1. Microbial Growth 

2. I. The Growth Curve

3. Closed system = Batch Culture
Closed culture vessel
One batch of culture medium
Different from continuous culture (see below)
Nutrients used up, culture eventually dies
Four stages of bacteria growth in batch culture

4. Lag Phase
A period of apparent inactivity in which the cells are adapting to a new environment and preparing for reproductive growth, usually by synthesizing new cell components
ATP
Ribosomal proteins
rRNA
tRNA
Co-factors
Enzymes

5. Varies in length depending upon the condition of the microorganisms and the nature of the medium
Assessment of medium – Receptors
DNA synthesized – initiation of cell division

6. Exponential phase (Log Phase)
Optimal growth rate and cell division dependent on medium, O2, temperature, pH, genetic composition
Regular, constant cell division (logarithmically)
Smooth curve – division not synchronous
Most useful phase for biochemical, physiological and DNA replication studies
Biotechnology applications – competent cells – uptake of plasmid DNA
Late log = optimal plasmid concentration
The population is most uniform in terms of chemical and physical properties during this period

7. Stationary Phase
When the population reaches ~109/ml (106 for protozoan and algal cultures), cell division = cell death (stasis)
Nutrients become scarce
O2 is depleted
Toxic waste accumulates
The number of viable microorganisms remains constant either because metabolically active cells stop reproducing or because the reproductive rate is balanced by the rate of cell death

8. Death Phase Viable cell mass decreases Often logarithmic
Cells not viable when inoculated into fresh medium
Cells have reached the carrying capacity of their environment

9. The mathematics of growth-microbial growth can be described by certain mathematical terms:
Mean generation (doubling) time (g) is the time required for the population to double
Mean growth rate constant is the number of generations per unit time, often expressed as generations per hour

10. Generation times vary markedly with the species of microorganism and environmental conditions
they can range from 10 minutes for a few bacteria to several days with some eukaryotic microorganisms
Population size = 2n where n = the number of generations

11. II. Measurement of Microbial Growth

12. Counting cells directly (live and dead)
Petroff-Hausser Counting Chamber
Slide with depressed etched grids (25 squares)
Covered with a coverslip
25 squares (area) = 1mm2
Depth = 0.02mm
Volume = 2 x 10-5 ml in 25 squares
Determination of cell numbers:
20 cells in one square x 25 squares/2 x 10-5 ml = 2.5 x 107 cells/ml

13. Electronic Counter Coulter counter
Measures electrical resistance as cells pass single file through a thin stream
RBC and WBC are counted
Less accurate with small cells
High interference
Clumping

14. Counting only live cells
Plating techniques (spread plate, pour plate) using serial dilutions
Colony forming units (CFU) usually arise from one organism (but may be several if clumpy)
Membrane filtration assay
Membrane traps bacterial on the surface
Membrane transferred to an agar plate
Colonies grow  counted
Can use selective media (e.g. Endo agar for coliform counts in contaminated water supplies)

15. Measurement of cell mass
Cell mass increases as cell number increases
Dry weight measurements
Growth, concentration, wash, dried, weighed
Spectrophotometric determination
Light is scattered and is proportional to cell number
Linear relationship between absorbance and cell density
Often written as % transmittance (as absorbance increases, transmittance decreases)
Requires cultures to be ~107/ml and upwards (slight turbidity)

16. III. Continuous Culture Techniques

17. Used to maintain cells in the exponential growth phase at a constant biomass concentration for extended periods of time
Conditions are met by continual provision of nutrients and removal of wastes = OPEN SYSTEM
Constant conditions are maintained

18. Chemostat
A continuous culture device that maintains a constant growth rate by:
supplying a medium containing a limited amount of an essential nutrient at a fixed rate
removing medium that contains microorganisms at the same rate
As fresh media is added to the chamber, bacteria are removed
Limiting nutrients control growth rates
Cell density depends on nutrient concentration

19. Turbidostat
A continuous culture device that regulates the flow rate of media through the vessel in order to maintain a predetermined turbidity or cell density
There is no limiting nutrient
Absorbance is measured by a photocell (optical sensing device)
The number of cells in culture controls the flow rate and the rate of growth of culture adjusts to this flow rate

20. Balanced and Unbalanced Growth

21. Balanced (exponential) growth occurs when all cellular components are synthesized at constant rates relative to one another
Unbalanced growth occurs when the rates of synthesis of some components change relative to the rates of synthesis of other components.
This usually occurs when the environmental conditions change

22. IV. Environmental Factors Affecting Microbial Growth

23. Solutes and Water Activity
Osmotic concentrations affect microbes (e.g. plasmolysis in hypertonic solutions)
Water activity (aw) = measurement of availability of water in particular environments
Aw = Psolution/Pwater (P = vapor pressure)
= inversely related to osmotic pressure
If the solution has a high osmotic pressure (high extracellular solute concentration), then its Aw = low

