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Microbial Nutrition and Growth Culturing Microorganisms Microbial Genetics Controlling Microbial Growth

CHNOPS

The source of common essential nutrients are:
Carbon
Hydrogen
Nitrogen
Oxygen
Phosphorus
Sulfur

Organic Nutrients

-Contain at least some combination of carbon AND hydrogen atoms
-Natural organic molecules are usually products of living things
-Simple to large polymers

Heterotrophs

Organisms that must obtain carbon in an organic form

Inoculation

Introductory of a sample into a container of media to produce a culture of observable growth

Isolation

Separating one species from another

Incubation

Under conditions that allow growth
Temperature controlled chamber (incubator)
-Microbe multiplies and produces macroscopically observable growth

Inspection

Observe colony morphology, staining properties, and bacterial morphology - macroscopic and microscopic
-Pure Culture - grows only single known species of microorganisms
-Mixed Cultures - hold 2 or more identified species or microorganisms
-Contaminated Culture - once pure or mixed culture that has unwanted microbes growing

Media

Providing nutrients in the laboratory: Classed according to 3 properties
-Physical State - liquid, semilsolid, solid
-Chemical Composition - Chemically defined and complex
-Functional Type - General purpose, enriched, selective, differential, anaerobic, transport, assay, enumeration

Synthetic (defined) Media

Contains pure organic and inorganic compounds in an exact chemical formula

Complex Media

Contains at least one ingredient that is not chemically definable

The 5 I's

Inoculation
Isolation
Incubation
Inspection
Identification

General Purpose Media

Grows a broad range of microbes, usually nonsynthetic

Enriched Media

Contains complex, organic substances such as blood, serum, hemoglobin, or special growth factors required by fastidious microbes

Capnophile

Organisms that survive in high concentrations of carbon dioxide

Barophile

Organisms that survive at high pressures

Microaerophile

Requires oxygen to survive, but requires environments containing low oxygen levels

Genetics

The study of heredity; The study of what genes are, how they carry information, how information is expressed, and how genetics are replicated

The science of genetics explores

Transmission of biological traits from parent to offspring
Expression and variation of those traits
Structure and function of genetic material
How this material changes

Gene

A segment of DNA that encodes a functional product, usually a protein

Genotype

The genes of an organism

Phenotype

Expression of the genes

Chromosomes

DNA complexed with protein
-Bacterial chromosomes are a single circular loop
-Eukaryotic chromosomes are multiple and linear

Structure of Prokaryotic Genomes: Prokaryotic Chromosomes

Main portion of DNA, along with associated proteins and RNA
Prokaryotic cells are haploid (single chromosome copy)
Typical chromosome is circular molecule of DNA associated with proteins in the nucleoid of the cell

Bacterial Genome: Plasmids

-Small Molecules of DNA that replicate independently of the chromosome
---Can be high copy number or low copy number
---Plasmids can be lost during cell division, if only one daughter cell gets all the copies of the plasmid - (when this happens, it is said to be "cured" of the plasmid
-Not essential for normal metabolism, growth, or reproduction
-Can confer survival advantages

Types of Plasmids

Fertility Factors
Resistance Factors
Bacteriocin Factors
Virulence Plasmids
Dissimilation Plasmids

Fertility Factors

Allow bacterium to undergo conjugation (bacterial sex) to exchange genes with other bacteria

Resistance Factors

Carry genes that make the bacteria resistant to antibiotics
-Enzymes that break down antibiotic
-Mutations that prevent antibiotic from entering the cell

Bacteriocin Factors

-Chemicals that kill other bacteria - one way to beat the competition for resources is to kill them
-Some antibiotics are bacteriocins from bacteria

Virulence Plasmids

-Make the bacterium better able to evade a host's immune system and cause disease
-An example would be a plasmid that allows the bacterium to make a capsule

Dissimilation Plasmids

Contain genes that allow bacteria to digest unconventional food sources

Eukaryotic Genome Structure: Nuclear Chromosomes

-Typically have more than once chromosome per cell
-Chromosomes are linear and sequestered within nucleus
-Eukaryotic cells are often diploid (two chromosome copies)

Eukaryotic Genome Structure: Extranuclear DNA of Eukaryotes

-DNA molecules of mitochondria and chloroplasts
--Resemble chromosomes of prokaryotes
--Only code for about 5% of RNA and proteins
-Some fungi an protozoa carry plasmids

Gene Function: Genotype

Set of genes in the genome

Gene Function: Phenotype

Physical features and functional traits of the organism

Gene Function: Transfer of genetic information

-Transcription - information in DNA copied as RNA
-Translation - Polypeptides synthesized from RNA
-Central Dogma of genetics
---DNA transcribed to RNA
---RNA translated to form polypeptides

