CULTURE MEDIA, CULTIVATION, STERILE TECHNIQUE

Transcription

1 CULTURE MEDIA, CULTIVATION, STERILE TECHNIQUE

2 saprotroph FUNGI ARE HETEROTROPHS! biotroph dead cells living cells

3 Important ecological roles as decomposers, mutualists, parasites

4 Fungus cells are totipotent. Fungi differentiate into a variety of structures including spores which enable continuation over space and time. The mechanisms for formation of each type of structure is largely determined by the perception of the environment by the thallus. Water and organic nutrients are particularly important for fungi, thus we would expect explosions of fungi in humid, energy rich habitats. However, a few fungi also tolerate extreme environments. Growth of most fungi is indeterminate. The diversity of stages of any one mycelium means that fungi, at any one time, may have hyphae in extension, productive, fruiting and senescing phases. The concept of a single response of a body, such as an animal, does not apply to the filamentous fungi.

5 Environmental Factors: Oxygen is used in respiration in most organisms. The fungi include species that are obligately aerobic or obligately anaerobic (eg rumen fungi). However many fungi are in between, with the capacity to function facultatively in aerobic and anaerobic conditions. CO 2 -The presence of carbon dioxide is also required for some fungi. Water availability has a major influence on the function of fungi. Most fungi require very high water availability (relative humidity), and rapidly dry out or senesce in dry conditions. ph - fungi can tolerate a wide range of ph, though most media used to culture fungi are acidic. Temperature - Fungi can normally tolerate the range of temperature of the environment from which they are taken. Their response to temperature is quite varied, however. Active growth will usually be associated with a limited range of temperatures. Those fungi that grow between 15 and 35 C are called mesophilic, and above thermophilic. Those that grow at freezing are called psychrophilic. Light has an important influence on fungal growth in specific cases. The effect of UV radiation on spore and sporocarp formation, and phototropic release are clear examples of light being important. UV radiation also reduces viability of spores especially in air. Overall, light does not play a major part in metabolism and growth of fungi.

6 As Heterotrophs Fungi typically require: Carbon Source - Glucose (dextrose) is the most widely utilizable carbon source, Fructose and mannose are the next most commonly utilized sugars by fungi. More complex sources in sugar alcohols, starches, cellulose, hemicellulose, and other complex carbohydrates. Nitrogen sources include peptone, yeast extract, malt extract, amino acids, ammonium and nitrate compounds. *Macro l nutrients: M, K, P, Mg, S, Ca. *Micronutrients: Fe, Cu, Mn, Zn, Mo. (*minerals) Fungi have natural deficiencies for vitamins that are satisfied at mm to nm concentrations. The most common naturally occurring vitamin deficiencies are two B vitamins thiamin and biotin. Other organic nutrients such as glucose are often contaminated with vitamins sufficient to supply the growth requirements of fungi.

7 Culture Medium - Potato dextrose (D-glucose or grape sugar )agar Percentage of U.S. Recommended Daily Allowances for a Medium Potato Protein... 6 % Vitamin C % Thiamin... 8 % Riboflavin... 2 % Niacin % Iron... 8 % Vitamin B % Folacin (folic acid). 8 % Phosphorus... 8 % Magnesium... 8 % Zinc... 2 % Copper % Pantothenic acid... 4 % Iodine % Calories Protein... 3 grams Carbohydrate grams Fat... 0 grams Dietary Fiber mg Sodium mg Potassium mg

8 Growth Media Liquid medium Malt Extract from malted grains Solid medium Agar-agar is a natural gelling substance from the cell walls of red algae, of, like Gelidium and Gracialaria.

9 Most culture media fit into one of three categories: (1) synthetic, (2) semisynthetic, and (3) natural. Synthetic media are composed of ingredients of known chemical composition and concentration. These media are useful in physiological or descriptive studies when it is necessary to duplicate exactly a previous batch of medium or to record the effects of the deletion or addition of a particular substance. Semi-synthetic media resemble synthetic media in containing a known set of ingredients, but differ in that at least some of the ingredients are of unknown or variable composition. We know that the yeast extract contains thiamine and other vitamins, but we do not know the exact amounts or what else might be present. The result is a medium of quite predictable composition but one not completely known chemically. Semi-synthetic media are widely used in routine work and offer something of a compromise between synthetic and natural media. Natural media are so called because they are partly or completely composed of natural materials. A slice of potato is a natural culture medium, as is a piece of meat or bread. Natural media are often very good and allow sporulation in fungi that may otherwise remain sterile. Their major disadvantage is that they may differ considerably from batch to batch and thus not yield reliable experimental results. Nevertheless, natural media are widely used in laboratory work.

