Microbial Viability Methods

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Questions and Answers

What does the term 'viable counting methods' refer to?

Counting the total number of cells including dead ones.

Methods that count all cells in a sample.

Methods that count only cells able to reproduce when cultured. (correct)

Techniques that only count non-reproducing cells.

Which technique is commonly used for counting the number of viable microbes in a sample?

Enzyme-linked immunosorbent assay.

Radioactive labeling.

Colorimetric analysis.

Spread plate technique. (correct)

What is the purpose of using nucleic acid fluorescent stains in the membrane filter technique?

To enhance visibility of dead cells.

To trap bacteria for easier counting.

To stain only the bacterial cell walls.

To stain and observe bacteria microscopically. (correct)

When is the Most Probable Number (MPN) test most useful?

When microbes cannot be cultured on solid media. Signup and view all the answers

How is microbial dry weight typically determined?

By centrifugation followed by drying and weighing. Signup and view all the answers

What measurement can indicate an increase in microbial population?

Total protein levels. Signup and view all the answers

Why might several hundred milliliters of culture be needed for determining microbial dry weight?

Because bacteria have low mass and require larger volumes to collect a measurable weight. Signup and view all the answers

Which of the following methods is NOT a viable counting method?

Microscopic direct count. Signup and view all the answers

What is the principle behind spectrophotometry for measuring cell mass?

It assesses the amount of light scattered by microbial cells. Signup and view all the answers

At what absorbance level is the relationship between absorbance and cell concentration considered almost linear?

0.5 Signup and view all the answers

Which energy currency is most commonly used by living organisms?

ATP Signup and view all the answers

What is required for microbial growth in terms of nutrients?

Energy and substrates for biosynthesis and metabolism Signup and view all the answers

Which process requires the transfer of anhydride bonds from ATP?

Biosynthetic polymerization Signup and view all the answers

Why must samples exceeding an absorbance of 0.5 be diluted before measurement?

To prevent light scattering from falsely elevating absorbance readings. Signup and view all the answers

What do microbial cells require to synthesize proteins and structural membranes?

Pre-formed building blocks and metabolic energy Signup and view all the answers

What is not a typical role of nutrients in microbial growth?

Cellular communication Signup and view all the answers

What is the primary function of catabolism?

To break down complex molecules into simpler compounds Signup and view all the answers

Which statement correctly describes the reduction of an atom?

It gains electrons and may bond to a hydrogen atom Signup and view all the answers

Which of the following compounds is an example of an oxidized form of carbon?

Carbon dioxide Signup and view all the answers

In a redox reaction, what is the term for the substance that gets oxidized?

Electron donor Signup and view all the answers

What role do cofactors like NAD and FAD play in redox reactions?

They shuttle electrons between different parts of the pathway Signup and view all the answers

What is an enzymatic pathway?

A series of enzymatically-catalyzed reactions from a raw material to a product Signup and view all the answers

How is the potential chemical energy of reduced forms of carbon compared to oxidized forms?

Reduced forms carry more potential energy Signup and view all the answers

What happens to an atom when it becomes more oxidized?

It decreases in energy content Signup and view all the answers

What type of microorganisms use light as their primary energy source along with CO2 as their carbon source?

Photolithoautotrophs Signup and view all the answers

Which nutritional class extracts electrons from reduced organic compounds?

Photoorganoheterotrophy Signup and view all the answers

Which of the following is an example of a chemolithoautotrophic microorganism?

Methanogens Signup and view all the answers

Which groups of microorganisms are classified under chemoorganoheterotrophy?

Most nonphotosynthetic microbes including pathogens Signup and view all the answers

What do lithotrophs utilize as their electron source?

Inorganic substances Signup and view all the answers

Growth factors are defined as organic compounds that are:

Essential cell components that cannot be synthesized by the organism Signup and view all the answers

What is a common characteristic of chemolithoheterotrophs?

They can use both organic and inorganic carbon sources Signup and view all the answers

Which of the following is NOT a representative microbe of photolithoautotrophy?

Methanogens Signup and view all the answers

What is the net yield of ATP per glucose molecule during glycolysis?

Two ATPs Signup and view all the answers

Which molecule is NOT produced during glycolysis?

Acetyl-CoA Signup and view all the answers

What role does oxygen play in anaerobic respiration?

It is not used at all Signup and view all the answers

What is the primary function of the pyruvate dehydrogenase complex?

To oxidize pyruvate to CO2 and acetyl-CoA Signup and view all the answers

Which molecules act as electron carriers in the mitochondrial electron transport chain?

NADH and FADH2 Signup and view all the answers

Which of the following statements about fermentation is true?

Reduced organic acids or alcohols are formed from pyruvic acid Signup and view all the answers

What is the substrate for the tricarboxylic acid (TCA) cycle?

Acetyl-CoA Signup and view all the answers

What is produced alongside NADH during the conversion of glyceraldehyde 3-phosphate to pyruvate?

Two ATPs Signup and view all the answers

Study Notes

Viable Counting Methods

Viable counting methods determine the number of viable microbes in a sample.
These methods are also known as standard plate counts (SPC).
They count only cells that can reproduce when cultured.
Two commonly used methods are spread plate and pour plate techniques.
Results are often expressed as colony forming units (CFU).

Membrane Filter Technique

This technique is used to determine the number of bacteria in aquatic samples.
Bacteria are trapped on membrane filters.
Bacteria are stained with fluorescent nucleic acid stains like acridine orange or DAPI.
Samples are observed microscopically.

Most Probable Number (MPN) Test

This test is used when plate counts aren't suitable.
It's used when microbes can't be cultured on solid media or large colonies overgrow the plate.
It determines the most probable number of viable cells in a sample.

