Module 2

Chapter 7: Microbial Metabolism

Metabolism and The Role of Enzymes

• Metabolism: Pertains to all chemical reactions and physical workings of the cell

• Anabolism:

  • Any process that results in the synthesis of cell molecules and structures

  • A building process that forms larger macromolecules from smaller ones

  • Requires the input of energy

• Catabolism:

  • Breaks the bones of larger molecules into smaller molecules

  • Releases energy

• Metabolism performs these functions:

  • Assembles smaller molecules into larger macromolecules - ATP (energy) is utilized to form bonds (anabolism)

  • Degrades macromolecules into smaller molecules, a process that yields energy (catabolism)

  • Stores energy in the form of ATP (adenosine triphosphate)

Enzymes: Catalyzing the Chemical Reactions of Life

• Enzymes

  • Chemical reactions of life cannot proceed without them

  • Are catalysts that increase the rate of chemical reactions without becoming part of the products or being consumed in the reactions

Concept Check - 1

Anabolism and catabolism constitute the sum of reactions in the cell known as _____.

A. Binary fission

B. Metabolism

C. Energy balance

D. Mutation

How Do Enzymes Work?

• Reactants are converted into products by bond formation or bond breakage

  • Substrate: Reactant molecules acted on by an enzyme

• Speed up the rate of reactions without increasing the temperature

• Much larger than substrates

• Have a unique active site on the enzyme that fits only the substrate

Checklist of Enzyme Characteristics

• Most composed of protein; may require cofactors

• Act as organic catalysts to speed up the rate of cellular reactions

• Have unique characteristics such as shape, specificity, and function

• Enable metabolic reactions to proceed at a speed compatible with life

• Have an active site for target molecules called substrates

• Are much larger than their substrates

• Associate closely with substrates but do not become integrated into the reaction products

• Are not used up or permanently changed by the reaction

• Can be recycled, thus functioning in extremely low concentrations

• Are greatly affected by temperature and pH

• Can be regulated by feedback and genetic mechanism

Conjugated Enzyme Structure

• Metallic cofactor

• Coenzyme

• Coenzyme and Metallic cofactor

  • All of them made apoenzymes

Cofactors: Supporting the Work of Enzymes

• The need of microorganisms for trace elements arises from their roles as cofactors for enzymes

  • Iron, copper, magnesium, manganese, zinc, cobalt, selenium, etc.

• Participate in precise functions between the enzyme and substrate

  • Help bring the active site and substrate close together

• Coenzymes

  • Organic compounds that work in conjunction with an apoenzyme

  • The general function is to remove a chemical group from one substrate molecule and add it to another substrate molecule

  • Carry and transfer hydrogen atoms, electrons, carbon dioxide, and amino groups

  • Many derive from vitamins

Enzyme-Substrate Interactions

• A temporary enzyme-substrate union must occur at the active site

  • Fit is so specific that it is described as a “lock-and-key” fit

• The bond formed between the substrate and enzyme are weak adn easily reversible

• Products are formed

• Enzyme is free to interact with another substrate

Concept Check - 2

What is true about enzymes? They _________

A. are proteins

B. speed up reactions

C. are specific to substrates

D. are not consumers in reactions

E. all

Concept Check - 3

Micronutrients such as Cu, Fe, Mn, and Zn, are essential for microbes because they function as ________

A. coenzymes

B. cofactors

C. active site

D. apoenzymes

Concept Check - 4

Apoenzymes are _______ part of a conjugated enzyme

A. protein

B. protein + inorganic adjuncts

C. protein + organic adjuncts

D. Substrates

E. all

Metabolic Pathways

• Often occur in a multistep series or pathway, with each step catalyzed by an enzyme

• Products of one reaction are often the reactant (substrate) for the next, forming a linear chain or reaction

• Many pathways have branches that provide alternate methods for nutrient processing

• Others have a cyclic form, in which the starting molecule is regenerates to initiate another turn of the cycle

• Do not stand alone; interconnected and merge at many sites

ATP: Metabolic Money

• Three-part molecule

  • Nitrogen base (adenine)

  • 5-carbon sugar (ribose)

