Experiment 13 Nutritional Requirements: Media for the Routine Cultivation of Bacteria ***the yeast extract is an example of an enriched medium and is used for the cultivation of fastidious microorganisms. Fastidious Microorganisms Organisms that have highly elaborate and specific nutritional needs. These bacteria do not grow or grow poorly on a basic artificial medium and require the addition of one or more growth-supporting substances – enrichments such as additional plant or animal extracts, vitamins, or blood. Questions: Why are complex media preferable to chemically defined media for routine cultivation of microorganisms? Complex media are preferable for routine cultivation of microorganisms than chemically defined media because it provides highly enriched sources such as plant or animal extracts, vitamins, minerals, protein, sugar, and or blood to the organism, supplying them specific nutritional substances needed for their growth. Chemically defined media on the other hand does not supply such a vast abundance of nutritional substances. Would you expect a heterotrophic organism to grow in an inorganic synthetic medium? Explain. No, I would not expect a heterotrophic organism to grow in an inorganic synthetic medium because an inorganic synthetic medium contains mostly the addition of salts which is not an efficient source of nutrition of a heterotrophic organism. A heterotrophic organism would grow more successfully in a complex media. They are capable of supporting growth of most hetertrophs through their abundant amino acid, sugar, vitamin, and mineral sources. Turbidity A measure of the cloudiness or haziness of a fluid caused by suspended solids. The more total suspended solids in the water, the murkier it seems and the higher the turbidity. Bausch & Lomb Spectronic 20 Spectrophotometer This instrument measures the amount of light transmitted (T) or absorbed (A). Questions: Explain the advantages of using A readings rather than percent T as a means of estimating microbial growth. The density of a cell suspension is expressed as absorbance (A) rather than percent T, since A is directly proportional to the concentration of cells, whereas percent T is inversely proportional to the concentration of suspended cells. Therefore, as the turbidity of a culture increases, the A increases and percent T decreases, indicating growth of the cell population in the culture. Explain the reason for the use of different medium blanks in adjusting the spectrophotometer prior to obtaining A readings. The spectrophotometer needs to be calibrated against a blank solution so that measurements after it can use the blank solution's absorbance as a zero reference. That way when the actual solution your measuring is placed in the spectrophotometer, the measurement given by the reading only reflects your solutions concentration. Chemically Defined Media Inorganic Synthetic Media This inorganic medium is prepared by incorporating the following salts per 1000 ml of water: Sodium Chloride (NaCl) Magnesium Sulfate (MgSO₄) Ammonium Dihydrogen Phosphate (NH₄H₂PO₇) Dipostassium Hydrogen Phosphate (K₂HPO₄) Atmospheric Carbon Dioxide (CO₂) Glucose Salts Broth This medium is composed of salts incorporated into the inorganic synthetic broth + glucose (5 g per liter), which serves as the sole organic carbon source. Complex Media Made of extracts of plant and animal tissue Most contain abundant amino acids, sugars, vitamins, and minerals They are capable of supporting the growth of most heterotrophs Nutrient Broth This basic complex medium is prepared by incorporating the following ingredients per 1000 ml of distilled water: Peptone – is a primarily a nitrogen source. Beef Extract – is a source of organic carbon, nitrogen, vitamins, and inorganic salts. Yeast Extract Broth This is composed of the basic ingredients used in the nutrient broth + yeast extract (5 g per liter), which is a rich source of vitamin B and provides additional organic nitrogen and carbon compounds.