Microbiology Cell Counting Techniques

Microbial Nutrition, Ecology, and Growth

• Microbial nutrition involves chemical analysis, sources of essential nutrients, and transport mechanisms.
• Macronutrients are required in large quantities and play roles in cell structure and metabolism (examples: proteins, carbohydrates).
• Micronutrients, or trace elements, are needed in small amounts for enzyme function and protein structure maintenance (examples: manganese, zinc, nickel).
• Inorganic nutrients contain atoms other than carbon and hydrogen (example: metals & salts, gases like oxygen, carbon dioxide, and water).
• Organic nutrients contain carbon and hydrogen atoms (example: methane, carbohydrates, lipids, proteins, and nucleic acids)
• Cytoplasm is 70% water and contains proteins, and 96% of the cell is composed of six elements (carbon, hydrogen, oxygen, nitrogen, phosphorous, and sulfur).
• Heterotrophs obtain carbon from organic molecules produced by other organisms (example sugars, proteins, lipids).
• Autotrophs use inorganic carbon dioxide (CO2) as a carbon source and are not dependent on living organisms.
• Nitrogen is a significant component of proteins, DNA, RNA and ATP.
• Some bacteria and algae use inorganic nitrogen nutrients (NO3-, NO2-, or NH3), and some can fix N2.
• Regardless of the nitrogen source, it must be converted to ammonia (NH3) for use in the cell.
• Oxygen is a component of carbohydrates, lipids, and proteins and plays a significant role in cell structural and enzymatic functions.
• Oxygen is also a component of inorganic salts (sulfates, phosphates, nitrates) and water, and makes up 20% of the atmosphere.
• It is essential to the metabolism of many organisms.
• Hydrogen is a key component in organic and several inorganic compounds.
• Roles include pH maintaining, forming hydrogen bonds, serving as a source of free energy in respiration.
• Phosphorous is mainly in the form of phosphate (PO4-3) derived from phosphoric acid (H3PO4) found in rocks and oceanic mineral deposits.
• It is a critical component of nucleic acids and essential to cellular genetics and energy transfer.
• Sulfur is widely found in the environment in rocks and sediments as sulfate, sulfides, hydrogen sulfide gas, and sulfur.
• It is an essential component of certain vitamins and amino acids (methionine and cysteine).
• Sulfur helps stabilize proteins by forming disulfide bonds.
• Additional important mineral ions include potassium, sodium, calcium, magnesium, and iron.
• Bacteria are composed of water (70%) and proteins (15%).
• A table shows chemical composition of an E. coli cell (including percentages by dry weight).

Sources of Essential Nutrients

• Essential nutrients are required for metabolism and growth, serving as carbon and energy sources.
• Carbon sources include organic molecules for heterotrophs and inorganic carbon dioxide (CO2) for autotrophs.
• Energy sources include chemoheterotrophs, photoautotrophs, and chemoautotrophs.

Growth Factors

• Essential organic nutrients.
• Not synthesized by the microbe and must be supplemented.
• Examples include amino acids and vitamins.

Chemoheterotrophs

• Organisms that derive both carbon and energy from organic compounds.
• Saprobic: decomposers of plant litter, animal matter, and dead microbes.
• Parasitic: live in or on the body of a host.
• Extracellular digestion occurs in saprobes with a cell wall (figure shown).

Photoautotrophs

• Organisms deriving energy from sunlight and converting light into chemical energy.
• Primary producers of organic matter and oxygen.
• Examples include algae, plants, and some bacteria.

Two Types of Autotrophs

• Chemoorganic autotroph: derive energy from organic compounds and carbon source from inorganic compounds.
• Lithoautotroph: derive energy and carbon entirely from inorganic sources.
• Methanogens are a type of chemoautotroph.

Summary of Different Nutritional Categories

• Nutritional categories of microbes are summarized based on energy and carbon sources (table shown).

Transport Mechanisms

• Passive transport does not require energy, substances move along a concentration gradient, and includes osmosis and diffusion.
• Facilitated diffusion uses a carrier protein.
• Active transport requires energy and carrier proteins and may move substances against a concentration gradient.
• Bulk transport (endocytosis, exocytosis, pinocytosis) involves transporting large molecules.

Osmosis

• Diffusion of water through a semipermeable membrane.
• Water moves towards higher concentrations of solute.
• Isotonic: solute concentrations inside and outside the cell are equal.
• Hypotonic: lower solute concentration outside the cell.
• Hypertonic: higher solute concentration outside the cell.