  • Chain of three phosphate groups bonded to ribose

  • Phosphate groups are bulky and carry negative charges, causing a strain between the last two phosphates

  • The removal of the terminal phosphate releases energy

The Metabolic Role of ATP

• ATP utilization and replenishment is an ongoing cycle

  • Energy released during ATP hydrolysis powers biosynthesis

  • Activates individual subunits before they are enzymatically linked together

• Used to prepare molecules for catabolism

• When ATP is utilized, the terminal phosphate is removed to release energy, and ADP is formed

  • Input of energy is required to replenish ATP

• In heterotrophs, catabolic pathways provide the energy infusion that generates the high-energy phosphate to form ATP from ADP

Getting Materials and Energy

• Nutrient processing in bacteria is extremely varied, but in most cases the nutrient is glucose

• Aerobic respiration

  • Conversions of glucose CO2 with the production of energy (ATP)

  • Utilizes glycosis, the Kerbs cycle, and the electron transport chain (ETC)

  • Relies on free oxygen as the final electron and hydrogen acceptor

  • Characteristics of many bacteria, fungi, protozoa/animals

• Anaerobic respiration

  • Used by strictly anaerobic organisms and those who are unable to metabolize without oxygen

  • Involves glycolysis, the Kerbs cycle, and the electron transport chain

  • Uses NO 3-, SO4 2-, CO3 3-, and other oxidized compounds as final electron acceptors

• Fermentation

  • Incomplete oxidation of glucose

  • Oxygen is not required

  • Organic compounds are final electron acceptors

Concept Check - 5

The energy molecule ATP is made up of

A. Proteins

B. Adenine

C. Ribose + Adenine + 3P

D. Adenine + 3P

E. All

Concept Check - 6

Aerobic & Anaerobic - both types of cellular respiration - involve

A. Glycolysis

B. Krebs Cycle

C. Electron Transport System

D. All above

E. Only B, C

Glycolysis

• Turns glucose into pyruvate, which yields energy in the pathways

The Krebs Cycle: A Carbon and Energy Wheel

• Takes place in the cytoplasm of bacteria and in the mitochondrial matrix of eukaryotes

  • A cyclical metabolic pathway begins with acetyl CoA

  • Transfer the energy stored in acetyl CoA to NAD+ and FAD by reducing them

  • NADH and FADH2 carry electrons to the electron transport chain

  • 2 ATPs are produced for each molecule of glucose through phosphorylation

Concept Check - 7

Glycolysis is the first step in catabolism that generates

A. Pyruvate

B. ATP

C. NADH

D. All above

E. Only B, C

Concept Check - 8

The Krebs Cycle starts with converting ______ into ______ that runs the wheel of releasing energy molecules.

A. Pyruvate, Acetyl CoA

B. ATP, NADH

C. NADH, Pyruvate

D. Acetyl, CoA, Pyruvate

The Respiratory Chain: Electron Transport

• A chain of special redox carriers that receives reduced carriers (NADH, FADH2) generated by glycolysis and the Krebs cycle

  • Passes them in a sequential and orderly fashion from one to the next

  • Highly energetic

  • Allows the transport of hydrogen ions outside of the membrane

  • In the final step of the process, oxygen accepts electrons and hydrogen, forming water.

• Primary compounds in the electron transport chain:

  • NADH dehydrogenase

  • Flavoproteins

  • Coenzyme Q (ubiquinone)

  • Cytochromes

• Cytochromes protein-metal ion complex involves accepting electrons and donating them to the next carrier in the series

The Respiratory (Electron Transport) Chain

• Reduced carriers (NADH, FADH) transfer electrons and H+ to first electron carrier in the chain: NADH dehydrogenase

  • These are then sequentially transferred to the four to six carriers with more positive reduction potentials.

    • The carriers are called cytochromes

• Extracellular space gets more + charged, acidic with protons and thus creates proton motive force

• Aerobic respiration yields a maximum of 3 ATPs per oxidized NADH and 2 ATPs per oxidized FADH

• Anaerobic respiration yields per NADH and FADH.