Mutations of Genes

-Change in the nucleotide base sequence of a genome
Rare event
-Can be deleterious, beneficial, or silent (has no impact whatsoever)
-Rarely leads to a protein that improves ability of organism to survive
-Wild Type (wild strain) - a natural, nonmutated characteristic

Mutant Strain

An organisms that has a mutation, showing variance in morphology, nutritional characteristics, genetic control mechanisms, resistance to chemicals, etc

Spontaneous Mutations

Random change in the DNA due to errors in the replication that occur without known cause

Induced Mutations

result from exposure to known mutagens, physical (primarily radiation) or chemical agents that interact with DNA in a disruptive manner

Types of Gene Mutations: Point Mutations

- most common
-One base pair is affected
---Insertions - one base pair added to the sequence of the DNA
---Deletions - One base pair removed from the sequence of the DNA
---Substitutions - a base is changed in the sequence of the DNA

Types of Gene Mutations: Frameshift Mutatiions

-Nucleotide triplets after the mutation are displaced
-Insertions and deletions of one or more nucleotide pairs

Point Mutations: Base Pair Substitutions

-Point Mutation
-Change in one base pair
-Does not change amino acid in protein

Point Mutations: Missense Mutation

-Change in one base
-Result in change in amino acid

Point Mutations: Nonsense Mutations

-Results in a nonsense codon
--a codon that should encode an amino acid is converted to a stop codon

Mutagens: Radiation

-Ionizing radiation
--Ionizing radiation (X rays and gamma rays) causes the formation of ions that can react with nucleotides and the deoxyribose-phosphate backbone
--Nucleotide excision repairs mutations
-Nonionizing radiation - ultraviolet radiation causes thymine dimers

Mutagens: Chemical Mutagens

-Nucleotide analogs
--Disrupt DNA and RNA replication
-Nucleotide-altering chemicals - Result in base pair substitutions and missense mutations
-Frameshift Mutagens - result in nonsense mutations

Frequency of Gene Mutations

-Mutations are rare events
--Otherwise organisms could not effectively reproduce
-Mutagens increase the mutation rate by a factor of 10 to 1000 times

Positive Effects of Mutations

-Organisms with mutations that are beneficial in their environment can readily adapt, survive, and reproduce
--these mutations are the basis of change in populations
-Any change that confers an advantage during selection pressure will be retained by the population

Negative Effects of Mutations

-Mutations leading to nonfunctional proteins are harmful, possibly fatal

Genetic Recombination and Transfer

Recombination -Exchange of nucleotide sequences often mediated by homologous sequences
Recombinants -Cells with DNA molecules that contain new nucleotide sequences

Genetic Recombination and Transfer: Vertical Gene Transfer

-Organisms replicate their genomes and provide copies to descendants
--Binary Fission
--Budding
--Fragmentation
--Sexual Reproduction

Genetic Recombination and Transfer: Horizontal Gene Transfer Among Prokaryotes

-Horizontal Gene Transfer -Donor cell contributes part of genome to recipient cell
-Three types
--Transformation
--Transduction
--Bacterial Conjugation

Transformation

-One of conclusive pieces of proof that DNA is genetic material
-Cells that take kup DNA are competent
--results from alterations in cell wall and cytoplasmic membrane that allow DNA to enter cell

Transduction

-Generalized transduction - transducing phage carries random DNA segment from donor to recipient
-Specialized transduction - only certain donor DNA sequences are transferred

Genome

of a cell or virus is its entire genetic complement, including its:
-Genes -specific sequences of nucleotides that code for polypeptides or RNA molecules
-Nucleotide sequences that connect genes to one another

Autoclave

An instrument that sterilizes by exposing instruments to steam under pressure

Halogens

-Iodine-containing organic compound found in such antiseptics as Betadine
-Elements such as iodine, chlorine and bromine are the basis for many effective antimicrobial agents

Aldhydes

Disinfectants that have the chemical group -CHO, which reacts with and damages both proteins and nucleic acids

Proteins

heavy metal and oxidizing agent disinfectants damage _____, interfering with microbial metabolism

Endospores

Sterilization procedures generally focus on inactivating or eliminating bacterial _____

Less Than

The amount of time needed to sterilize materials using moist heat is _____ the time needed to sterilize using dry heat

BSL-4

The containment level appropriate when handling highly contagious deadly microbes

Thermal Death Point

The lowest temperature that kills all cells in a broth in 10 minutes is known as

Pasteurization

The process of heating milk or fruit juice to levels that kill any pathogenic microbes present

Osmotic pressure

The use of high levels of salt or sugar in the preservation of foods relies on the phenomenon of

Less Effectively

Ultraviolet light penetrates materials _____ than gamma rays

Triclosan

A phenolic antimicrobial compound that has been incorporated into consumer items such as garbage bags and diapers

Lyophilization

The process of freeze-drying microbes to preserve them

Kill microbes

The suffix -cide means to...