Diffusion

• Net movement of molecules from a high concentration area to a low concentration area.
• No energy is expended.
• Affected by concentration gradient and permeability.

Facilitated Diffusion

• Transport of polar molecules and ions across the membrane.
• No energy is expended.
• Carrier protein facilitates the binding and transport of molecules.
• Specific to one type of molecule.
• The rate of transport is limited by the number of binding sites on the carrier protein.

Active Transport

• Transport of molecules against a gradient.
• Requires energy (active).
• Examples include permeases and protein pumps that transport sugars, amino acids, organic acids, phosphates, metal ions.
• Group translocation chemically alters the transported molecules.

Endocytosis

• Substances are taken in, but not transported through the membrane.
• Requires energy.
• Common for eukaryotes.
• Examples include phagocytosis and pinocytosis.

Environmental Factors

• Factors influencing microbial growth: temperature, gas, pH, osmotic pressure, other factors, and microbial association.

Temperature

• Optimal growth and metabolism for various microbes
• Psychrophiles (0 to 15°C), mesophiles (20 to 40°C), thermophiles (45 to 80°C), effects on growth & metabolism.

Gas

• Key gases in microbial growth - oxygen (respiration, oxidizing agent), carbon dioxide (various metabolic needs)
• Three bacterial categories based on oxygen requirements: obligate aerobes, facultative anaerobes, obligate anaerobes
• Types of toxic oxygen metabolites

pH

• Ideal pH for growth (6.5 - 7.5); exceptions exist for acidophiles and alkalinophiles
• Some bacteria tolerate or thrive in acidic environments.
• Mold and yeasts prefer intermediate pH levels.

Osmotic Pressure

• Hypertonic environments (high solute concentrations outside the cell) cause plasmolysis.
• Halophiles require high salt concentrations, while facultative halophiles can tolerate high osmotic pressure
• Microbes have adaptations for survival in hypertonic environments.

Other Factors

• Radiation (UV, X-rays, cosmic rays), pressure (barophiles), desiccation (spores and cysts).

Ecological Association

• Symbiotic relationships (mutualism, commensalism, parasitism)
• Non-symbiotic relationships (synergism, antagonism).

Interrelationships between Microbes and Humans

• Human body is a rich environment for symbiotic bacteria (e.g., skin, gastrointestinal tract)
• Can be (commensal, parasitic, and synergistic).
• Examples: E. coli and lactobacillus.

Microbial Growth

• Growth as the acquisition of biomass leading to cell division or reproduction.
• Requirements include nutrients and favorable environmental factors.
• Growth occurs on two levels: synthesis of cell components and increase in cell numbers.

Binary Fission

• Division of a bacterial cell where the parental cell enlarges, duplicates its DNA, creates a septum dividing the cell into two chambers, leading to two identical daughter cells.

Generation Time

• Time needed for a complete bacterial division cycle (doubling time).
• Ranges from 10 minutes in some bacteria to several days in other microorganisms.
• Growth rate is measurable by generation time.

Mean Generation Time and Growth Rate

• Mean generation time = doubling time.
• Expressed in minutes.
• Can be determined directly from a semilog plot of bacterial concentration versus time after inoculation.

Growth Curve

• The standard bacterial growth curve depicts various stages of growth of a pure culture in a closed system (batch culture), including lag, log, stationary, and death (numerical display shown, tables too)

Phases of Growth

• Lag phase: initial adjustment to new conditions
• Log phase: maximum exponential growth rate
• Stationary phase: rate of cell growth equals the rate of cell death (nutrient exhaustion, waste buildup, etc.)
• Death phase: majority of cells die exponentially due to unfavorable conditions.

Measurement of Bacterial Growth

• Turbidity
• Direct cell count
• Automated devices (Coulter counter, flow cytometer, real-time PCR)
• Serial dilution and colony counting

Standard Plate Count

• Serial dilution of cultured sample for plating on nutrient agar media.
• Incubation and colony counting
• Calculation for determining cell numbers in original sample
• Commonly-used method for determining microbial populations.

MPN (Most Probable Number)

• Method to estimate microbial numbers in a sample
• Uses dilutions of the sample to assess growth in broth media.
• Statistical method to infer population size of a sample from growth data.

Other Measurements of Microbial Growth

• Membrane filtration
• Direct microscopic count
• Mass determination

This quiz covers essential methods in microbiology for counting cells, including direct cell count limitations and the role of Coulter counters. It explores colony forming units (CFU) in serial dilution and the use of etched slides for microorganism observation. Test your knowledge on these crucial techniques!