1. Lecture 4 Nutrition and Growth (Text Chapters: 5.1-5.3; 6.1; 6.4-6.8; 6.10-6.15)

2. Microbial Nutrition Why is nutrition important? –The hundreds of chemical compounds present inside a living cell are formed from nutrients. Macronutrients : elements required in fairly large amounts Micronutrients : metals and organic compounds needed in very small amounts

3. Main Macronutrients Carbon (C, 50% of dry weight) and nitrogen (N, 12% of dry weight) Autotrophs are able to build all of their cellular organic molecules from carbon dioxide Nitrogen mainly incorporated in proteins, nucleic acids Most Bacteria can use NH 3 and many can also use NO 3 - Nitrogen fixers can utilize atmospheric nitrogen (N 2 )

4. Other Macronutrients Phosphate (P), sulfur (S), potassium (K), magnesium (Mg), calcium (Ca), sodium (Na), iron (Fe) Iron plays a major role in cellular respiration, being a key component of cytochromes and iron-sulfur proteins involved in electron transport. Siderophores : Iron-binding agents that cells produce to obtain iron from various insoluble minerals.

5. Representative Siderophore Ferric enterobactin Aquachelin

7. Micronutrients Need very little amount but critical to cell function. Often used as enzyme cofactors

8. Growth factors Organic compounds, required in very small amount and then only by some cells

9. Culture Media: Composition Culture media supply the nutritional needs of microorganisms – defined medium : precise amounts of highly purified chemicals –complex medium(or undefined) : highly nutritious substances. Inclinical microbilogy, –Selective : contains compunds that selectively inhibit –Differential: contains indicator –terms that describe media used for the isolation of particular species or for comparative studies of microorganisms.

10. Culture Media: Physical Properties Liquid –Bouillon or broth Solid –Addition of a gelling agent (typically 1% agar) to liquid media –Immobilize cells, allowing them to grow and form visible, isolated masses called colonies (Figure 5.2). Semisolid –Reduced amount of agar added –Allows motile microorganism to spread

11. Bacterial Colonies on Solid Media S. Marcescens (Mac) P. aeruginosa (TSA) S. Flexneri (Mac)

12. Laboratory Culture of Microorganisms Microorganisms can be grown in the laboratory in culture media containing the nutrients they require. Successful cultivation and maintenance of pure cultures of microorganisms can be done only if aseptic technique is practiced to prevent contamination by other microorganisms.

13. Microbial Growth Binary fission

14. Microbial growth involves an increase in the number of cells. Growth of most microorganisms occurs by the process of binary fission Cell Growth and Binary Fission

15. Microbial Growth Peptidoglycan layer

16. Microbial populations show a characteristic type of growth pattern called exponential growth, which is best seen by plotting the number of cells over time on a semi- logarithmic graph. Microbial Growth pattern

17. Growth Curve Microorganisms show a characteristic growth pattern (Figure 6.8) when inoculated into a fresh culture medium.

18. Measuring Microbial Growth Growth is measured by the change in the number of cells over time. –Cell counts done microscopically (Figure 6.9) measure the total number of cells in a population –whereas viable cell counts (plate counts) (Figures 6.10, 6.11) measure only the living, reproducing population.

19. Total Cell Count

20. Viable Cell Count : Determination of Colony Forming Units

21. Serial Dilution of Cells

22. Indirect Cell Number Measurement : Turbidity Turbidity measurements are an indirect but very rapid and useful method of measuring microbial growth (Figure 6.12). However, to relate a direct cell count to a turbidity value, a standard curve must first be established.

23. Turbidity Measurements of Microbial Growth

24. Environmental Effects on Bacterial Growth Temperature pH Osmotic pressure Oxygen classes

25. Temperature and Microbial Growth Cardinal temperatures –minimum –optimum –maximum Temperature is a major environmental factor controlling microbial growth.

26. Classification of Microorganisms by Temperature Requirements

27. Temperature Classes of Organisms Mesophiles –Midrange temperature optima –Found in warm-blooded animals and in terrestrial and aquatic environments in temperate and tropical latitudes Psychrophiles –Cold temperature optima –Most extreme representatives inhabit permanently cold environments Thermophiles –Growth temperature optima between 45ºC and 80ºC Hyperthermophiles –Optima greater than 80°C –These organisms inhabit hot environments including boiling hot springs, as well as undersea hydrothermal vents that can have temperatures in excess of 100ºC

28. Heat-Stable Macromolecules Thermophiles and hyperthermophiles produce heat-stable macromolecules, such as Taq polymerase, which is used to automate the repetitive steps in the polymerase chain reaction (PCR) technique.

30. pH and Microbial Growth The acidity or alkalinity of an environment can greatly affect microbial growth. Most organisms grow best between pH 6 and 8, but some organisms have evolved to grow best at low or high pH. The internal pH of a cell must stay relatively close to neutral even though the external pH is highly acidic or basic. –Acidophiles : organisms that grow best at low pH –Alkaliphiles : organismsa that grow best at high pH

32. Osmotic Effects on Microbial Growth Osmotic pressure depends on the surrounding solute concentration and water availability Water availability is generally expressed in physical terms such as water activity Water activity is the ratio of the vapor pressure of the air in equilibrium with a substance or solution to the vapor pressure of pure water.

34. Halophiles and Related Organisms In nature, osmotic effects are of interest mainly in habitats with high salt environments that have reduced water availability Halophiles : have evolved to grow best at reduced water potential, and some (extreme halophiles) even require high levels of salts for growth. Halotolerant : can tolerate some reduction in the water activity of their environment but generally grow best in the absence of the added solute Xerophiles : are able to grow in very dry environments

36. Oxygen and Microbial Growth Aerobes : –Obligate : require oxygen to grow –Facultative : can live with or without oxygen but grow better with oxygen –Microaerphiles : require reduced level of oxygen Anaerobes : –Aerotolerant anaerobes : can tolerate oxygen but grow better without oxygen. –Obligate : do not require oxygen. Obligate anaerobes are killed by oxygen

38. Test for Oxygen Requirements of Microorganisms Thioglycolate broth : contains a reducing agent and provides aerobic and anaerobic conditions a)Aerobic b)Anaerobic c)Facultative d)Microaerophil e)Aerotolerant

39. Toxic Forms of Oxygen and Detoxifying EnzymesHydrogenperoxide Superoxide

40. Announcement All lecture resources will be posted on http://www.calstatela.edu/faculty/hpark8/