Premed · Premed · Microbiology
Lecture 5: Microbial Growth and Nutrition
Microbiology
Learning Objectives
By the end of this lecture, students will be able to:
- Define microbial growth and describe the requirements for microbial nutrition
- Classify microorganisms based on their carbon, energy, and electron sources
- Explain the environmental factors that influence microbial growth (temperature, pH, osmolarity, oxygen)
- Describe the phases of the bacterial growth curve
- Explain methods for measuring microbial growth
- Define and differentiate between different culture media types
Lecture Content
I. Nutritional Requirements of Microorganisms
In microbiology, growth refers to an increase in the number of cells within a population, not an increase in the size of individual cells. All microorganisms share a common set of nutritional requirements. They need a carbon source to build the structural backbone of organic molecules, an energy source to drive biosynthetic reactions, and electron (hydrogen) donors for reduction reactions. Nitrogen is essential for amino acids and nucleotides and can be obtained from ammonium ions, nitrate, molecular nitrogen, or organic nitrogen compounds. Phosphorus is required for nucleic acids, phospholipids, and ATP, while sulfur is needed for certain amino acids (cysteine and methionine) and coenzymes. Trace elements such as iron, manganese, zinc, cobalt, copper, and molybdenum serve as cofactors for enzymes, and water is the universal solvent for all biochemical reactions. Some organisms also require growth factors -- organic compounds needed in small amounts that the cell cannot synthesize on its own, including vitamins, amino acids, purines, and pyrimidines. Organisms dependent on externally supplied growth factors are called auxotrophs.
II. Nutritional Classification of Microorganisms
Microorganisms are classified nutritionally along three axes. Based on their carbon source, they are either autotrophs (using CO2 as the sole carbon source) or heterotrophs (using organic compounds). Based on their energy source, they are phototrophs (using light) or chemotrophs (using chemical energy from oxidation reactions). Based on their electron donor, they are lithotrophs (using inorganic electron donors such as H2, H2S, NH3, or Fe2+) or organotrophs (using organic electron donors).
The most common combinations encountered in microbiology include chemoorganoheterotrophs -- the category encompassing most medically important bacteria, fungi, and protozoa, which use organic compounds for carbon, energy, and electrons. Photoautotrophs such as cyanobacteria, algae, and plants use light energy and fix CO2. Chemolithoautotrophs like nitrifying and sulfur-oxidizing bacteria oxidize inorganic compounds for energy while fixing CO2. Photoheterotrophs, exemplified by purple non-sulfur bacteria, use light energy but require organic carbon sources.
III. Culture Media
The choice of culture medium depends on the organism of interest and the purpose of the investigation. Defined (synthetic) media have a precisely known chemical composition, making them useful for nutritional studies. Complex media contain nutrients of undefined exact composition, such as peptone, yeast extract, and beef extract; common examples include nutrient broth, tryptic soy broth (TSB), and brain heart infusion (BHI).
Selective media contain agents that inhibit the growth of certain organisms while permitting others. MacConkey agar uses bile salts and crystal violet to inhibit Gram-positive bacteria, thereby selecting for Gram-negatives. Mannitol salt agar (MSA) contains 7.5% NaCl, which selects for halotolerant staphylococci. Eosin methylene blue (EMB) agar also selects for Gram-negative organisms. Differential media allow organisms growing on the same plate to be distinguished by their metabolic reactions. MacConkey agar doubles as a differential medium: lactose fermenters produce pink or red colonies (such as E. coli) while non-fermenters remain colorless (such as Salmonella). Blood agar differentiates hemolytic patterns -- alpha hemolysis produces partial, greenish clearing, beta hemolysis produces complete clearing, and gamma hemolysis indicates no hemolysis at all. MSA differentiates mannitol fermenters, which produce a yellow halo (as does S. aureus), from non-fermenters.
Enrichment media promote the growth of a particular organism from a mixed sample, such as selenite broth for Salmonella or alkaline peptone water for Vibrio cholerae. Transport media maintain organism viability without allowing multiplication during specimen transit (examples include Stuart's, Amies, and Cary-Blair media). Anaerobic culture techniques employ anaerobic jars, anaerobic chambers (glove boxes), or GasPak systems that generate hydrogen and carbon dioxide while removing oxygen.
IV. Environmental Factors Affecting Growth
A. Temperature
Every microorganism has a minimum, optimum, and maximum growth temperature, and organisms are classified accordingly. Psychrophiles grow optimally at 0--15 degrees C and inhabit the deep ocean and polar regions. Psychrotrophs can grow at 0 degrees C but thrive at 20--30 degrees C and are significant causes of food spoilage. Mesophiles grow best at 25--40 degrees C and include most human pathogens, with 37 degrees C being the typical optimum. Thermophiles prefer 50--60 degrees C, as found in hot springs, and hyperthermophiles -- mostly archaea -- grow optimally above 80 degrees C, thriving at deep-sea hydrothermal vents.
B. pH
Most bacteria, including human pathogens, are neutrophiles with an optimal pH of 6.5--7.5. Acidophiles prefer pH values below 5.5 (examples include Lactobacillus and Acidithiobacillus; Helicobacter pylori tolerates gastric acid). Alkaliphiles thrive at pH above 8.5, inhabiting soda lakes. Microorganisms maintain their internal pH near neutrality using proton pumps and buffering systems.
C. Osmotic Pressure and Water Activity
Halophiles require elevated NaCl concentrations for growth, ranging from mild halophiles (1--6% NaCl) to moderate halophiles (6--15%) to extreme halophiles (15--30%, primarily archaea such as Halobacterium). Halotolerant organisms such as Staphylococcus aureus can tolerate moderate salt but do not require it. Osmotolerant organisms withstand high sugar concentrations. In hypertonic solutions, cells undergo plasmolysis, while in hypotonic solutions they risk lysis unless protected by a cell wall.