Inhibits growth but does not completely destroy

The suffic -static means to...

False

The duration of time required for sterilization of liquid media is always the same when using moist heat sterilization, regardless of the quantity of media being sterilized

"waterless" hand gel containing 70% isopropanol

-The alcohol of the waterless hand cleaner is a germicide that disrupts cytoplasmic membranes and denatures proteins.
-Innefective against bacterial endospores or fungal spores
-limited effect on non-evneloped viruses
-Alcohol evaporates quickly, so the germicidal effect is short term
-Intermediate level disinfectant & not an effective degermer

Hand Soap Containing Triclosan

-Contains a phenolic
-Damages cytoplasmic membranes and denatures proteins
-Effective on the same range of microbes as the alcohol. -Phenolics are intermediate-level disinfectants that persist on surfaces for long periods of time, providing extended disinfection
- If used with running water and the hands are vigorously rubbed, the hand soap can be an effective degermer

Wipe that lists benzethonium chloride (a synthetic quaternary ammonium salt)

-Synthetic "quats" (quaternary ammonium compounds) disrupt cell membranes
-Effective against fungi, enveloped viruses, and most bacteria, -Ineffective against nonenveloped viruses and endospores. -Quats are low-level disinfectants that are germicidal for some microbes

The cellular structures or processes that can be targets of antimicrobial agents

-1) A variety of cellular molecules and structures that make up the cell: cytoplasmic membrane and the cell wall, either of which can be damaged by various types of antimicrobial agents
-2) Proteins and nucleic acids - denatured proteins, mutated DNA molecules

The In-Use Test

-A method of evaluating antimicrobial agents such as disinfectants or antiseptics
-Specimens are collected from objects that need to be disinfected, both before and after the disinfecting agent is applied.
-Then the specimens are inoculated into growth media, and the presence or absence of growth is an indicator of the effectiveness of the agent
-Makes use of microbes that are actually found in the area of concern, and it gives a "real-life" picture of how the antimicrobial agent will work in that situation.

A broth containing 1 million bacterial cells is treated with an antimicrobial agent that kills 90% of the cells in 1 minute. What is the minimum amount of time it will take before all the cells in the broth are dead?

7 minutes
Because 90% of the cells present are killed each minute, the decline of the bacterial population gives a straight line when the experimental values are plotted on a semilogarithmic graph. The last cell will be killed at some point between 6 and 7 minutes of exposure.

An instrument coming into contact with only the skin of an immunocompromised patient should be treated with which of the following?

intermediate-level germicide
An item that would need to be treated with only a low-level germicide in the case of a healthy patient would need to be sterilized with a high-level germicide if used with an immunocompromised patient.

Which of the following could be used to sterilize an object?
ethelene oxide, 100% alcohol, orthophenlyphenol, or silver nitrate

ethylene oxide
100% alcohol, orthophenylphenol, and silver nitrate all have antimicrobial activity to varying degrees, but they do not sterilize.

Which radiation types are classed as non-ionizing radiation?

UV light, visible light, infrared radiation, and radio waves are all types of nonionizing radiation

Antimicrobial agents that damage the viral envelope

Prevent attachment of the virus to its target cell.
Viral envelopes allow the viruses to attach to cells, so that they can replicate.

osmotic pressure

Damage to the cell wall will adversely effect a bacterial cell by enhancing the damaging effects of which of the following?

Catalase in human tissues neutralizes it

Hydrogen peroxide does not make a good antiseptic for open wounds because

odophors and chloramines are similar in that they are both halogen-containing compounds that

slowly release their active ingredients
Iodophors and chloramines are so effective because they release their active ingredients more slowly than do the more elemental forms of these halogens.

exponential growth

Microbial death is defined as the permanent loss of reproductive ability due to the importance of which of the following microbial activities?

organic materials can interfere with these procedures.

