Chapter 6: Microbial Growth

1. Chapter 6:Microbial Growth

2. Microbial Growth • Microbial growth = growth in population • Increase in number of cells, not cell size • Two main categories of requirements for microbial growth: • Physical requirements (environmental conditions) • Temperature, pH, osmotic pressure • Chemical requirements

3. Physical Requirements for Growth: Temperature • Temperature • Minimum growth temperature • Optimum growth temperature • Maximum growth temperature • Three main classifications • Psychrophiles (optimum ~120C) • Psychrotrophs (optimum ~230C) • Mesophiles (optimum ~370C) • Thermophiles (optimum above 500C)

4. Physical Requirements for Growth: Temperature Refrigeration Cause majority of food spoilage Figure 6.1

5. Hansen’s Disease(Leprosy) • Mycobacterium leprae • Optimal growth temperature: 30°C • Grows in peripheral nerves, nasal mucosa and skin cells Figure 22.8

6. Physical Requirements for Growth: pH • pH • Most bacteria grow between pH 6.5 and 7.5 • Molds and yeasts grow optimally between pH 5 and 6 • Acidophiles grow in acidic environments (pH<5.5) • Alkaliphiles grow in basic environments (pH>8.5) • Acidic foods (pickles, sauerkraut) preserved by acids from bacterial fermentation • Growth media used in the laboratory contain buffers

7. Physical Requirements for Growth: Osmotic Pressure • Osmotic Pressure • Hypertonic environments (=high osmotic pressure), increased salt or sugar, cause plasmolysis • Obligate halophiles require high osmotic pressure • Facultative halophiles tolerate high osmotic pressure(>2% salt) • Nutrient agar has a high percentage of water to maintain low osmotic pressure (bacterial cells are 80-90% water) Low osmotic pressure High osmotic pressure Water flow High solute concentration/ Low water concentration Low solute concentration/ High water concentration

8. Physical Requirements for Growth: Osmotic Pressure • Plasmolysis: cell growth is inhibited when the plasma membrane pulls away from the cell wall • Added salt or sugar is another method of preserving food Hypertonic solution (high osmotic pressure) Isotonic solution Figure 6.4

9. Chemical Requirements for Growth • Carbon • Structural organic molecules, energy source • Heterotrophs use organic carbon sources • Autotrophs use CO2 • Nitrogen, Sulfur, Phosphorus • For synthesis of amino acids, nucleotides, vitamins, phospholipids • Most bacteria decompose proteins to obtain N • Inorganic ions are sources for these elements (NH4+, NO3-, PO43-, SO42-)

10. Chemical Requirements for Growth • Trace Elements (Iron, Copper, Zinc) • Inorganic elements required in small amounts, usually as enzyme cofactors • Often present in tap water • Organic Growth Factors • Organic compounds obtained from the environment (i.e. the organism cannot synthesize them) • Vitamins, amino acids

11. Chemical Requirements for Growth: Oxygen • Oxygen (O2)

12. Chemical Requirements for Growth: Oxygen • Aerotolerance of individual organisms depends on their ability to handle oxygen toxicity • Oxygen radical species: O2-, O22-, OH • Presence/lack of enzymes that neutralize toxic oxygen species • SOD (Superoxide dismutase) • Catalase/peroxidase .

13. Chemical Requirements for Growth: Oxygen • Oxygen (O2) Express SOD and catalase Require oxygen, but at lower levels than in the air Tolerate oxygen (express SOD/catalase) but incapable of using it for growth Don’t express SOD/catalase

14. Culture Media • Culture Medium: Nutrients prepared for microbial growth • Source of energy, carbon, nitrogen, sulfur, phosphorus, trace elements and organic growth factors • Sterile: No living microbes • Inoculum: Introduction of microbes into medium to initiate growth • Culture: Microbes growing in/on culture medium

15. Culture Media:Agar • Complex polysaccharide • Used as solidifying agent for culture media in Petri plates, slants, and deeps • Generally not metabolized by microbes • Agar is not a nutrient • Liquefies above 100°C • Can incubate at a wide range of temperatures

16. Culture Media

17. Anaerobic Culture Media:Broth cultures • Reducing broth media • Contain chemicals (thioglycollate) that combine with dissolved O2 to deplete it from the media

18. Anaerobic Culture Methods:Agar Cultures • Anaerobic jar • Oxygen and H2 combine to form water Figure 6.5

19. Culture Media:Selective and Differential Media • Selective media: suppress growth of unwanted microbes and encourage growth of desired microbes • Differential media: make it easy to distinguish colonies of different microbes Enterobacter aerogenes on EMB E. coli on EMB Figure 6.9b, c

20. 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 (identical) cells • One colony arises from one colony-forming unit (CFU) • Specimens (pus, sputum, food) typically contain many different microorganisms • Common way to isolate a single species from a mixture of microorganisms: Streak plate method

21. Streak Plate Method for Isolation of a Pure Species • Use loop to pick colony • Inoculate broth • Pure culture Figure 6.10a, b

22. Microbial Growth in Hosts:Biofilms Microbial communities 3-dimensional “slime” i.e. dental plaque, soap scum Share nutrients Sheltered from harmful factors Cell-to-cell communication: quorum sensing Figure 6.5 Bacterial biofilm growing on a micro-fibrous material

23. Microbial Growth in Hosts:Biofilms & Quorum Sensing • Quorum sensing allows a form of bacterial communication • Individual cells can sense the accumulation of signaling molecules (autoinducers) • Informs individual cells about surrounding cell density • May change the behavior (gene expression) of individual cells • Results in a coordinated response by the whole population http://biofilmbook.hypertextbookshop.com/public_version/

24. Prokaryotic Reproduction:Binary Fission Figure 6.11

25. Reproduction in Prokaryotes:Generation Time Generation time: the time required for one population doubling • Varies with species and environmental conditions

26. Reproduction in Prokaryotes:Generation Number • Generation number: the number of times a cell population has doubled in a given time under given conditions Figure 6.12b

27. Reproduction in Prokaryotes:Growth Plot Logarithmic Arithmetic Figure 6.13

28. Bacterial Growth Curve • Lag: little/no cell division • Adapting to new medium • *Metabolically active* • Log: exponential growth • Most metabolically active • Gen. time at constant minimum • Stationary: equilibrium phase • Growth rate = death rate • Nutrients exhausted, waste accumulation, pH changes • Death: logarithmic decline Figure 6.14

29. Measuring Microbial Growth • To determine the size of a bacterial population in a specimen, cell counting techniques are used • Often there are too many cells per ml or gram of specimen… • A small proportion of the specimen (a dilution) is counted • The number of cells in the original specimen can be calculated based on the count in the small dilution

30. Direct Measurements of Microbial Growth:Viable Cell Count • Plate Counts: Perform serial dilutions of a sample • How many cells are in 1 mL of original culture? DF=1 10-5 DF: 10-3 10-4 10-2 10-1 Figure 6.15, top portion

31. Direct Measurements of Microbial Growth:Viable Count • Inoculate one agar plate with each serial dilution Figure 6.16

32. Direct Measurements of Microbial Growth:Viable Count • After incubation, count colonies on plates that have 30-300 colonies (CFUs) Figure 6.15

33. Direct Measurements of Microbial Growth • Filtration • Ideal when microbial density is low in a sample Figure 6.17a, b

34. Direct Measurements of Microbial Growth Disadvantages: -Likely to count dead cells -Motile cells can be difficult to count Figure 6.19