D. Oxygen Requirements
Microorganisms vary dramatically in their relationship with oxygen. Obligate aerobes such as Mycobacterium tuberculosis and Pseudomonas aeruginosa require oxygen as the terminal electron acceptor in aerobic respiration. Obligate anaerobes like Clostridium and Bacteroides are killed by oxygen because they lack superoxide dismutase (SOD) and catalase, rendering them unable to detoxify reactive oxygen species. Facultative anaerobes such as E. coli and S. aureus grow with or without oxygen, using aerobic respiration when it is available and switching to fermentation or anaerobic respiration when it is not. Aerotolerant anaerobes like Lactobacillus and Streptococcus pyogenes grow equally well regardless of oxygen but do not use it metabolically, relying exclusively on fermentation. Microaerophiles such as Helicobacter pylori and Campylobacter jejuni require oxygen at reduced levels (2--10%). Capnophiles like Neisseria and Haemophilus require elevated CO2 concentrations (5--10%).
The toxic forms of oxygen include the superoxide radical (O2-), detoxified by superoxide dismutase (SOD), hydrogen peroxide (H2O2), detoxified by catalase, and the hydroxyl radical (OH.), the most reactive species, for which no specific enzyme exists -- damage is prevented by upstream detoxification via SOD and catalase.
<image>A diagram showing oxygen requirements of different bacterial groups. Panel A: Thioglycolate broth tubes showing characteristic growth patterns for obligate aerobes (growth at top only), obligate anaerobes (growth at bottom only), facultative anaerobes (growth throughout, heaviest at top), aerotolerant anaerobes (growth evenly distributed throughout), and microaerophiles (growth in a band below the surface). Each tube is clearly labeled. Panel B: A table listing the presence or absence of superoxide dismutase and catalase for each group, explaining their oxygen tolerance.</image>
V. Bacterial Growth Curve
When bacteria are inoculated into fresh liquid medium, their population dynamics follow a predictable sequence of phases in batch culture.
During the lag phase, there is no increase in cell number. The cells are metabolically active, synthesizing enzymes and adapting to their new environment. The duration of the lag phase depends on the age and size of the inoculum and on how different the new medium is from the previous one.
In the log (exponential) phase, cells divide at a constant, maximal rate, and the generation (doubling) time is constant. Under optimal conditions, E. coli has a generation time of about 20 minutes. Cells in this phase are most metabolically active and most susceptible to antibiotics. Population growth follows the equation N(t) = N(0) x 2^(t/g), where g is the generation time.
The stationary phase is reached when the growth rate equals the death rate, causing the cell number to plateau. This equilibrium results from nutrient depletion, accumulation of waste products, and spatial constraints. During this phase, cells may produce secondary metabolites such as antibiotics and toxins, some cells enter dormancy as persister cells, and quorum sensing signals accumulate.
In the death (decline) phase, the death rate exceeds the growth rate and the viable cell count decreases exponentially, although some resistant cells may persist for extended periods.
<image>A graph of the bacterial growth curve plotting log of cell number (y-axis) versus time (x-axis). Four phases are clearly demarcated and labeled: lag phase (flat line), log/exponential phase (steep upward slope), stationary phase (plateau), and death phase (downward slope). Key features annotated include: generation time (doubling time) marked on the exponential phase, a note about nutrient depletion and waste accumulation at the transition to stationary phase, and the formula N(t) = N0 x 2^(t/g) shown near the exponential phase.</image>
VI. Measuring Microbial Growth
Direct methods include plate counts (viable count / CFU), in which serial dilutions of a culture are plated on agar and colonies are counted after incubation, with each colony representing one colony-forming unit. This method measures only viable, culturable cells. The direct microscopic count uses a Petroff-Hausser counting chamber to count all cells (live and dead) quickly, though it does not distinguish viability. Membrane filtration passes a liquid sample through a 0.45 micrometer filter, which is then placed on agar for incubation -- a technique used for counting bacteria in water and other dilute samples.
Indirect methods include turbidity (spectrophotometry), which measures optical density (OD) at 600 nm; more cells produce more light scattering and a higher OD reading. This approach is quick and non-destructive but does not distinguish live from dead cells and must be correlated with plate counts for accuracy. Dry weight measurements are used for filamentous organisms and dense cultures, and metabolic activity assays measure oxygen consumption, CO2 production, or ATP levels as proxies for growth.
VII. Continuous Culture
A chemostat is a continuous culture device that maintains cells in exponential phase by adding fresh medium at a constant rate while removing culture at the same rate. The growth rate is controlled by the dilution rate and the concentration of a limiting nutrient. Chemostats are used in industrial microbiology and in research on steady-state physiology. A turbidostat takes a different approach, adjusting flow rate based on optical density readings to maintain constant turbidity.
VIII. Biofilms
In nature, most bacteria grow not as free-floating planktonic cells but as biofilms -- structured communities attached to surfaces. Biofilm formation proceeds through a series of stages: reversible attachment to a surface, irreversible attachment mediated by pili and adhesins, maturation involving the production of an extracellular polymeric substance (EPS) matrix composed of polysaccharides, proteins, and DNA, development of a three-dimensional architecture with water channels, and ultimately dispersal, in which cells detach to colonize new surfaces.
Biofilms are of enormous clinical significance. Bacteria within biofilms are 100 to 1,000 times more resistant to antibiotics than their planktonic counterparts. Biofilm-associated infections include endocarditis, chronic lung infections in cystic fibrosis patients, and infections of medical devices such as catheters and implants. Dental plaque is a classic example of a biofilm. Quorum sensing -- cell-to-cell signaling through small diffusible molecules -- plays a central role in regulating biofilm formation.
