Microbiology Cell Counting Techniques

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Questions and Answers

What is the main limitation of direct cell count methods?

They can only count living cells.

They are not applicable to all microorganisms.

They do not differentiate between living and dead cells. (correct)

They require specialized growth media.

What is the purpose of using a Coulter counter in cell counting?

To count cells as they flow through an orifice. (correct)

To assess the metabolic activity of cells.

To identify species of microorganisms.

To differentiate between viable and non-viable cells.

In the process of serial dilution and colony counting, what does the Colony Forming Units (CFU) assay measure?

Only the rate of binary fission.

Overall biomass of the microorganisms.

Total cell population including dead cells.

Reproductively active cells that form colonies. (correct)

What is a key factor in determining the concentration of bacteria from the original sample in colony counting?

The number of colonies on plates with 25 – 250 colonies. Signup and view all the answers

Which method involves counting microorganisms by observing them on etched slides?

Microscopic cell count method. Signup and view all the answers

What phase follows the exponential growth phase in a bacterial culture?

Stationary phase Signup and view all the answers

How does turbidity serve as an indicator of bacterial growth?

It indicates light scattering proportional to cell concentration. Signup and view all the answers

Which of the following methods is primarily used for direct cell counts in bacterial cultures?

Petroff-Hausser counting chamber Signup and view all the answers

What limiting factor contributes to the death phase in bacterial growth?

Oxygen depletion Signup and view all the answers

Which method is best for estimating microbial mass when direct measurement is impractical?

Biochemical determinations Signup and view all the answers

What is the relationship between turbidity and bacterial population size?

Greater turbidity indicates a larger population size. Signup and view all the answers

Which of the following statements about binary fission is incorrect?

It happens only during the stationary phase. Signup and view all the answers

Which of these factors is NOT a method of measuring bacterial growth?

Cellular respiration measurement Signup and view all the answers

What is the viable cell count determined by when there are multiple plates within the statistically valid range?

Using the formula involving colonies and dilution factors Signup and view all the answers

Which method is used to count low microbial concentrations through membrane filtration?

Membrane filtration Signup and view all the answers

What volume of sample is typically filtered in the membrane filtration method?

1 to 100 ml Signup and view all the answers

In the direct microscopic count, how are cells examined?

Using counting chambers or slides Signup and view all the answers

What is NOT considered an indirect method of measuring microbial growth?

Direct microscopic count Signup and view all the answers

What is the purpose of mass determination in measuring microbial growth?

To determine wet and dry mass of the cells Signup and view all the answers

When using the Most Probable Number (MPN) method, how many different volumes of sample are typically assessed?

Five repetitions per volume Signup and view all the answers

Which technique is applicable for detecting the presence of E.coli in water samples?

Membrane filtration Signup and view all the answers

Study Notes

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

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Description

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!