The Terminal Step

• Aerobic respiration

  • Catalyzed by cytochrome oxidase

  • Adapted to receive electrons from cytochrome c, pick up hydrogens from solution, and react with oxygen to form

  • 2H+ + 2e- + ½ 02 → H2O

After Pyretic Acid III: Fermentation

• Fermentation

  • The incomplete oxidation of glucose or other carbohydrates in the absence of oxygen

  • Uses organic compounds, such as pyretic Acid, as the terminal electron acceptors

  • Yields a small amount of ATP

  • Used by organisms that do not have an electron transport chain

Products of Fermentation in Microorganisms

• Alcoholic beverages:

  • Ethanol

  • CO2

• Solvents:

  • Acetone

  • Butanol

• Organic acids:

  • Lactic acid

  • Acetic acid

• Vitamins, antibiotics, and hormones

Concept Check - 9

Which of the following pathways produces maximum ATPs?

A. Aerobic respiration

B. Anaerobic respiration

C. Fermentation

D. Glycolysis

Concept Check - 10

In aerobic respiration, electrons are finally accepted by _______, which functions as an electron sink

A. Pyruvic acid

B. Oxygen molecule

C. Nitrate

D. Sulfate

Concept Check - 11

Electron Transport Chain is missing in

A. Aerobic organisms

B. Anaerobic organisms

C. Fermenting organisms

D. All

Chapter 8: Introduction to Genes

Introduction to Genetics and Genes

• Genetics: The study of inheritance, or heredity of living things;

• Explores;

  • The transmission of biological properties (traits) from parent to offspring

  • The structure and function of the genetic material

  • How genetic material changes

The Nature of Genetic Material

• Genome: The sum total of the genetic material of an organism

  • Most of the genome exists in the form of chromosomes

  • Some appear as plasmids or in mitochondria/chloroplasts of eukaryotes

  • Genome of cells composed entirely of DNA

  • Genome of viruses can contain either DNA or RNA

• Chromosome: Discrete cellular structure composed of a neatly packaged DNA molecule

• Prokaryotic chromosomes

  • DNA condensed into a packet utilizing histone-like proteins

  • Single, circular chromosome

• Eukaryotic chromosomes

  • DNA wound around histone protein

  • Located in the nucleus

  • Diploid (paired set) or haploid (single set)

  • Linear appearance; Nos. variable

• Gene

  • Classical genetics: The fundamental unit of heredity responsible for a given trait in an organism

  • A certain segment of DNA that contains the necessary code to make a protein or RNA molecule

• Three categories of genes:

  • Structural genes: Code for proteins

  • Genes that code for RNA machinery used in protein production

  • Regulatory genes: Control gene expression

• Genotype: The sum of all alleles (alternative forms of a gene); an organism’s distinctive genetic makeup

• Phenotype

  • The expression of certain traits (structures or functions)

  • All organisms contain more genes in their genotypes than are manifested in the phenotype at any given time

Concept Check - 1

A gene present in the cell generally refers to a segment of

A. DNA carrying no codes/function

B. RNA carrying codes/function

C. DNA carrying codes/ functions

D. RNA carrying no codes/functions

Locations and Forms of the Genome in Cells and Viruses

• Eukaryotic (composite) cells

  • Plasmids (in some fungi and protozoa)

  • Chromosomes

  • Nucleus

  • Chroloroplast

  • Mitochondrion

• Prokaryote cells

  • Chromosome

  • Plasmids

• Viruses

  • DNA

  • RNA

The Size and Packaging of Genomes

• Genome of E. Coli

  • Single chromosome (1 mm long) contains 4,288 genes

  • 1 mm if unwound and stretched linearly:

    • 1000 times longer than the cell (1 micron)

  • Takes up 1/3 to ½ of the cell volume

  • DNA is supercoiled and packaged tightly inside the cell

• Human genome

  • 22,000 genes on 46 chromosomes

  • If all 46 chromosomes were unraveled and laid out end to end, it would measure 6 feet long

Concept Check - 2

The sum total of genetic materials present in a cell constitutes a

A. Gene

B. Genome

C. Genotype

D. Phenotype

The Significance of DNA Structure

• How can an apparently complex genetic language be based on just four nitrogen base “letters?”