10 SYNTHETIC Czapek's Solution Agar Sucrose 30 g NaNO3 3.0 g K2HPO4 1.0 g MgSO4.7H2O 0.5 g KCl 0.5 g FeSO4.7H2O 0.01 g Agar 15 g Distilled water 1000 ml Czapek's Solution Agar is a synthetic medium widely used in mycological laboratories, particularly for the identification of species of Aspergillus and Penicillium. Many moulds produce very characteristic colonies on it and may also exude pigmented substances. Aerial growth is often suppressed and sporulation may be enhanced.

11 Semi-Synthetic Medium Potato Dextrose Agar Thinly sliced, peeled white potatoes 500 g Glucose 20 g Agar 15 g Distilled water 1000 ml Heat potatoes at 60 C for 1 hour and filter through cheesecloth. Make up volume to 1000 ml and add other ingredients. Cook 1 hour and then sterilize. Potato Dextrose Agar, or PDA, as it is usually called, is an old formula used by plant pathologists and many mycologists for general laboratory use.

12 Natural Media

13 To transfer the culture we do the following: 1.Take an inoculating needle, usually a thin needle or wire at the end of a long pencil-like handle, and heat it in an alcohol or gas flame until it glows bright red (Figure 10A). 2.Allow the needle to cool for about 15 seconds. (A hot needle will kill the mold that is to be transferred). 3.Open the Petri dish containing the culture just wide enough to allow entry of the needle. 4.With the heat-sterilized needle, cut out a small portion of the colony margin. Hyphal tip transfers work best as they are usually the most active parts of the culture; in addition, transfers from the heavily sporulating central portions will result in spores being spread into the air. Especially in medical work, hyphal tip transfers are essential. The excised colony margin should be only about 1 mm square (Figure 10B). 5.Transfer the square of colony margin to the sterile plate, making sure that the lid is opened only wide enough to admit the needle and make the transfer. Place the block at the center, withdraw the needle and flame it until it is red hot, to kill all adhering spores and hyphae (Figure 10C,D). 6.Close the lid; label the plate with a marking pen, including name of culture and date. We usually wrap a thin strip of paraffin film around the sides of the plate to cover the opening, but this is not absolutely necessary; just a couple of pieces of masking tape to hold the lid down will do. 7.Leave the culture to grow in a protected place that has as little air movement as possible. Figure 10. Steps in sterile technique. Inoculating needle is heated in an alcohol flame (A). Small piece of colony is removed from Petri dish (B) and transferred to a new dish of agar (C), yielding a plate containing a piece of the old culture at its center (D).

14 In vivo culture with host plant Obligate Symbiotic Fungi During the 1980s, the improvement of technologies related to the cultivation of excised roots allowed the successful cultivation of some AM fungi strains. In vitro culture on excised roots

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17 Malt agar Spawn on grain! plugs

18 I. Growth parameters for mushroom cultivation 1. Moisture humidity and substrate 2. Air Exchange - CO2 3. Temperature 4. Lighting II. Three main stages in Mushroom growth and reproduction 1. Spawn Run mycelium growing in substrate 2. Primordia Formation - initiation of mushrooms 3. Fruitbody Development ending in cropping

19 Creating Conditions for Mushroom Fruiting High humidity. Most species like 80 to 95% humidity. Ideal temperature for fruiting varies with species and strain. Oyster and shiitake have cold and warm weather strain Good air exchange ventilation or fan, low CO2 levels Enough light. Indirect sunlight for most species. The button mushroom, Agaricus bisporus an exception prefers darkness Enoki, low light, high C02

20 Under high CO2 levels or with less frequent ventilation, mushrooms produce long stipes with small caps, while they produce short stipes with broad caps under low CO2 levels or frequent ventilation.