1. 1 Chapter 6 Microbial Growth

2. 2 Growth vs. Reproduction Growth: an orderly and permanent increase in the mass of protoplasm of an organism or population. "orderly" - a proportionate increase in all constituents Reproduction: an increase in the numbers of individuals and can be independent of growth cells may grow without reproducing or may reproduce without growth

3. 3 Effects of Temperature on Bacterial Growth Temperature range for any organism : minimum temperature: lowest where growth occurs optimum temperature: where a species grows best maximum temperature: highest where growth is possible Psychrophiles: "cold-loving", grow at 0 o C to 20 o C, optimum 15 o C Mesophiles: "middle" loving, grow at 20 o C to 45 o C, optimum 20 o C to 37 o C Thermophiles: "heat-loving”, grow at 37 o C to 65 o C

4. 4 cold loverscold growers

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7. 7 pH and Bacterial Growth For most organisms, optimum pH is generally between pH 6.5 - pH 7.5 Acidophiles (acid loving) have optimums between pH 0 - 5.5 Basophiles: (basic loving) optimum from pH 8.5 to 11.5 organisms can change the culture media due to their own waste products (generally acidic), may be toxic due to pH change

8. 8 Osmotic Pressure & Bacterial Growth Cells are 80-90% water Isotonic solution (80-90% water): no net movement of water Hypotonic: water will move into cell (cell rounds up and can burst) Hypertonic: water moves out of cell (cell shrinks, plasmolysis) Some organisms can tolerate high osmotic pressure conditions Halophiles: grow in high salt conditions (obligate halophile requires high salt concentrations)

9. 9 Chemical Requirements for Bacterial Growth

10. 10 Nutrients Required for Growth Growth requires availability of macronutrients, trace elements, and growth factors Carbon: structural backbone of many molecules Nitrogen, Sulfur, Phosphorus required items for production of proteins as well as DNA/RNA (requires N & P) Organic growth factors: vitamins, some amino acids, purines, pyrimidines Trace elements – often needed for proper enzymatic functions ANY of these nutrients can be limiting factors in growth

11. 11 Oxygen Effects on Growth organisms can produce more energy when growing in oxygen, but toxic byproducts of oxygen can be fatal to cells (Table 6.1) Toxic byproducts from use of O 2 Superoxide Radical – extremely toxic to living cells. This harmful molecule is neutralized by the enzyme superoxide dismutase (SOD) which is produced by all organisms growing in the presence of oxygen. SOD converts superoxide radical to hydrogen peroxide & O 2 note: hydrogen peroxide is still toxic, this will be dealt with shortly

12. 12 Toxic Byproducts of O 2 continued Hydrogen Peroxide, toxic to cells can be neutralized two different enzymes Catalase 2 H 2 O 2  2 H 2 0 + O 2 you can detect presence of this enzyme in an organism by applying hydrogen peroxide to a culture (on solid media) and observing presence of bubbles Peroxidase H 2 O 2 + 2 H +  2 H 2 0 note: since this reaction doesn’t produce gaseous oxygen you can not see bubbling when applying hydrogen peroxide.

13. 13 Classification of Organisms by O 2 Requirements Aerobes: require oxygen for growth Facultative Anaerobes: can grow with or without oxygen Anaerobes: cannot tolerate molecular oxygen Microaerophiles: require oxygen, but can only grow at low oxygen tension

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15. 15 Isolation of Pure Cultures Most microorganisms in nature are found in mixed cultures. Isolation of colonies is done using the streak plate technique. Each isolated colony begins as a single bacterium. A pure culture is a culture coming from an isolated colony on a plate containing only one colony type.

16. 16 Other culture techniques Spread plate – a diluted liquid inoculum is spread over surface of a plate (fig 6.16 part b) – another isolation technique Pour plate – a diluted liquid inoculum is mixed with melted nutrient agar and colonies are allowed to grow – another isolation technique Broths - propagation (rapid growth) of cultures Slants – maintenance of cultures

17. 17 Types of Media Culture media: nutrient material prepared for the growth of microorganisms in the laboratory (must initially be sterile) Synthetic (or Defined) Medium: made of known amounts of chemicals example: E. coli minimal salts medium Complex medium: some ingredients are of unknown composition or amount (extracts of plants, yeast or meat)  Nutrient broth  Tryptic soy broth

18. 18 Solid, Semi-Solid, Liquid? Solid media: a synthetic or complex medium can be solidified using agar. Agar: from red algae, sulfated polymer of D-galactose, 3,6 anhydro-L- galactose, and D-glucuronic acid, is not degraded by most microorganisms. Agar melts in boiling water, solidifies when cooled to about 40 o C, does not melt again until 80 - 90 o C Solid media: 1.5% agar, give surface for growing bacteria (often used to maintain cultures) Semi-solid media: usually 0.5% agar, used for motility studies Liquid media: no agar, used for propagation of cultures

19. 19 General or Specific Media? General medium - provides basic needs of organisms Selective medium - encourages the growth of certain organisms while discouraging the growth of others. examples: crystal violet or basic fuschin dyes selective for Gram- (against G+) MacConkey, EMB Differential medium - distinguishes between different groups of bacteria medium contains constituents which cause an observable change (color or pH change) example: MacConkey contains lactose and neutral red, lactose fermenters appear pink; Blood agar - hemolysis

20. 20 Cultivation Of Anaerobes Special media/conditions necessary may include : reducing media (chemicals to remove oxygen, ex. sodium thioglycollate) non-oxygen atmosphere (candle jar or anaerobic jar) Enrichment medium: provide conditions which will only allow bacteria with a specific desired trait to survive and grow (particular carbon source, growth conditions, etc.). example: Sabouraud’s dextrose agar (fungi)

21. 21 Microbial Growth Bacterial division: binary fission, two identical cells, each with one full chromosome Generation time: the time required for cells to divide (and thus double the population), can be as short as 20 minutes or longer than a day Calculation of generation time (use logarithmic plot), fig. 6.13 and 6.14

22. 22 Excess media Sufficient media to support cells present Insufficient media to support life. Metabolic waste buildup Microbial Growth Curve

23. 23 Measuring Growth Direct methods: 1. Plate counts (serial dilutions) increase in number of bacteria with time (viable count), each colony represents one bacterium 2. Filtration – known volume of bacterial culture collected on a filter disk then the disk is placed in media and allowed to grow (fig 6.17 p 177) each colony represents one bacterium in original sample 3. Most probable number (MPN): statistical method 4. Direct microscopic count: Petroff-Hausser counter

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26. 26 explanation of MPN Most Probable Number Method uses statistics to provide an estimate of number of bacteria in a given sample. In the book’s example you inoculate three sets of five tubes Our original sample has 100ml total volume. Question, how many bacteria does this contain? Series 1 tubes each get 10 ml of the original sample. Series 2 tubes each get 1 ml of original sample. Series 3 tubes each get 0.1 ml of original sample

27. 27 How many tubes in each series had bacterial growth? (indicating presence of at least one bacterium in our inoculation) Series 1 = all five tubes have growth Series 2 = three tubes Series 3 = only one tube Interpretation of results. 5 – 3 – 1 (find in table) 95% of the time, a sample giving this pattern will have at least 40 bacteria in original sample possibly as many as 300. The average number of bacteria in a sample with this pattern is 110

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29. 29 Measuring Growth Indirect methods: 1.increase in turbidity with time (cloudier media = more bacteria) 2. metabolic products – measurable metabolic products assumed to increase as numbers of bacteria increase 3. dry weight – bacteria separated by centrifuge and then weighed

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