• Consider a segment of DNA that is 1,000 nucleotides long

  • 4^1,000 different sequences possible

  • This equals 1.5 × 10^602, a number that provides nearly endless degrees of variation

Concept Check - 3

Each nucleotide in DNA is made up of

A. Ribose sugar + base

B. Deoxyribose sugar + base + P

C. Ribose sugar + base + P

D. Deoxyribose sugar + P

DNA Replication: How DNA Duplicates?

• Overall replication process is semiconservative replication

  • DNA double helix unzips

  • Each old strand serves as a template for a new strand, thus producing two complete daughter molecules

  • Each daughter DNA molecule has one old and another new strand

DNA Synthesis Progresses From The Origin of Replication

• Origin of replication = a particular DNA sequence (unidirectional/bidirectional)

  • Prokaryotes →. Only one origin (AT-rich segment) is attached to the cell membrane

  • Eukaryotes →. Many origins

How does DNA synthesis proceed?

• Initiator proteins (start untangling and unzipping DNA helix)

• DNA polymerase starts synthesis at replication forks

• Replication fork allows the synthesis of:

  • Leading strand DNA

    • Lagging strand DNA

DNA Replication

1. The origin of replication is a short sequence rich in adenine and thymine bases held together by only two hydrogen bonds rather than three. Because the origin of replication is AT-rich, less energy is required to separate the two strands than would be necessary if the origin were rich in guanine and cytosine

   1. During replication topoisomerase unwinds the DNA helix, giving access to helices (unzipping enzymes) to bind to the dsDNA at the origin

2. Helicases break the hydrogen bonds holding the two strands together, resulting in two separate strands.

3. Single-stranded binding proteins keep the strands apart

4. DNA polymerase III adds nucleotides following the template pattern

   1. Because DNA polymerase is correctly oriented for synthesis only in the 5’ to 3’ direction of the new molecule (blue) strand, only one strand, called the leading strand, can be synthesized as a continuous, complete strand. The strand lagging strand. On this strand, the polymerase adds nucleotides a few at a time in the direction away from the fork (5’ to 3’). As the fork opens up a bit, the next segment is synthesized backward to the point of the previous segment, a process repeated until synthesis is complete. In this way, the DNA polymer can synthesize the two new strands simultaneously. This manner of synthesis produces one strand containing short fragments of DNA (100 to 1,000 bases long) called Okazaki fragments. These fragments are attached to the growing end of the lagging strand by another enzyme called DNA ligase.

5. In all cases, the initiation of DNA synthesis requires “jump-starting” with a length of RNA manufactured by RNA primase. DNA polymerase can then add DNA nucleotides to that primer sequence. The primer sequence is later removed through enzymatic action.

   1. The RNA primer is not seen on the leading strand as it appeared closer to the origin, which is not pictured. RNA primers are required at the beginning of each fragment of DNA synthesized on the lagging strand.

Completion of Chromosome Replication in Bacteria

• DNA is always synthesized in the 5’ to 3’ direction

• Continuous DNA synthesis is the Leading strand by DNA polymerase III

• DNA synthesis in small fragments (Okazaki fragments) at a lagging strand

• “Jumpstarting” of DNA synthesis occurs with RNA primer made by RNA primase

• DNA polymerase then extends the promised segments, forming Okazaki fragments

• DNA ligase joins small fragments at the Lagging strand

Enzymes and their Functions

• Helicase

  • Unzipping the DNA helix

• Primase

  • Synthesizing an RNA primer

• DNA polymerase III

  • Adding bases to the new DNA chain; proofreading the chain for mistakes

• DNA polymerase I

  • Removing primer, closing gaps, repairing mismatches

• Ligase

  • Final binding of nicks in DNA during synthesis and repair

• Topoisomerase I and II

  • Supercoiling and untangling

Concept Check - 4

What is the meaning of the semiconservative mode of DNA replication? the synthesis of

A. one new + one old strands

B. Both new strands

C. Both old strands

D. Only one strand

Concept Check - 5

What is true?