Objects to be disinfected or sterilized need to be cleaned first because

Least to Most resistant

Enveloped Viruses
Gram-Negative Bacteria
Mycobacteria
Prions

Phenol Coefficient

is the LEAST useful indicator of an antimicrobial agent's effectiveness

increased pressure

the most important part of sterilization using an autoclave

degerming

A nurse preparing a section of skin for an injection is an example of

antiseptic

An antimicrobial chemical used on the skin is usually called

increased time and higher temperature

Compared to moist heat methods, dry heat needs what in order to sterilize effectively?

low-level germicides

-Items that come into contact with only the skin of a patient can be disinfected with
-kills vegetative bacteria, fungi, protozoa, and some viruses

ultrahigh-temperature sterilization

-Milk that can be stored for months at room temperature has been treated by which method?
-Ultrahigh-temperature sterilization kills all forms of living microbes: UHT sterilized milk can be kept at room temperature indefinitely without spoilage, although flavor changes may occur.

quats

Low-level disinfectant

the Kelsey-Sykes capacity test

-the preferred method of determining the efficacy of an antimicrobial chemical in the European Union?
-it reveals the minimum amount of time required for a particular disinfectant to be effective.

5 hours

particular species of bacteria has a generation time of 20 minutes. A microbiologist inoculates a broth with 1,000 cells of this bacterium. Under ideal growth conditions, what is the minimum amount of time required before the broth contains more than 10,000,000 cells?

pH

Fermented foods such as sauerkraut or pickles are kept from spoiling by the effects of which of _____

30 minutes

If it takes 2 hours for a population of bacteria to grow from 100 cells to 1,600 cells, what is the generation time of this bacterial species?

a fungus

A microbe is growing on Sabouraud dextrose agar. Which of the following is this microbe likely to be?

hydroxyl radical: catalase

Which of the following toxic forms of oxygen is improperly paired with the detoxifying enzyme or molecule?

Why would a particular microbe need to be cultured inside an animal?

-Standard laboratory culture techniques have proven inadequate.
-Animals can be used to culture pathogenic microbes when the growth conditions for the microbe are unknown or unachievable under normal laboratory conditions.

Blood agar is an example of which of the following? I. a differential medium II. a complex medium III. a selective medium ____________________.

-I and II
-Blood agar is a complex medium, because of the large amount of specialized nutrients it contains. It is also a differential medium because different bacterial species produce various hemolysis patterns as they grow on it.

osmotic pressure

-Crenation can result from a change in which of the following?
-Cells placed in hypertonic solutions will lose water, resulting in the shriveling of the cytoplasm known as crenation.

obligate anaerobes

Media containing sodium thioglycollate are used for the growth of _____

log

-Microbes in the __________ phase of the microbial growth curve are most susceptible to antimicrobial drugs
-The rapid growth of cells during the log phase means they are more susceptible to antimicrobial drugs during this phase than in any other phase; many of these drugs interfere with the metabolism of the cell or with the synthesis of important cell structures.

log phase

Microbial growth is at its maximum rate during which of the following phases of the microbial growth curve?

proteins and nucleotides.

Nitrogen is a growth-limiting nutrient because cells need it to manufacture _____

membranes become rigid and fragile

-Which of the following does NOT happen at the maximum growth temperature of a microorganism?
-At temperatures at or near the maximum growth temperature, the lipids of the cell become extremely fluid, which makes the cell's membranes too fluid to contain the cell or its organelles. Additionally hydrogen bonds break at high temperatures, causing proteins to become denatured.

a photoheterotroph

-does NOT use an inorganic source of carbon as its sole source
-Autotrophs use inorganic sources of carbon, whereas heterotrophs use organic sources of carbon.

redox reactions; organic

A chemoheterotroph obtains its energy from __________ and its carbon from __________ sources.

A colony-forming unit is a

single cell or group of related cells that gives rise to a colony

turbidity

A spectrophotometer is useful for which of the following methods of measuring microbial growth?

streak-plate method

The most commonly used isolation technique in microbiology laboratories is the ____

membrane filtration

Which of the following techniques is most useful when the density of a microbial population is very small?

