Culture Media Culture Medium Nutrients prepared for microbial

Culture Media • Culture Medium: Nutrients prepared for microbial growth • Sterile: No living microbes • Inoculum: Introduction of microbes into medium • Culture: Microbes growing in/on culture medium

Agar • Complex polysaccharide • Used as solidifying agent for culture media in Petri plates, slants, and deeps • Generally not metabolized by microbes • Liquefies at 100°C • Solidifies ~40°C

Culture Media • Chemically Defined Media: Exact chemical composition is known • Complex Media: Extracts and digests of yeasts, meat, or plants – Nutrient broth – Nutrient agar

Culture Media Table 6. 2 & 6. 4

Anaerobic Culture Methods • Reducing media – Contain chemicals (Thioglycollate or oxyrase) that combine O 2 – Heated to drive off O 2

• Fluid Thioglycollate broth is a reducing medium. It contains sodium thioglycollate , which reacts with molecular oxygen keeping free oxygen levels low. • The sodium thioglycollate in the broth creates a redox potential in the tube, with higher levels of oxygen at the top of the tube, and a complete absence of oxygen at the bottom of the tube. • Fluid thioglycollate broth also typically contains a redox potential indicator such resazurin , which produces a pink color in an oxidized environment.

Thioglycollate Broth Tube 1 : Obligate Anaerobe -- note the absence of growth in the top portion of the broth where oxygen is present. Tube 2 : Obligate Aerobe -- note the growth is only in the top portion of the tube where oxygen is present. Tube 3 : Aerotolerant -- note the uniform growth from top to bottom. Tube 4 : Facultative -- note the uneven distribution of growth from top to bottom (more growth at the top. ( Tube 5 : Obligate Aerobe -- note the growth is only in the top portion of the tube where oxygen is present.

Anaerobic Culture Methods • Anaerobic jar anaerobic jar is an instrument used in the production of an anaerobic environment. This method of anaerorbios is as others is used to culture bacteria which die or fail to grow in presence of oxygen Sodium bicarbonate and sodium borohydride are mixed with a small amount of water to produce CO 2 and H. + A palladium catalyst in the jar combines with the O 2 in the jar and the H +to remove O. 2 Figure 6. 5

Anaerobic Culture Methods • Anaerobic chamber Figure 6. 6

Capnophiles require high CO 2 • Candle jar • CO 2 -packet Figure 6. 7

Selective Media • Suppress unwanted microbes and encourage desired microbes. Figure 6. 9 b, c

Differential Media • Make it easy to distinguish colonies of different microbes. Figure 6. 9 a

Enrichment Media • Encourages growth of desired microbe • Assume a soil sample contains a few phenol -degrading bacteria and thousands of other bacteria – Inoculate phenol-containing culture medium with the soil and incubate – Transfer 1 ml to another flask of the phenol medium and incubate – Only phenol-metabolizing bacteria will be growing

• A pure culture contains only one species or strain • A colony is a population of cells arising from a single cell or spore or from a group of attached cells • A colony is often called a colony-forming unit (CFU)

Streak Plate Figure 6. 10 a, b

Industrial Media and the Nutrition of Industrial Organisms

THE BASIC NUTRIENT REQUIREMENTS OF INDUSTRIAL MEDIA • Carbon or energy requirements • Nitrogen is found in proteins including enzymes as well as in nucleic acids • Minerals • Growth Factors

Average composition of microorganisms (% dry weight)

CRITERIA FOR THE CHOICE OF RAW MATERIALS USED IN INDUSTRIAL MEDIA • • Cost of the material Ready availability of the raw material Transportation costs Ease of disposal of wastes resulting from the raw materials Uniformity in the quality of the raw material and ease of standardization Adequate chemical composition of medium Presence of relevant precursors Satisfaction of growth and production requirements of the microorganisms

SOME RAW MATERIALS USED IN COMPOUNDING INDUSTRIAL MEDIA • • Corn steep liquor Pharmamedia Distillers soluble Soya bean meal Molasses Sulfite liquor Other Substrates (alcohol, acetic acid, methanol, methane, and fractions of crude petroleum)

Corn steep liquor • This is a by-product of starch manufacture from maize. • As a nutrient for most industrial organisms corn steep liquor is considered adequate, • rich in carbohydrates, nitrogen, vitamins, and minerals. • highly acidic, it must be neutralized (usually with Ca. CO 3) before use.

Approximate composition of corn steep liquor (%)

Pharmamedia • Yellow fine powder made from cotton-seed embryo. • It is used in the manufacture of tetracycline and some semi-synthetic penicillins. • rich in protein, (56% w/v) and contains 24% carbohydrate, 5% oil, and 4% ash • rich in calcium, iron, chloride, phosphorous, and sulfate.

Distillers soluble • By-product of the distillation of alcohol from fermented grain. (maize or barley) • It is rich in nitrogen, minerals, and growth factors.

Composition of maize distillers soluble

Soya bean meal • The seeds are heated before being extracted for oil that is used for food, as an antifoam in industrial fermentations, or used for the manufacture of margarine. • The resulting dried material, soya bean meal, has about 11% nitrogen, and 30% carbohydrate and may be used as animal feed. • Its nitrogen is more complex than that found in corn steep liquor • Not readily available to most microorganisms, except Actinomycetes. • It is used particularly in tetracycline and streptomycin fermentations.

Molasses

Sulfite Liquor • Sulfite liquor (also called waste sulfite liquor, ) is the aqueous effluent resulting from the sulfite process for manufacturing cellulose or pulp from wood. • During the sulfite process, hemicelluloses hydrolyze and dissolve to yield the hexose sugars, glucose, mannose, galactose, fructose and the pentose sugars, xylose, and arabinsoe. • Used as a medium for the growth of microorganisms after being suitably neutralized with Ca. CO 3 and enriched with ammonium salts or urea, and other nutrients. • It has been used for the manufacture of yeasts and alcohol. • Some samples do not contain enough assaimilable carbonaceous materials for some modern fermentations. • They are therefore often enriched with malt extract, yeast autolysate, etc.

GROWTH FACTORS • Not synthesized by the organism • Must be added to the medium. • Function as cofactors of enzymes and may be vitamins, nucleotides etc. • The pure forms are usually too expensive for use in industrial media • Growth factors are required only in small amounts.

Some sources of growth factors