A. DNA always synthesizes in 5’ to 3’ direction

B. Okazaki fragments synthesized at lagging strand

C. Continuous DNA synthesis at Leading strand

D.” Jumpstarting” of DNA synthesis occurs with RNA primer made by RNA primase

E. All of the above

Gene Expression: Protein Synthesis

• The central dogma identifies the flow of genetic information

  • DNA → RNA → Protein

  • Transcription - Translation

Transcription and Translation

• Transcription: DNA master codes transferred into mRNA codes

• Translation: Transcribed RNA used to produce protein

• Exceptions to this pattern

  • RNA viruses convert RNA to other RNA

  • Retroviruses convert RNA to DNA

What Transcription needs:

• RNA polymerase:

  • Large, complex enzyme that directs the conversion of DNA into RNA

• Template strand:

  • Only one strand of DNA that contains meaningful instructions for the synthesis of a functioning polypeptide

Transcription copies genetic information into RNA

• Direction of movement ←

• RNA polymerase

• DNA template strand

• mRNA transcript

• Inactive DNA strand

Three Types of RNA Result From Transcription

• Messenger RNA (mRNA):

  • Carried message from the gene

• Ribosomal RNA (rRNA):

  • A platform for protein synthesis

• Transfer RNA (tRNA):

  • Transfer amino acids from cytosol to ribosomes

Transfer RNA (rRNA)

• A copy of a specific region of DNA

  • It differs from mRNA

• The size is 75-95 nucleotides long

• Contain sequences of bases that form hydrogen

• Bends back by itself into several hairpin loops

  • Gives it a secondary cloverleaf structure that folds even further into a complex, three-dimensional helix

• An adaptor that converts RNA language into protein language

• Designates the specificity of the tRNA and complements mRNA’s codons

• A binding site for the amino acid that is specific for tRNA’s anticodon

Messenger RNA (mRNA)

• A transcript (copy) of a structural gene or genes in the DNA

• The message of this transcribed strand is later read as a series of codons

• The length of the mRNA varies from about 100 nucleotides to several thousand

• Carries the sequence that will dictate the eventual amino acid sequence of the protein

The Master Genetic Code:

• The triplet message (the language of DNA base) encoded in mRNA

• Triplet code & 4 types of bases → 64 codons

• Genetic code is redundant (i.e. more than one code for each amino acid)

• Start codon → AUG

• Stop codons → UGA, UAG, UAA

• Codes are universal for all organisms

Concept Check - 1

Which is the key enzyme in initiating transcription?

A. DNA polymerase

B. RNA polymerase

C. Ligase

D. All

Who cracked the Genetic Code?

1. M. W. Nirenberg

2. H. G. Khorona

3. R. W. Holley

• These scientists were awarded the 1968 Noble Prize in Physiology & Medicine

Translation is the process of making protein

• In bacteria, translation of mRNA starts while transcription is still occurring

3-STEPS:

1. Chain initiation

2. Chain elongation

3. Chain termination

• Translation involves:

  • Codons (mRNA)

  • Anticodons (tRNA)

  • Ribosomes (rRNA) - Polyribosomes

• Translation: All of the elements needed to synthesize a protein are brought together on the ribosomes

Concept Check - 2

In the expression of a gene, mRNA codons stand (code) for

1. Lactose

2. Glucose

3. Lipid

4. Amino acids

Concept Check - 3

Which of the following types of RNA contains hairpin loops and is involved in carrying amino acids to the ribosome during translation?

A. Messenger RNA

B. Regulatory RNA

C. Ribosomal RNA

D. Transfer RNA

E. Primer RNA

Concept Check - 4

Anticodons are associated with

1. tRNA

2. DNA

3. rRNA

4. mRNA

Concept Check - 5

Synthesis of mRNA on DNA template is called

1. Translation

2. Replication

3. Transcription

4. DNA synthesis

Mutations: Changes in the Genetic Code

• Genetic change by mutation is the driving force of evolution

• Any change to the nucleotide sequence. in the genome is a mutation

  • Gross at the level of a phenotype

  • Cryptic at the level of a base pair

• Wild type: A microorganism that exhibits a natural, non-mutates characteristic

• Mutant strain: Shows variance in one or more of the following

  • Morphology

  • Nutritional characteristics

  • Genetic control mechanisms

  • Resistance to chemicals

  • Temperature preference

Causes of Mutations

• Spontaneous mutation: A random change in the DNA arising from errors in replication

• Induced mutations: Result from exposure to known mutagens (physical or chemical agents) that disrupt DNA

