my innoculation loop ripped right into the agar.

Then I tried a different recipe as per Kaiser which was 3-4 grams agar in 100ml.. quite drastically more. This agar came out very very hard. I plated some yeast on these last night. I am worried though that the agar might be too hard to allow the yeast to receive moisture.

Yet another source (Rajotte's book) tells me to use yet another amount (1.2g agar per 100ml wort).

Control questions:

1. Types of the nutrient media

2. Methods of sterilization of the nutrient media

3. Methodology of nutrient media preparation.