40°C

Which of the following would be the maximum growth temperature for a mesophile?

nucleotide analog

5-bromouracil mimics the chemical structure of thymine, making it a(n) _

Frameshift Mutation

A mutation that changes the reading frame of a gene

Euchromatin

Loosely packed regions of eukaryotic chromosomes

Transduction

Transfer of DNA between cells by viruses

Nucleosomes

DNA wraps around histones (in eukaryotes) forming ___ which clump together to form chromatin

Heterochromatin

Tightly packed chromatin is called _____ and is associated with areas of the DNA that is not active

Euchromatic

_____ is loosely packed and associated with areas of the DNA that are being expressed

Nonsense Mutation

If a mutation causes a codon that should encode an amino acid to be converted to a stop codon, it's called a _____

Mutagens

Factors that increase the rate of mutation in an organism to higher than the normal background mutation rate

Diploid

a cell that has two copies of each chromosome

Haploid

a cell that has one copy of each chromosome

Point mutation

results in the change of a single base pair

Genotype

The set of genes an organism carries in its genome

Phenotype

The physical manifestation of the expression of genes

Nucleoid

Although they lack a nucleus, the DNA of prokaryotic cells tends to be localized in one region known as the ___

Missense Mutation

A mutation to a gene causes a valine amino acid to be converted to a phenylalanine amino acid

Plasmids

Circular, extra-chromosomal pieces of DNA found in prokaryotes

False

Plasmids are only found in prokaryotes

True

Eucharyotes and archaea both have histones in their chromatin
Bacteria have non-histone proteins in their chromatin

True

Bacteriocins, chemical compounds that kill bacteria, produced by a particular species of bacteria, will kill bacteria of the same or similar species

False

All mutations are detrimental to an organism

False

Only eukaryotes have more than one chromosome

False

In eukaryotic cells, all the DNA within the cell is located in the nucleus

Types of factors (genes) that are carried on plasmids.

fertility factors, resistance factors, bacteriocin factors, virulence factors.

Horizontal Gene Transfer vs. Vertical Gene Transfer

-Vertical gene transfer involves the passing of genetic material from one generation to the next through sexual or asexual reproduction.
-Horizontal gene transfer involves an existing organism gaining new genetic material and does not result in the formation of progeny.

A point mutation can be completely harmless, or it can result in the death of a cell or organism. Explain why these types of mutations can have such varying effects.

A point mutation is a single base change in the nucleotide sequence of a cell's genome. The effects of a point mutation can depend on its location. Point mutations in noncoding regions of the genome are usually harmless. Even in coding regions, point mutations can be harmless if they result in silent mutations. Silent mutations preserve the sense of the amino acid code because of the concept of "wobble," in which two codons can code for the same amino acid by varying only at the third base of the codon. If the point mutation has occurred at this third base, then the amino acid sequence of the protein will remain unchanged. Point mutations occurring at the first or second base of the codon are almost always much more serious because they change the codon to a completely different amino acid. This type of point mutation is known as a missense mutation. (The only exception to this occurs when the new amino acid is chemically similar to the previous amino acid, in which case the missense mutation usually causes little or no change in the overall structure or function of the protein.) Finally, one of the most serious types of point mutations is a nonsense mutation in which the codon has been changed to a stop codon. These types of mutations result in the abnormal termination of a protein sequence. In all such cases, if the protein affected by the mutation is an enzyme or some other vital protein required for proper cellular function, then the cell and/or organism may die as a result of these relatively simple mutations.

How are bacterial conjugation, transformation, and transduction similar? How do these processes differ?

-Bacterial conjugation, transformation, and transduction are all forms of horizontal gene transfer in bacteria. They increase the genetic diversity of prokaryotic species that only reproduce by asexual reproduction.
-Bacterial conjugation is the exchange of genetic material between two bacterial cells. This process involves the presence of fertility factors in one of the bacteria. The bacterium carrying the fertility factor will form a sex pilus which will connect it to a bacterium that does not contain the fertility factor. The pillus will pull the bacteria close together and genetic information will be exchanged between the two bacteria.
-Transformation refers to the uptake of naked DNA by a bacteria cell. Competent bacteria can take up naked DNA from their environment, this DNA typically comes from cells that have died and released their cellular contents into the environment. Can also be used in laboratory settings to introduce plasmids to bacteria.
-Transduction involves horizontal gene transfer via a viral intermediate. In transduction bacteriophage viruses accidentally package bacterial DNA into their viral capsules instead of viral DNA. When the virus carrying bacterial DNA infects a new host it injects a piece of bacterial DNA into the host cell, the DNA can be incorporated into the host genome by recombination.

List the three types of chemical mutagens and briefly describe how they interact with DNA and how they cause mutations.

- Nucleotide analogs
--replace the normal base (A, T, G, or C) they are similar to -during DNA replication
--cause base pair mismatches in subsequent rounds of -DNA replication
-- Nucleotide altering chemicals
--- physically alter existing nucleotides into a different form
---result in point mutations in subsequent rounds of DNA -replication
--Frameshift mutagens
---insert in between bases on a strand of DNA creating a bulge
---DNA polymerase cannot read the DNA that has the bulge and may insert of delete base pairs when replicating the DNA

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