  • They are deliberate mutations resulting from a mutagen

    • Radiation: UV light, X-rays

    • Chemicals: Nitrous acid

• Point mutations: Small mutations that affect only a single base on a gene

  • Base-pair substitution

  • Base-pair deletion or insertion

• Silent mutation: Alters the abuse but does not change the amino acid

• Missense mutation: Leads to a different amino acid

  • Created a faulty, nonfunctional, or less functional protein

• Nonsense mutation:

  • Changes a normal codon stop into a stop codon

  • almost always results in a nonfunctional protein

• Back mutation: Occurs when a gene that mutates back to its original base composition

• Frameshift mutation

  • Occurs when one or more bases are inserted into or deleted from a DNA strand

  • The reading frame of mRNA has been changed

  • Leads to a more dramatic effect - nearly always results in a nonfunctional protein

Concept Check - 1

• A wild-type organism may differ from its mutant strain in the following

  1. Morphology

  2. Biochemical properties

  3. Nutritional characteristics

  4. Resistance to chemicals

  5. All

  6. Only 1,2

Repair of Mutations

DNA has a proofreading mechanism to repair mistakes in replication

• The cell has additional systems for finding and repairing DNA that has been damaged

• Excision repair

  • Enzymes break the bonds between the bases

  • A different enzyme removes the defective bases one at a time

  • The remaining gap is filled in by DNA polymerase I and ligase

Excision Repair of Mutation by Enzymes

1. The first enzyme complex recognizes one or several incorrect bases and removes them

2. The second complex (DNA polymerase I and ligase) places the correct bases and seals the gaps

3. Repaired DNA

Positive and Negative Effects of Mutations

• Mutations are permanent and heritable

• Most spontaneous mutations are not beneficial

• A small number create variant strains that more readily adapt, survive, and reproduce

• When the environment changes, some mutants will be equipped to survive in the new environment

• Acquired drug resistance is a clear model for this type of selection and adaption

Concept Check - 2

What are the physical mutagens?

A. X-rays

B. UV light

C. Temperature

D. A and B

E. None

Concept Check - 3

A type of mutation in which a normal codon changes into a stop codon resulting in a nonfunctional protein. This is a type of _____ mutation.

A. Nonsense

B. Missense

C. Frameshift

D. Silent

Concept Check - 4

Which of the following types of mutations will have the most devastating effect on a cell?

A. Point mutation

B. Frameshift mutation

C. Missense mutation

D. Silent mutation

E. Back mutation

Concept Check - 5

What are the enzymes involved in the excision repair of DNA?

A. RNA polymerase

B. DNA polymerase I

C. Ligase

D. B, and C

E. None

Chapter 9: Physical and Chemical Control of Microbes

Controlling Microorganisms

• Limiting body exposure to pathogens

  • A monumental challenge

• The methods of microbial control:

  • Sterilization

  • Disinfection

  • Decontamination

    • Also called sanitization

  • Antisepsis

Sterilization

• Definition

  • Process that destroys or removes all viable microorganisms (including viruses)

• Key points

  • Generally reserved for inanimate objects

  • Common uses:

    • Surgical instruments

    • Syringes

    • Commercially packaged food

• Examples of Agents:

  • Heat (Autoclave)

  • Sterilants (chemical agents capable of destroying spores)

Disinfection

• Definition

  • Physical process or a chemical agent to destroy vegetative pathogens. but not bacterial endospores

• Key points

  • Normally used on inanimate objects

  • Common uses:

    • Boiling food utensils

    • Applying 5% bleach solution to an examining table

    • Immersing thermometers in an iodine solution between uses

• Examples of Agents

  • Soaps

  • Detergents

  • heat (boiling)

Decontamination/Sanitization

• Definition

  • Cleansing technique that mechanically removes microbes to reduce contamination to safe levels

• Key points

  • Important to restaurants/dairies/breweries, Soiled utensils/containers

  • Common uses:

    • Cooking utensils

    • Dishes

    • Bottles

    • Cans must be sanitized for reuse

• Examples of Agents

  • Soaps

  • Detergents

  • Commercial

  • Dishwashers

Antisepsis/Degermatation

• Definition

  • Reduces the number of microbes on the human skin

  • A form of decontamination on living tissues

• Key points

  • Involves scrubbing the skin (mechanical friction) or immersing

  • It is in chemicals (or both)

