Chapter 4

Microbial Growth and Its Control

Cell Nutrition and Essential Elements

Microbial cells require a variety of nutrients for growth, which are classified as macronutrients and micronutrients. The chemical makeup of a cell is dominated by a handful of elements, and the macromolecular composition reflects the cell's functional needs.

• Macronutrients: Required in large amounts; include carbon (C), oxygen (O), nitrogen (N), hydrogen (H), phosphorus (P), and sulfur (S).

• Micronutrients: Needed in minute amounts; include trace metals and growth factors.

• Macromolecules: Proteins, lipids, polysaccharides, lipopolysaccharides, and nucleic acids make up most of the cell's dry weight.

• Elemental composition: Proteins and RNA are the most abundant macromolecules in bacterial cells.

• Carbon sources: Heterotrophs require organic carbon, while autotrophs synthesize organics from CO2.

• Nitrogen sources: Can be obtained from proteins, ammonia, nitrate, or nitrogen gas.

• Other essential elements: Phosphorus (nucleic acids, phospholipids), sulfur (amino acids, vitamins), potassium, magnesium, calcium, and sodium.

Additional info: The periodic table in the image highlights elements essential for microorganisms, and the macromolecular composition chart shows the relative abundance of cell components.

Micronutrients: Trace Metals and Growth Factors

Micronutrients are required in small amounts and often serve as cofactors for enzymes. Growth factors are organic micronutrients, such as vitamins, amino acids, purines, and pyrimidines.

Trace Element	Function
Boron (B)	Autoinducer for quorum sensing; found in some antibiotics
Cobalt (Co)	Vitamin B12; transcarboxylase
Copper (Cu)	Respiration, cytochrome c oxidase; photosynthesis
Iron (Fe)	Cytochromes, catalases, peroxidases, nitrogenases
Manganese (Mn)	Activator of enzymes; photosystem II
Molybdenum (Mo)	Flavin-containing enzymes; nitrate reductases
Nickel (Ni)	Hydrogenases; coenzyme F430
Selenium (Se)	Formate dehydrogenase; selenocysteine
Tungsten (W)	Formate dehydrogenases; oxotransferases
Vanadium (V)	Vanadium nitrogenase; bromoperoxidase
Zinc (Zn)	Carbonic anhydrase; nucleic acid polymerases

Growth factors: Vitamins and other organics, most function as coenzymes.

Growth Media and Laboratory Culture

Microbes are grown in nutrient solutions called culture media, which can be defined or complex. Media are sterilized to prevent contamination, and solid media use agar as a gelling agent.

• Defined media: Exact chemical composition is known.

• Complex media: Composed of digests of microbial, animal, or plant products.

• Selective media: Inhibit growth of some microbes but not others.

• Differential media: Contain indicators to detect metabolic reactions.

• Colony morphology: Used to identify microorganisms and assess purity.

• Aseptic technique: Prevents contamination during transfer.

Additional info: The images show colony morphology and the steps of aseptic technique, including streaking for isolation.

Measuring Microbial Growth

Microscopic Counts

Microscopic cell counts involve observing and enumerating cells using counting chambers. Stains can differentiate live and dead cells and provide phylogenetic information.

Viable Counting

Viable counts measure living, reproducing cells using plate count methods. Serial dilutions are used for dense cultures, and results are reported as colony-forming units (CFU).

• Spread-plate method: Sample spread on agar surface.

• Pour-plate method: Sample mixed with molten agar.

• Applications: Used in food, dairy, medical, and aquatic microbiology.

• Plate count anomaly: Direct counts often reveal more organisms than plate counts due to differing growth requirements.

Turbidimetric Measures

Cell suspensions scatter light, and turbidity measurements (optical density) are used to estimate cell numbers. A spectrophotometer measures OD, which is proportional to cell number within limits.

Binary Fission and Microbial Growth Cycle

Microbial growth is an increase in cell number, typically by binary fission. The growth cycle in batch culture includes lag, exponential, stationary, and death phases.

• Binary fission: Cell divides after enlarging to twice its original size; each daughter cell receives a chromosome and cell constituents.

• Generation time: Time required for cells to double in number; varies by species and conditions.

• Growth curve phases: Lag (adjustment), exponential (rapid growth), stationary (nutrient depletion), death (decline).

Quantitative Aspects of Microbial Growth

Exponential growth is characterized by cell numbers doubling at regular intervals. The mathematics of bacterial growth allows calculation of generation time and specific growth rate.

• Exponential growth equation: $N = N_0 \times 2^n$

• Generation time (g): $g = \frac{t}{n}$

• Specific growth rate (k): $k = \frac{\ln N - \ln N_0}{t}$

Additional info: Initial increases are slow but can result in large populations over time.