24. Energy is required by microbes to tolerate low aw because in order to keep water, solute concentration inside of cells must be kept high
= Osmotolerance
S. aureus can tolerate up to 3M NaCl
Archaebacteria halophiles tolerate M NaCl (Great Salt Lake, Dead Sea)
Avoidance of plasmolysis

25. pH (Log scale of 0 – 14; each pH unit = 10x change)
pH is the negative logarithm of the hydrogen ion concentration

26. Acidophiles grow best between pH 0 and 5.5
Neutrophiles grow best between pH 5.5 and 8.0
Alkalophiles grow best between pH 8.5 and 11.5

27. Extreme alkalophiles grow best at pH 10.0 or higher
Despite wide variations in habitat pH, the internal pH of most microorganisms is maintained near neutrality either by proton/ion exchange or by internal buffering
Sudden pH changes can inactivate enzymes and damage PMs
Reason for buffering culture medium, usually with a weak acid/conjugate base pair (e.g. KH2PO4/K2HPO4 – monobasic potassium/dibasic potassium)

28. Temperature Microorganisms are sensitive to temperature changes
Usually unicellular and poikilothermic
Enzymes have temperature optima
If temperature is too high, proteins denature, including enzymes, carriers and structural components
Temperature ranges are enormous (-20 to 100oC)

29. Organisms exhibit distinct cardinal temperatures (minimal, maximal, and optimal growth temperatures)
If an organism has a limited growth temperature range = stenothermal (e.g. N. gonorrhoeae)
If an organism has a wide growth temperature range = eurythermal (E. faecalis)

30. Psychrophiles can grow well at 0C, have optimal growth at 15C or lower, and usually will not grow above 20C
Arctic/Antarctic ocean
Protein synthesis, enzymatic activity and transport systems have evolved to function at low temperatures
Cell walls contain high levels of unsaturated fatty acids (semi-fluid when cold)

31. Psychrotrophs (facultative psychrophiles) can also grow at 0C, but have growth optima between 20C and 30C, and growth maxima at about 35C
Many are responsible for food spoilage in refrigerators
Mesophiles have growth minima of 15 to 20C, optima of 20 to 45C, and maxima of about 45C or lower
Majority of human pathogens

32. Thermophiles have growth minima around 45C, and optima of 55 to 65C
Hot springs, hot water pipes, compost heaps
Lipids in PM more saturated than mesophiles (higher melting points)
Hyperthermophiles have growth minima around 55C and optima of 80 to 110C
Sea floor sulfur vents

33. Oxygen concentration
Obligate aerobes are completely dependent on atmospheric O2 for growth
Oxygen is used as the terminal electron acceptor for electron transport in aerobic respiration
Facultative anaerobes do not require O2 for growth, but do grow better in its presence
Aerotolerant anaerobes ignore O2 and grow equally well whether it is present or not

34. Obligate (strict) anaerobes do not tolerate O2 and die in its presence
Microaerophiles are damaged by the normal atmospheric level of O2 (20%) but require lower levels (2 to 10%) for growth

35. Oxygen tolerance is determined by an organism’s ability to destroy toxic oxidizing products of oxygen reduction
Remember, because oxygen has two unpaired outer orbital electrons, it accepts electrons readily

36. Toxic compounds
Superoxide radical:
O2 + e-  O2•-
Hydrogen peroxide:
O2•- + e- + 2H+  H2O2
Hydroxyl radical:
H2O2 + e- + H+  H2O + OH•
These compounds are used deliberately by phagocytic WBC to break down intracellular microbes (respiratory burst)

37. Solution used by obligate aerobes and facultative anaerobes:
Produce enzymes that convert these toxic oxidizing products to non-toxic compounds
Superoxide dismutase
2O2•- + 2H+  O2 + H2O2
Catalase
2H2O2  2H2O + O2
Aerotolerant microbes have SOD; Obligate anaerobes lack SOD and catalase or have low concentrations

38. Laboratory considerations
Aerobic cultures
Shaken or sterile air introduced to medium

39. Anaerobic cultures Remove oxygen
Include reducing agents in medium (e.g. thioglycollate or cysteine
Dissolved oxygen is destroyed
Growth beneath surface
Replace oxygen with nitrogen gas and CO2 gas

40. Gas-Pak jar
H2 + palladium catalyst + O2  H2O
Bags or pouches  CaCO3  CO2 rich atmosphere

41. Pressure
Barotolerant organisms are adversely affected by increased pressure, but not as severely as are nontolerant organisms
Barophilic organisms require, or grow more rapidly in the presence of, increased pressure

42. Radiation
Ultraviolet radiation damages cells by causing the formation of thymine dimers in DNA
Photoreactivation repairs thymine dimers by direct splitting when the cells are exposed to blue light
Dark reactivation repairs thymine dimers by excision and replacement in the absence of light

43. Ionizing radiation such as X rays or gamma rays are even more harmful to microorganisms than ultraviolet radiation
Low levels produce mutations and may indirectly result in death
High levels are directly lethal by direct damage to cellular macromolecules or through the production of oxygen free radicals