• Examples of Agents

  • Alcohol or Iodine wash

  • Surgical hand scrubs

Microbial Control Methods

• Physical agents:

  • Heat

    • Dry

      • Incineration - Sterilization

      • Dry oven - Sterilization

    • Moist

      • Steam under pressure - Sterilization

      • Boiling water, hot water, pasteurization - Disinfection

  • Radiation

    • Ionizing

      • X-ray, cathode, gamma - Sterilization

    • Non-ionizing

      • UV - Disinfection

• Chemical agents:

  • Gases - Sterilization, Disinfection

  • Liquids

    • On animate objects - Antisepsis

    • On inanimate objects - Disinfection, Sterilization

• Mechanical Removal Agents

  • Air - Decontamination

  • Liquids - Sterilization

• Terms & Definitions

  • Disinfection: The destruction or removal of vegetative pathogens but not bacterial endospores. Usually used only on inanimate objects

  • Sterilization: The complete removal or destruction of all viable microorganisms. Uses on inanimate objects

  • Antisepsis/Degermatation: Chemicals applies to body surfaces to destroy or inhibit vegetative pathogens

  • Decontamination/Sanitization: The mechanical removal of most microbes

Concept Check - 1

The destruction or removal of vegetative pathogens but nor bacterial endospores. Usually used only on inanimate objects.

A. Disinfection

B. Antisepsis

C. Sterilization

D. Sanitization

Relative Resistance of Microbial Forms

• Primary targets — organisms capable of causing infection or spoilage in the environment or on the human body

• The targeted population: mixture of microbes with extreme differneces in resistance and harmfulness

• Bacterial endospores considered the most resistant microbial entities

• The goal of any sterilization process is the destruction of bacterial endospores

  • Any process that kills endospores will invariably kill all less resistant microbial forms

Relative Resistance of Different Microbial Types to Microbial Control Agents

Less resistant → More resistant

• Enveloped viruses

• Most gram-positive bacteria

• Nonenveloped viruses

• Fungi and fungal spores

• Most gram-negative bacteria

• Protozoan trophozoites

• Protozoan cysts

• Staphylococcus and Pseudomonas

• Mycobacterium

• Bacteria endospores

• Prions

Concept Check - 2

The complete removal or destruction of all viable microorganisms. Used on inanimate objects.

A. Disinfection

B. Antisepsis

C. Sterilization

D. Sanitization

Differentiation: Agents vs. Processes

• Sepsis: The growth of microorganisms in the blood and other tissues

• Aseptic

  • Practices that prevent the entry of infectious agents into sterile tissues and thus prevent infections

  • Aseptic techniques: Practiced in healthcare; range from sterile methods to antisepsis

• Antiseptics: Chemical agents applies directly to exposed body surfaces (skin and mucous membranes), wounds, and surgical incisions to prevent vegetative pathogens

  • Preparing the skin before surgical incisions with iodine compounds

  • Swabbing an open root canal with hydrogen peroxide (H202)

  • Ordinary hand washing with a germicidal soap

• Stasis and static mean “to stand still”

• Bacteriostatic: Chemical agents that prevent the growth of bacteria on tissues or on objects in the environment

• Fungistatic: Chemicals that inhibit fungal growth

• Antiseptics and drugs often have mircrobiostatic effects because microbicidal compounds can be toxic to human cells

• Even -cidal agent doesn’t necessarily result in sterilization, depending on how it is used

Concept Check - 3

The use of iodine compounds to prepare the skin for surgery is known as

A. Disinfection

B. Antisepsis

C. Sterilization

D. Sanitization

E. Degermatation

Practical Matters in Microbial Control

• Does the application require sterilization, or is disinfection adequate?

• Is the item to be reused or permanently discarded?

• If it will be reused, can the item withstand heat, pressure, radiation, or chemicals?

• Is the control method appropriate for a given application? In the case of a chemical, will it leave an undesirable residue?

• Will the agent penetrate to the necessary extent?

• Is the method cost- and labor-efficient and is it safe?

