Culture Media Laboratory Reagents Biology and Biotechnology department

Culture Media & Laboratory Reagents Biology and Biotechnology department Miss. Aya Z. Alajrami

Objectives • Be able to introduce of culture media for fungi. • Be able to introduce of laboratory reagents.

Introduction • Microorganisms need nutrients as a source of energy and certain environmental conditions in order to grow and reproduce. • In the environment, microbes have adapted to the habitats most suitable for their needs. • In laboratory, however these requirements must be met by a culture medium. • To culture fungi in the laboratory, it is necessary to furnish in the medium all essential nutrients for the synthesis of protoplasm.

• A wide range of media are used for growing fungi. Most mycologists develop preferences for certain types of media based on experience and peculiarities of the type of fungi that are routinely grown. • Media will affect colony morphology and color, whether particular structures are formed or not, and may affect whether the fungus will even grow in culture. • For example, some fungi lack the necessary enzymes to utilize different carbon sources. All fungi require several specific elements for growth and reproduction. • The requirements for growth are generally less stringent than for sporulation, so it is often necessary to try several types of media when attempting to identify a fungus in culture.

Constituents of Media • Media generally contain a source of carbon, nitrogen and vitamins. Glucose (dextrose) is the most widely utilizable carbon source, and hence is the most commonly used in growth media. Fructose and mannose are the next most commonly utilized sugars by fungi and are found in media from natural sources. Sucrose (table sugar) may be used in some media. Carbon source Nitrogen source Salts Vitamins

• Nitrogen sources include peptone, yeast extract, malt extract, amino acids, ammonium and nitrate compounds. • Salts, including Fe, Zn and Mn, are often added to ‘defined’ media, but are usually not added to the common media used for routine culture. • Fungi have natural deficiencies for vitamins that are satisfied at m. M to n. M concentrations. • The most common naturally occurring vitamin deficiencies are thiamin and biotin. • Deficiency of both is quite common among the Ascomycota.

Categories of Culture Media Enrichment media Defined media Complex media Depending on the type and combination of nutrients, different categories of media can be made: Semi- Synthetic media Differential media Selective media

v Complex media: An undefined medium, its consist from water and natural components for growing many types of fungi. v. Defined media (also known as chemically defined media, synthetic): all the chemicals used are known and its useful to know the requirements for fungal growth. v Semi- Synthetic media: The chemicals used in culture media are partially known. v. Enrichment media: Similar to selective media but designed to increase the numbers of desired fungus to a detectable level without stimulating the rest of the other fungi and bacterial population.

v Selective media: Suppress unwanted fungi, or encourage desired fungi. üHint: The choices of the media with v. Differential media: Distinguish colonies of specific fungus from another growing in the same media. This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators added to the medium to visibly indicate the defining characteristics of a microorganism. solid or broth is depended on the aim of experiments e. g. solid media uses for getting spores more than getting mycelia but broth media uses for getting mycelia more than getting spores to detection of the dry weight of fungi or other applications.

Isolation Media � Water Agar (WA): use for isolating fungi from surface-sterilized substrates. Antibiotic Agar (AA): use for isolating fungi from substrates not readily surface-sterilized, or to clean up a culture contaminated with bacteria. � Acidified Cornmeal Agar (ACMA): use for isolating fungi from substrates that are likely to be contaminated with bacteria. Not a substitute for AA, but the acidity inhibits bacteria and the medium supports the growth of a wide range of fungi. � Cornmeal Agar (CMA): use for growing a wide range of fungi, particularly members of the Imperfect fungi; provides a good balance of mycelial growth and sporulation.

v Potato Dextrose-Yeast Extract Agar (PDYA): good for growing cultures derived from mushrooms. � v Potato Carrot Agar (PCA): considered a relatively weak medium somewhat comparable to CMA, good for some imperfect fungi. � v Sabouraud Dextrose Agar (SAB): for recovery and maintenance of a wide variety of fungi commonly isolated in the clinical laboratory. v Malt Agar (MA): lacks peptone, and is useful for culturing many Ascomycota; sporulation in some species is inhibited by peptone. v Malt Extract Agar (MEA): a good growth medium for soil fungi, fungi isolated from wood, Basidiomycetes, etc. An all-purpose type of medium. v Potato Dextrose Agar (PDA): a relatively rich medium for growing a wide range of fungi.

Preparation of media

Semi- synthetic media (PDA) • Materials Procedure: 1. Add 400 ml of distilled water to a Erlenmeyer flask. Weigh out 250 g of potato after cutting them into small pieces; boil these pieces for 30 min. After that filter the results of boiling by gose and then added to the solution 20 g of dextrose and 15 g of agar; dissolve these in solution of boiled potato by stirring with a magnetic stirrer and complete this solution into 1000 ml. (Note: Heating may be necessary). 2. Add antibiotic into the solution. 3. Autoclave at 121°c, 1. 5 atm for 15 - 20 min. 4. After sterilization dispense the nutrient medium into Petri dishes

Preparation of solid nutrient media in plate (Petri dishes)

PDA powder bottle

PDA manual

Laboratory Reagents o Antibiotics uses in nutrient media An antibiotic is a drug that kills or slows the growth of bacteria in laboratory of fungi. Some of these antibiotics sensitive to heat and its belong into non- autoclavable and the others are autoclavable antibiotics. § Non- autoclavable antibiotics ü Penicillin: for gram positive bacteria which kills them by interfering with the ability to synthesize cell wall. ü Streptomycin: for gram positive and negative bacteria which kills them by preventing initiation of protein synthesis of microbial cells. Hint: These antibiotics added to nutrient media after sterilization. § Autoclavable antibiotics ü Chloramphenicol: for gram positive and negative bacteria. Kills them by inhibiting bacterial protein synthesis. ü Gentamycin: for gram negative bacteria. Kills them by suppressing protein synthesis and growth.

o Antimicrobial stain q Rose Bengal: A fluorescein compound is not used in nutrient media to inhibit the activity of rapid growth of fungi but also against the bacterial growth and uses for isolate fungi from soil and air. q Acridine Orange: for gram positive and negative bacteria. o Staining of Fungi q Lactophenol Cotton Blue Stain: is formulated with lactophenol, which serves as a mounting fluid, and cotton blue. Organisms suspended in the stain are killed due to the presence of phenol. The high concentration of the phenol deactivates lytic cellular enzymes thus the cells do not lyse. Cotton blue is an acid dye that stains the chitin present in the cell walls of fungi.

Reagent Formula ü Safranin: Dissolve 2. 5 g safranin O Phenol 200. 0 gm Killing agent Cotton Blue 0. 5 gm Stain Glycerol 400. 0 ml Wetting agent Lactic Acid 200. 0 ml Preservative Deionized Water 200. 0 m Solvent Certistain in 100 ml of 96% ethanol. For use, 10 ml of solution should be diluted with 90 ml of distilled water. ü Malachite Green: Dissolve 8 g malachite green powder in 100 ml D. W.

q Hint: for staining fungi: Place one drop of stain to the slide then transfer fugal cells by using sterile loop or needle according of your study examination.