Chapter 6

1. Chapter 6 Microbial Growth

2. Lab 2 Goals and Objectives: Lecture: Chapter 6 (Microbial Growth) Learn aseptic technique and pure culture isolation • Exercise 9: Aseptic Technique • Exercise 10: Pure Culture Technique • Turn in cultures from home for incubation • Finish microscope worksheet if necessary

3. Microbiologyis the study of life and organisms that are too small to be seen with the naked eye. • Microbial growth - an increase in the number of cells in a population , not increase in size.

4. Growing microorganisms 1. Culture Medium: Nutrients prepared for microbial growth • Sterile: No living microbes 2. Inoculation: Introduction of microbes into medium Inoculum: The material used in an inoculation 3. Culture: Microbes growing in/on culture medium

5. Culture Media • Nutrientbroth- liquid form • Nutrientagar- solid form • 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

6. Reproduction of Prokaryotes - Binary fission

7. Bacterial growth in culture • Bacteria multiply by binary fission • The population grows in geometric progression • 12 • 2 4 • 3 8 • 4 16 • 5 32 • 6 64 • 7 128 • 8 256 • 9 518 • 10 1036 • 11 2064 • 12 4128 • 13 8256 • ……… -Lag phase: initial period of little to no cell division as bacteria acclimate to new media -Log phase: period of exponential growth with a constant generation time -Stationary phase: cell growth is equal to cell death -Death phase: cell death exceeds cell growth

8. Number of generation

9. Generation time • Generation time - the time required for a cell to divide, to undergo one round of binary fission • If 100 cells growing for 5 hours produced 1,720,320 cells: • E. coli has a generation time of 20 min • Common bacterial generation times range 1-3 hrs

10. Quantifying Microbial Growth • Direct Measurements • Plate Counts • Filtration • Most Probable Number (MPN) • Direct Microscopic Count • Indirect Estimations • Turbidity • Metabolic Activity • Dry Weight

11. Microbial Growth • Physical • Temperature • pH • Osmotic pressure • Chemical • Oxygen • Carbon • Nitrogen • Sulfur • Phosphorus • Trace elements • Organic factors • The requirements for microbial growth

12. Physical Requirements -Temperature • Minimum growth temperature • the lowest temperature at which the species will grow • Optimum growth temperature • species grow best • Maximum growth temperature • the highest temperature at which growth is possible.

13. Physical Requirements - pH • Most bacteria grow between pH 6.5 and 7.5 • Molds and yeasts grow between pH 5 and 6 • Acidophiles grow in acidic environments

14. Physical Requirements - Osmotic Pressure • Osmotic pressure is the hydrostatic pressure produced by a difference in concentration between solutions on the two sides of a surface such as a semipermeable membrane. • Anisotonic environment for a cell is created when the solution outside of the cell is isotonic (having equal accent )with the cytoplasm of the cell. • Hypotonic environment - the solution outside of the cell is hypotonic in comparison to the cytoplasm of the cell. • cause cell lyses for some cells and nothing to bacteria, because of cell wall . • Hypertonic environments - hypertonic solution has more dissolved solute than the cytoplasm of the cell (increase salt or sugar) • cause plasmolysis ( shrink the cells ) • Facultative halophiles tolerate high osmotic pressure • Extreme or obligate halophiles require high osmotic pressure

15. Chemical Requirements • Carbon • Structural organic molecules, energy source • Autotrophs use CO2 • Chemoheterotrophs use organic carbon sources • Nitrogen • In amino acids, proteins • Most bacteria decompose proteins • Some bacteria use NH4+ or NO3 • A few bacteria use N2 in nitrogen fixation • Sulfur • In amino acids, thiamine, biotin • Most bacteria decompose proteins • Some bacteria use SO42 or H2S • Phosphorus • In DNA, RNA, ATP, and membranes • PO43is a source of phosphorus • Inorganic elements required in small amounts- potassium, magnesium and calcium • Trace Elements - iron, copper, molybdenum and zinc - Usually as enzyme cofactors

16. Chemical Requirements • Organic Growth Factors • Essential organic compounds an organism is unable to synthesize • Organic compounds obtained from the environment • Vitamins, amino acids, purines, pyrimidines

17. Chemical Requirements - Oxygen (O2)

18. Toxic Forms of Oxygen • Singlet oxygen: O2 boosted to a higher-energy state • Superoxide free radicals: O2 • Peroxide anion: O22 • Hydroxyl radical (OH)

19. Biofilms • Microbial communities • Form slime or hydrogels • Bacteria attracted by chemicals via quorum sensing Clumps of bacteria adhering to surface Migrating clump of bacteria Surface Water currents

20. Culture Media • Complex Media: Extracts and digests of yeasts, meat, or plants • Chemically Defined Media: Exact chemical composition is known • Differential Media: • Make it easy to distinguish colonies of different microbes. • Enrichment Media Encourages growth of desired microbe • Reducing media for anaerobic culture, Contain chemicals (thioglycollate or oxyrase) that combine O2 Heated to drive off O2

21. Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates. Clamp with clamp screw Lid with O-ring gasket Envelope containing sodium bicarbonate and sodium borohydride Palladium catalyst pellets Anaerobic indicator (methylene blue) Petri plates

22. Obtaining Pure Cultures • 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) • The streak plate method is used to isolate pure cultures

23. Exercise 9: Aseptic Technique • Each person make 3inoculations: • Broth to broth - Escherichia coli- 37°C • Slant to slant - E. coli- 30°C 3. Plate to slant - Serratia marcescens- 30°C (changed) • Exercise 10: Pure Culture Technique • Each person make 2 streak plates: Quadrant Streak Method B, page 82. • Incubate at 25°C • Turn in cultures from home for incubation • Finish microscope worksheet if necessary

24. Streak Plate Flame the loop, cool it Flame the loop, cool it Flame the loop, cool it Figure 6.10a, b

25. Each pair will need: 1 broth culture Escherichia coli, 1 slant culture Escherichia coli 1 plate culture Serratia marcescens Each person will need: 1 Nutrient Broth/BHI tubes, 2 Nutrient Agar/BHIA slants • Exercise 9: Aseptic Technique • Each person make 3inoculations: • Broth to broth - E. coli - 37°C • Slant to slant - E. coli - 37°C 3. Plate to slant - S. marcescens - 30°C (changed) • Exercise 10: Pure Culture Technique • Each person make 2 streak plates • Quadrant Streak Method B, page 85. • Incubate plates at 25°C Each pair will need: 1 mixed culture (which contains: Escherichia coli, Serratia marcescensand Micrococcus luteus) Each person will need: 2 Nutrient Agar/BHIA plates Get help now, today, if you are having any difficulty with oil immersion lens use or specimen measurements using the ocular micrometer!!!