1. Preparation of growth medium- PDA and Vogel’s minimal medium (Standard).
Take 200 g of potatoes and cut small pieces after peeling. Boil the pieces of potato in 500 ml water. After the potatoes are soft filter with miracloth (any strainer, or cloth). Remove the pieces and take the nutrient broth. Add 20 g of dextrose (sugar) and 15 g of Agar1. Boil and make up the volume 1000 ml. For liquid medium agar should not be added. Pour the medium in test tubes or flasks and autoclave2.
2. Study of hyphal structure, morphology and conidiation – by cellophane method
Prepare 200 ml PDA and autoclave. Pour in a glass trey and allow it to solidify. Cover the layer of medium with autoclaved film of cellophane (cut circles and boil in water). Inoculate Neurospora conidia over the cellophane. Incubate between 20-350C (optimum 340C). After growth of culture small pieces of cellophane can be cut and observed under microscope for studying the hyphal morphology and conidiation.
3. Determination of growth rates
Pore PDA in a covered glass dish and allow it to solidify. Cover with lid. Inoculate Neurospora at one end. Mark the growing edge after every 12 or 24 hours. Plot the growth curve and calculate the growth rate.
1. From soil – Heat treatment of soil
Suspend about 100-200 mg soil in 2 ml sterilized water in a capped tube. Heat at 600C (or boil over light flame for some times) for 30 minutes. Plate over PDA plates containing Rose Bengal (50 mg / liter).
2. From air – by
plate exposure and by baiting with bread
Expose the PDA plates to air for five minutes. Incubate at 300C.
Keep a moist piece of bread in warm and humid container. Give intermittent exposure to air.
1. Use of corn cobs as medium
Take the corn cob and remove the kernels. The cobs can be dried in sunlight and stored in dried state for years.
In order to study the sexual stage of Neurospora or makeing crosses take a piece of cob, boil it or burn over flame. Keep in a trey and moisten with boild water, cover the trey with polythene (holes punched for aeration). Inoculate with Neurospora strains of opposite mating types. Incubate at 20-250C for a week.
2. Preservation of perithercia formed in formalin
Keep in cob piece with Neurospora in 5% formaldehyde solution with little acetic acid. It can be used for studying sexual stage of Neurospora any time.
Dissection of perithecia to study asci bearing ascospores
Pick up the perithecia form the cob with the help of forceps. Keep over slide. Put one drop of water and split into two halves with the help of two needles. Observe under low power. Remove debris, and cover with cover glass and observe under high power.
Additional notes by Tony Griffiths:
Alternatives to autoclaves for sterilization of equipment, glassware and media.
The same methods used by cooks to bottle/can food work quite well. A pressure cooker is useful if available. Put the materials to be sterilized on a metal tray above about 2 cm of water inside the cooker. For liquids such as growth medium, never fill more than half the capacity of the vessel. Cook at top pressure for about 15 minutes, take the cooker off the heat and allow the pressure to drop naturally (slowly).
An oven is useful for sterilizing empty glassware and metalware. Plug tubes and flasks with a porous plug such as cotton or aluminum foil. Wrap small dry goods in brown paper or foil to keep it sterile when it has cooled.
Liquid media etc can be sterilized by placing the flask of medium on a metal support inside a large cooking pot with about 2 cm in the bottom, and whose mouth is covered by a lid or a sheet of foil. Boil the water for about an hour or until the medium is molten.
Commercial agar is very expensive to buy. Unfortunately there really aren’t any good alternatives that are readily available. Nevertheless, here are some ideas.
1. Cooking gelatin is quite expensive but does set clear and firm. Some claim that the dessert called Jello works, but it has a lot of sugar and who knows what else – try it!
2. If you live near the ocean, collect some seaweed and make your own agar (it comes from sea weed). Collect about a bucketful of seaweed, cut it up finely and boil it in water for about 30 minutes; filter off the solid material through a wide mesh type of cloth such as cheese cloth and let it set in a tray. If a nice gel develops, let it dry out in the sun or in a low temperature oven, then grind up the dry agar in a food grinder and use as necessary. Agar can be “washed” to get rid of soluble impurities by reheating the powder in water, and then re-drying and grinding (a tedious and time-consuming process).
3. Neurospora will grow well on slices of ripe melon. Try to keep sterile everything used to cut the melon, and the melon surface. (This can be done with alcohol.) Firm-fleshed melons work best (i.e. not watermelon). Of course Neurospora grows on bread too. If you want to just see it grow and form conidia, inoculate melon or bread and put it under a “bell jar” made of a cut in half 2 litre plastic soda bottle taped back together. If necessary, the whole set-up can then be bagged and thrown out.
Alternatives to laboratory equipment etc.
There really is no substitute for test tubes, but luckily these are quite cheap and the glass ones can be rewashed and re-sterilized. Small commercial glass drink bottles with metal lids can be treated like large test tubes that have the advantage of standing upright (sterilize with the lids loose, or replace lids with wads of cotton with foil covers).
Larger glass drink bottles can be used for making and sterilizing medium, much as one would use an Erlenmeyer (conical) flask. However, note that the glass used in drink bottles is not tempered and is more sensitive to sudden temperature changes than “Pyrex-style” glassware. Canning/bottling jars are designed to be more tolerant to heat changes. Always keep lids loose when sterilizing. Do not heat glass jars etc. over any kind of burner – use steam or water sterilization/cooking as above, and heat them up and cool them down slowly. Lidded saucepans are an alternative for medium making if the medium must be heated on a gas or electric burner. A turkey baster can be used to fill smaller vessels from the pan.
Test tube racks can be inexpensively made using “hardware cloth”, a square metal mesh available in hardware stores. It comes in several sizes. Just cut and bend to the right shape. Blocks of wood drilled with holes are also useful but do not stand up well to water or steam used in sterilizing.
Lidded Pyrex-style glass baking dishes come in all sizes and can be used in much the same manner as Petri dishes.
Sterile microbiological techniques require a small flame to sterilize isolation needles and to flame the necks of tubes etc. A spirit lamp or a fondue lamp works fine. A propane torch set very low is useful. At a pinch, a wax candle could be used, although most candles deposit carbon on the flamed object - if possible use the smoke-free type.
Inoculation tools can be made as twists of wire or wire loops mounted in a wooden handle. Wire flattened at the end by hammering makes a useful spade-like mini-tool for picking up fragments of colonies. Sewing needles mounted in wooden handles make suitable tools for isolating individual ascospores. Heating the points to sterilize between isolations makes them blunt before too long, but they can be resharpened on fine sandpaper or sharpening stones.