Measurement of Cell Mass

Microbial dry weight can be determined by collecting cells from a liquid medium, centrifuging, washing, drying in an oven, and weighing them.
This method is useful for measuring filamentous fungi growth.
It is time-consuming and not very sensitive.
Cell mass can also be estimated by measuring cellular substance concentrations (e.g., total protein, nitrogen).

Spectrophotometry

This method measures cell mass by measuring light scattering.
Microbial cells scatter light proportionally to their biomass and indirectly related to cell number.
Increased cell concentration leads to greater turbidity, reducing light transmission.
A spectrophotometer measures light scattering, known as absorbance or optical density.
Absorbance is almost linearly related to cell concentration.
Samples exceeding 0.5 absorbance must be diluted before measurement.

Microbial Metabolism

Microbial growth requires the polymerization of biochemical building blocks (proteins, nucleic acids, polysaccharides, and lipids) into complex molecules.
These building blocks are either preformed in the growth medium or synthesized by the growing cells.
Biosynthetic demands are met by enzymes requiring cofactors for catalytic activity.
Biosynthetic polymerization reactions require ATP.
Growth requires metabolic energy for synthesizing anhydride bonds and maintaining transmembrane ion and metabolite gradients.
ATP is often referred to as the cell's energy currency.

Nutrients

Microorganisms, like all living cells, require energy and nutrients for growth.
Nutrients provide nourishment for life and growth.
Nutrients are used for biosynthesis and energy production, essential for microbial growth.
Phototrophs use light as their energy source.
Chemotrophs obtain energy from oxidizing chemical compounds (organic or inorganic).
Microorganisms use two sources for electrons.
Lithotrophs use reduced inorganic substances as electron sources.
Organotrophs extract electrons from reduced organic compounds.
Based on their primary carbon, energy, and electron sources, most microorganisms can be classified into five nutritional types: photolithoautotrophy, photoorganoheterotrophy, chemolithoautotrophy, chemolithoheterotrophy, and chemoorganoheterotrophy.

Growth Factors

Growth factors are organic compounds essential for cell components or precursors that cannot be synthesized by the organism.

Catabolism

Breaks down complex organic molecules into simpler compounds.
Releases energy.

Anabolism

Builds complex organic molecules from simpler ones.
Requires energy.

Reduction and Oxidation

An atom becomes more reduced when it gains electrons, bonds to a less electronegative atom, and often bonds to hydrogen.
An atom becomes more oxidized when it loses electrons, bonds to a more electronegative atom, and often bonds to oxygen.
The oxidation state of carbon is important in metabolic pathways.
Reduced forms of carbon carry potential chemical energy.
Oxidized forms of carbon carry little potential chemical energy.
Reduction and oxidation occur simultaneously in redox reactions.
The substance that gets oxidized is called the electron donor.
The substance that gets reduced is called the electron acceptor.

Enzymatic Pathways for Metabolism

Metabolic reactions occur in a step-wise fashion.
Each step requires its own enzyme.
Enzymatic pathways (or metabolic pathways) are sequences of enzymatically catalyzed steps.

Cofactors for Redox Reactions

Enzymes catalyzing redox reactions require cofactors to shuttle electrons.
NAD and FAD are the two main redox cofactors.

Glycolysis

This is the first stage of catabolism, where glucose is broken down into pyruvate.
In the six-carbon stage, two ATPs are used to form fructose 1,6-bisphosphate.
For each glyceraldehyde 3-phosphate transformed into pyruvate, one NADH and two ATPs are formed.
Two glyceraldehyde 3-phosphates arise from one glucose.
The three-carbon stage generates four ATPs and two NADHs per glucose.
Subtracting the ATP used in the six-carbon stage from that produced in the three-carbon stage results in a net yield of two ATPs per glucose.
The breakdown of glucose to pyruvate in glycolysis can be represented by the following equation: Glucose + 2ADP + 2Pi + 2NAD+ → 2 pyruvate + 2ATP + 2NADH + 2H+

Tricarboxylic Acid Cycle (TCA Cycle, Citric Acid Cycle, Krebs Cycle)

This is the second stage of catabolism and provides much more energy than the glycolytic pathway.
The pyruvate dehydrogenase complex oxidizes pyruvate to CO2 and acetyl coenzyme A (acetyl-CoA).
Acetyl-CoA is further degraded in the TCA cycle.
Acetyl-CoA is the substrate for the TCA cycle.

Electron Transport Chain (ETC)

The mitochondrial ETC is a series of electron carriers that transfer electrons from donors (NADH and FADH2) to acceptors (like O2).

Anaerobic Respiration

Electrons released by oxidation are passed down an ETC, but oxygen is not the final electron acceptor.
Alternative electron acceptors include:
- Nitrate (NO3-) → Nitrite (NO2-)
- Sulfate (SO24-) → Hydrogen Sulfide (H2S)
- Carbonate (CO24-) → Methane (CH4)

Fermentation

This is an anaerobic process that doesn't use the ETC.
It involves incomplete oxidation of a carbohydrate that becomes the final electron acceptor.
It is essentially glycolysis with an additional step.

Features of Fermentation Pathways

Pyruvic acid is reduced to form organic acids or alcohols.
The final electron acceptor is a reduced derivative of pyruvic acid.
NADH is oxidized to form NAD, essential for glycolytic pathway operation.
Oxygen is not required.
No additional ATP is made.
Gases (CO2 and/or H2) may be released.

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Description

Explore various methods for determining the viability of microbes in samples, including standard plate counts, membrane filter techniques, and the most probable number test. This quiz delves into how each method works and the significance of results expressed as colony forming units. Test your knowledge of these essential microbiological techniques!