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.