Continuous Culture and Chemostats

Continuous culture maintains microbial populations in exponential phase. The chemostat is a device that controls growth rate and yield by regulating dilution rate and limiting nutrient concentration.

• Steady state: Cell density and substrate concentration remain constant.

• Experimental uses: Study physiology, ecology, evolution, and enrichment of bacteria.

Biofilm Growth

Biofilms are communities of microbes attached to surfaces and enmeshed in a polysaccharide matrix. They form in stages and have distinct properties from planktonic cells.

• Stages: Attachment, colonization, development, dispersal.

• Medical and industrial relevance: Implicated in infections, device contamination, pipe fouling.

Alternatives to Binary Fission

Some bacteria divide by budding or hyphal growth, producing cells with different morphologies. Actinomycetes form mycelia and arthrospores for survival.

Environmental Effects on Growth

Temperature

Microorganisms are classified by their temperature optima: psychrophiles (cold), mesophiles (moderate), thermophiles (hot), and hyperthermophiles (very hot).

• Cardinal temperatures: Minimum, optimum, and maximum for growth.

• Extremophiles: Grow in extreme conditions; adaptations include specialized enzymes and membrane lipids.

pH

Microbes are classified as neutrophiles, acidophiles, or alkaliphiles based on their pH optima. Most maintain cytoplasmic pH near neutrality.

Physiological Class	Approximate pH Optimum	Example Organism
Neutrophile	7	Escherichia coli
Acidophile	5	Rhodopila globiformis
Acidophile	3	Acidithiobacillus ferrooxidans
Acidophile	1	Picrophilus oshimae
Alkaliphile	8	Chloroflexus aurantiacus
Alkaliphile	9	Bacillus firmus
Alkaliphile	10	Natronobacterium gregoryi

Osmolarity

Water activity affects microbial growth. Halophiles require high salt concentrations, while halotolerant, osmophilic, and xerophilic organisms tolerate or require other solutes.

Material	Water Activity (aw)	Example Organisms
Pure water	1.000	Caulobacter, Spirillum
Human blood	0.995	Streptococcus, Escherichia
Seawater	0.980	Pseudomonas, Vibrio
Bread	0.950	Most gram-positive rods
Maple syrup, ham	0.900	Staphylococcus
Salami	0.850	Saccharomyces rouxii
Fruit cake, jams	0.800	Zygosaccharomyces bailii, Penicillium
Salt lakes, salted fish	0.750	Halobacterium, Halococcus
Cereals, candy, dried fruit	0.700	Xeromyces bisporus

Oxygen

Microorganisms are classified by their oxygen requirements: obligate aerobes, facultative aerobes, microaerophiles, aerotolerant anaerobes, and obligate anaerobes. Oxygen can be toxic due to reactive byproducts, and microbes possess enzymes to neutralize these.

Group	Relationship to O2	Type of Metabolism	Example	Habitat
Obligate Aerobe	Required	Aerobic respiration	Micrococcus luteus	Skin, dust
Facultative Aerobe	Not required, but better with O2	Aerobic/anaerobic respiration	Escherichia coli	Large intestine
Microaerophile	Required at low levels	Aerobic respiration	Spirillum volutans	Lake water
Aerotolerant Anaerobe	Not required, no better with O2	Fermentation	Streptococcus mutans	Oral cavity
Obligate Anaerobe	Harmful/lethal	Fermentation/anaerobic respiration	Methanobacterium formicicum	Sewage sludge

Controlling Microbial Growth

Heat Sterilization

Heat is the most widely used method for sterilization. The autoclave uses steam under pressure to kill endospores, while pasteurization reduces microbial load in heat-sensitive liquids.

• Decimal reduction time (D): Time required at a given temperature to reduce viability by 90%.

• Thermal death time: Time to kill all cells at a given temperature.

Radiation and Filtration

Ultraviolet and ionizing radiation are used to decontaminate surfaces and materials. Filtration is used for heat-sensitive liquids and gases.

• UV radiation: Damages DNA; useful for surfaces.

• Ionizing radiation: Produces ions and reactive molecules; used for sterilizing medical supplies and food.

• Depth filters: Trap particles in fibrous sheets.

• Membrane filters: Used for liquid sterilization; pores allow passage of liquid but not microbes.

Chemical Control

Chemical agents can kill or inhibit microbial growth. They are classified as -cidal (kill), -static (inhibit), or -lytic (cause lysis). Minimum inhibitory concentration (MIC) assays determine effectiveness.

• Antiseptics: Applied to living tissue; kill or inhibit microbes.

• Disinfectants: Used on surfaces; kill microbes but not necessarily endospores.

• Sanitizers: Reduce microbial numbers; less harsh.

• Sterilants: Destroy all microorganisms, including endospores.

Additional info: Disk diffusion and dilution assays are used to test antimicrobial susceptibility.