What is Microbial Death?

• Death: Permanent termination of an organism’s vital processes

  • Microbes have no conspicuous vital processes, there fore death is difficult to determine

  • Permanent loss of reproductive capability, even under optimum growth condition:

    • The accepted microbiological definition of death

Factors Affecting Death Rate

• The number of microbes

  • Higher load of contaminants takes longer to destroy

• The nature of the microorganisms in the population

  • Target population is usually a mixture of bacteria, fungi, spores, and viruses

• Temperature and pH of the environment

• The concentration (dose, intensity) of the agent

  • UV radiation is most effective at 260 nm

  • Most disinfectants are more active at higher concentrations

• The mode of action of the agent

  • How does it kill or inhibit the microorganism?

• The presence of solvents, interfering organic matter, and inhibitors

  • Saliva, blood, and feces can inhibit the action of disinfectants and even the action of heat

Modes of Action of Antimicrobial Agents

• Antimicrobials have a range of cellular targets

  • Least selective agents tend to be effective against the widest range of microbes (heat and radiation)

  • Selective agents such as drugs

  • Target only a single cellular component

• Cellular targets of physical and chemical agents

  • Cell wall

  • Cellular synthetic processes

  • Cell membrane

  • Proteins

Concept Check - 4

Permanent loss of reproductive capability of an organisms, even under optimum growth condition, is called

A. Stasis

B. Growth inhibition

C. Death

D. Sanitization

E. Degermatation

Actions of Various Physical and Chemical Agents Upon the Cell

• Cell wall

  • Effects of Agents:

    • Chemical agents can damage the cell wall by:

      • Blocking its synthesis

      • Digesting the cell wall

  • Examples of Agents Used

    • Chemicals

    • Detergents

    • Alcohol

• Cell membrane

  • Effects of Agents

    • Agents physically bind to lipid layer of the cell membrane, opening up the cell membrane and allowing injurious chemicals to enter the cell and important ions to exit the cell

  • Examples of Agents Used

    • Detergents

• Cellular Synthesis

  • Effects of Agents

    • Agents can interrupt the synthesis of proteins via the ribosomes, inhibiting proteins needed for growth and metabolism and preventing multiplication

    • Agents can also change the genetic codes (mutation)

  • Examples of Agents Used

    • Formaldehyde

    • Radiation

    • Ethylene oxide

• Proteins

  • Effects of Agents

    • Some agents are capable of denaturing proteins (breaking of protein bonds, which results in breakdown of the protein structure)

  • Examples of Agents Used

    • Moist heat

    • Alcohol

    • Phenolics

Concept Check - 5

Selective antimicrobial agents like drugs target

A. Multiple cellular components

B. Indigenous flora

C. Only protozoans

D. Single cellular component

Methods of Physical Control: Heat

• Elevated temperatures are microbicidal

• Lower temperatures are microbiostatic

• Moist heat: Hot water, boiling water, or steam

  • Between 60 celsius and 135 celsius

• Dry heat: Hot air or an open flame

  • Ranges from 160 celsius to thousands of degrees

Mode of Action and Relative Effectiveness of Heat

• Moist heat

  • Operates at lower temperatures and shorter exposure times to achieve the same effectiveness as dry heat

  • Microbicidal effect is the coagulation and denaturation of proteins

• Dry heat

  • Dehydrates the cell, removing water necessary for metabolic reactions

  • Denatures proteins

  • At very high temperatures, oxidizes cells, burning them to ashes

Temperature and Time to Sterilize

Moist heat

• 121 - 15 min

• 125 - 10 min

• 134 - 3 min

Dry heat

• 121 - 600 min

• 140 - 180 min

• 160 - 120 min

• 170 - 60 min

Moist Heat Methods

• Boiling

  • Useful in the home for disinfection of water, materials in babies, food and utensils, bedding, and clothing from the sickroom

• Pasteurization: Disinfection of beverages

  • Milk, wine, beer, other beverages

• Tenderization (Non pressured Steam)

  • Selected substances that cannot withstand the high temperature of the autoclave can be subjected to intermittent sterilization

  • Usually 3 rounds of 15-20 min of boiling in three days in a row'; also effective in spore removal