1. Cultivation of Microorganisms

2. KEY TERMS Obligate aerobe Obligate anaerobe Aerotolerant anaerobe
Facultative anaerobe
Microaerophilic
Mesophile
Thermophile
Psychrophile
Halophilic
Osmophilic
Medium

3. Bacterial Requirements for Growth
Nutrients
Oxygen (or absence)
Energy
Optimal temperature
Optimal pH

4. SOURCES OF METABOLIC ENERGY
The three major mechanisms for generating metabolic energy are
Fermentation
Respiration
Photosynthesis
At least one of these mechanisms must be employed if an organism is to grow

5. Fermentation
The formation of ATP in fermentation is not coupled to the transfer of electrons
Fermentation is a substrate phosphorylation, an enzymatic process in which a pyrophosphate bond is donated directly to ADP (adenosine diphosphate) by a phosphorylated metabolic intermediate
The phosphorylated intermediates are formed by metabolic rearrangement of a fermentable substrate such as glucose, lactose, or arginine
Example: fermentation of a molecule of glucose (C6H12O6) yields a net gain of two pyrophosphate bonds in ATP and produces two molecules of pyruvic acid (C3H6O3)

6. Fermentation Reaction

7. Respiration
Respiration is chemical reduction of an oxidant (electron acceptor) through a specific series of electron carriers in the membrane establishes the proton motive force across the bacterial membrane
Reductants (electron donor)
may be organic or inorganic: For example, lactic acid serves as a reductant for some organisms, and hydrogen gas is a reductant for other organisms
Oxidants
Gaseous oxygen (O2) often is employed as an oxidant, but alternative oxidants that are employed by some organisms include carbon dioxide (CO2), sulfate (SO42–), and nitrate (NO3−)

8. Respiration in Eukaryotic Cell
36ATP

9. Respiration in Eukaryotic Cell

10. Photosynthesis
Photosynthesis is similar to respiration in that the reduction of an oxidant via a specific series of electron carriers establishes the proton motive force
Difference in the two processes
in photosynthesis the reductant and oxidant are created photochemically by light energy absorbed by pigments in the membrane
Photosynthesis can continue only as long as there is a source of light energy

11. Photosynthesis

12. Environmental Factors Affecting Growth
Nutrients
Hydrogen ion concentration (pH)
Temperature
Aeration
Ionic strength & osmotic pressure

13. Nutrient Requirements
Carbon
Nitrogen
Sulfur
Phosphorus
Metal ions (e.g. iron)
Growth factors

14. Carbon Source Autotroph Heterotroph CO2 + Sunlight Photosynthesis
CO2+H+SO42-
Chemolithotroph
Heterotroph
Organic carbon
eg., glucose
Fermentation
Respiration

15. Nitrogen Source
The end products are NH4+ or NH3

16. Sulfur Source
Most microorganisms can use sulfate as a sulfur source, reducing the sulfate to the level of hydrogen sulfide (H2S)
Some microorganisms can assimilate H2S directly from the growth medium, but this compound can be toxic to many organisms

17. Phosphorus Source
Phosphate (PO43−) is required as a component of ATP, nucleic acids, and such coenzymes as NAD, NADP, and flavins
Many metabolites, lipids (phospholipids, lipid A), cell wall components (teichoic acid), some capsular polysaccharides, and some proteins are phosphorylated
Phosphate is always assimilated as free inorganic phosphate (Pi)

18. Mineral Sources
In formulating a medium for the cultivation of most microorganisms, it is necessary to provide sources of potassium, magnesium, calcium, and iron, usually as their ions (K+, Mg2+, Ca2+, and Fe2+)
Many other minerals (eg, Mn2+, Mo2+, Co2+, Cu2+, and Zn2+) are required; these frequently can be provided in tap water or as contaminants of other medium ingredients

19. Growth Factors
A growth factor is an organic compound which a cell must contain in order to grow but which it is unable to synthesize
Some amino acids
Some purines and pyrimidines
Vitamins
Different species of bacteria needs different growth factors

20. Nutrient Requirements
Carbon
Nitrogen
Sulfur
Phosphorus
Metal ions (e.g. iron)
Growth factors

21. Environmental Factors Affecting Growth
Nutrients
Hydrogen ion concentration (pH)
Temperature
Aeration
Ionic strength & osmotic pressure

22. Hydrogen Ion Concentration (pH)
Many grow best at neutral pH
Some can survive/grow
- Acid
Alkali

23. Hydrogen Ion Concentration (pH)
Most organisms (neutralophiles) grow best at a pH of 6.0–8.0, although some forms (acidophils) have optima as low as pH 3.0 and others (alkaliphiles) have optima as high as pH 10.5
Microorganisms regulate their internal pH over a wide range of external pH values
Acidophiles maintain an internal pH of about 6.5 over an external range of 1.0–5.0
Neutralophiles maintain an internal pH of about 7.5 over an external range of 5.5–8.5
Alkaliphiles maintain an internal pH of about 9.5 over an external range of 9.0–11.0

24. Temperature
Psychrophilic forms grow best at low temperatures (15–20oC)
Mesophilic forms grow best at 30–37oC
Most organisms are mesophilic
30oC is optimal for many free-living forms, and the body temperature of the host is optimal for symbionts of warm-blooded animals
Thermophilic forms grow best at 50–60oC

25. Optimal Growth Temperature
Mesophile
human body temperature
pathogens
opportunists
Pyschrophile
close to freezing
Thermophile
close to boiling

26. Heat Shock / Cold Shock
Microorganisms share with plants and animals the heat-shock response, a transient synthesis of a set of “heat-shock proteins,” (HSP) when exposed to a sudden rise in temperature above the growth optimum
HSPs appear to be unusually heat-resistant and to stabilize the heat-sensitive proteins of the cell
Bacteria also exhibit a phenomenon called cold shock: the killing of cells by rapid cooling
For example, the rapid cooling of Escherichia coli from 37 oC to 5oC can kill 90% of the cells
A number of compounds protect cells from either freezing or cold shock: glycerol, dimethyl sulfoxide (DMSO)

27. Aeration Obligate aerobe Facultative anaerobe Obligate anaerobe
specifically requiring oxygen as hydrogen acceptor
Facultative anaerobe
able to live aerobically or anaerobically
Obligate anaerobe
requiring a substance other than oxygen as hydrogen acceptor and being sensitive to oxygen inhibition

28. Obligate Aerobes No fermentation Oxidative phosphorylation
Grow in presence of oxygen
No fermentation
Oxidative phosphorylation

29. Obligate Anaerobes Fermentation No oxidative phosphorylation
Killed by oxygen
The natural by-products of aerobic metabolism are the reactive compounds hydrogen peroxide (H2O2) and superoxide (O2-)
Lack certain enzymes
superoxide dismutase
O2-+2H H2O2
catalase
H2O H20 + O2
peroxidase
H2O H2O /NAD NADH

30. Facultative Anaerobes
Fermentation
Aerobic respiration
Survive in oxygen

31. Microaerophilic Bacteria
Grow
low oxygen
Killed
high oxygen

32. Ionic Strength and Osmotic Pressure
For most organisms, the properties of ordinary media are satisfactory; however, for marine forms and organisms adapted to growth in strong sugar solutions, these factors must be considered
Halophilic Organism: requires high salt concentrations
Osmophilic Organism: requires high osmotic pressures

33. Cultivation Methods Two problems will be considered
Media: the choice of a suitable medium
Cultivation Methods: the isolation of a bacterial organism in pure culture

34. Cultivation Medium: Based on Purpose
Basic medium
Enrichment medium
Selective medium
Differentiation medium
Anaerobic medium

35. Cultivation Medium: Based on physical properties
Liquid medium
Solid medium
Semi-Solid medium

36. Medium Applications Liquid Medium Pure Culture Amplifying culture
Semi-solid medium
Motion: flagellum
Solid Medium
Pure culture
Isolation culture
colony

37. Colony Attributes