Classifications of Microorganisms

Question 1

What is Taxonomy?

Answer 1

Taxonomy: The science of classifying organisms according to how similar they are to other organisms
-These similarities are due to relatedness which reflects evolutionary relationships

Question 2

What is Phylogeny? What does it comprise of?

Answer 2

Phylogeny (or systematics); is the study of the evolutionally history of organisms
Phylogeny uses the tools of taxonomy to clarify the evolution of organisms, as well as their interrelationships
-Grouping organisms according to common properties implies that they Evolved from a common ancestor; each species retaining some of the ancestor’s characteristics

Question 3

Differentiate between Taxonomy and Phylogeny.

Answer 3

**Taxonomy proudces a HIERARCHY; while Phylogeny produces a PHYLOGENETIC TREE or Cladogram

Question 4

Where does some information for phylogenetic relationships come from? Do Fossils of bacteria exist?

Answer 4

In higher organisms, some of the information used to determine phylogenetic relationships comes from FOSSILS
Yes, fossils of bacteria exist (they are Rare)

Question 5

What are examples of Fossilized microorganisms?

Answer 5

Fossilized microorganims
-Their structures are NOT readily fossilized
Some exceptions (examples)
* A. Fossilized colonies of a marine protist from the White cliffs of Dover, England
B. Fossilized bacterial communities. Limestone buildups of cyanobacterial mats (and other microbes) that form in shallow water called stromatolites (as old as 3.5 billion years)

-since fossil evidence is Not available for most prokaryotes, their phylogeny must be based on other evidence

Question 6

What Is a stromatolite?

Answer 6

Stromatolite: rock structures formed by layers of bacteria like, cyanobacteria and other microbes (algae?)

Question 7

What is the Three-Domain System and how is it classified? What evidence supports this classification? Where did mitochondria and chloroplast originate from?

Answer 7

The Three-Domain system: composed of 3 domains, Bacteria, Archaea and Eukarya
** In 1990, Carl Woese Elevated the three cell types to a level ABOVE the kingdom
-There was no history of classification for domain. The domains were classified **Based on similarities on rRNA **
-Mitochondria and Chloroplasts originated from Bacteria

Question 8

Why were Archaea initially thought to be the most primitive?

Answer 8

Archaea initially thought to be most primitive b/c they were EXTREMOPHILES

Question 9

Which organisms are a part of each domain?

Answer 9

Bacteria Doman: Mitochondria, proteobacteria, cyanobacteria, chloroplasts, gram-positive bacteria, and Thermotoga
Archaea domain: methanogens, hyperthermophiles, extreme halophiles
Eukarya Domain: animals, fungi, amebae, slime molds, plants, ciliates, green algae, dinoflagellates, diatoms, euglenozoa, gird (protozoan parasites), mitosomes (mitochondria degenerates)

Question 10

Which gene are transferred horizontally in phylogenetic tree of the Three domains ? what does analysis of genome show?

Answer 10

Genera near the origin or “root” of the evolutionary tree appear to have transferred genes horizontally.
-The analysis of completed genomes show that each domain showers genes with other domains
For example, the bacterium Thermatoga acquired 1/4 of its genes form an archaeon.

Question 11

Discuss the cellular characteristics of the 3 domains; including cell type, cell wall, membrane lipids, first Amon acid in protein synthesis, antibiotic sensitive, tRNA loop, common arm of tRNA

Answer 11

Cellular characteristics of the 3 domains:
Archaea:
Cell type: Prokaryotic
Cell Wall: Varies in composition; contains NO Peptidoglycan
Membrane lipids: composed of Branched carbon chains attached to glycerol by Ether linkage
First amino acid in synthesis: Methionine
***Antibiotic Sensitivity: NO
**tRNA loop: Lacking
**Common Arm of tRNA: Lacking
ex: Sulfolobus sp.
Bacteria:
Cell Type: Prokaryotic
Cell wall: contains peptidoglycan
Membrane Lipids: composed of Straight carbon chains attached to glycerol by Ester linkage
First Amino Acid in Protein synthesis; Formylmethoinine
**Antibiotic Sensitivity: YES
**rRNA loop: PRESENT
**Common Arm of tRNA; PRESENT
ex: E.coli
Eukarya:
Cell type: Eukaryotic
Cell wall: Varies in composition; contains carbohydrates
Membrane Lipids: composed of Straight carbon chains attached to glycerol by Ester linkage
First Amino acid in protein synthesis: Methionine
Antibiotic sensitivity: NO
rRNA loop; Lacking
Common Arm of tRNA: PRESENT
ex. Amoeba sp
**the rRNA loop is only in Bacteria

Question 12

Describe the features of the Hairpin loop seen in Bacteria? What structure does Bacteria and eukaryotes have in common
Compare and contrast the membrane lipids of bacteria, Archaea, and eukarya domains.

Answer 12

Hairpin loop
-seen in bacteria (ex; E. coli)
contains 16s rRNA
- Bacteria and Eukaryotes BOTH have common arm of TRNA
-that are sequences of bases in tRNA, guanine-thymine-pseudoridine-Cytoisne guanine
-Both Bacteria and Eukarya have membrane lipids that are composed of STRAIGHT carbon chains attached to Glycerol by ESTER linkages
-Meanwhile, Archaea have membrane lipids that are composed of BRANCHED carbon chains attached to glycerol by ETHER linkages

Question 13

What are the different parts of tRNA structure

Answer 13

tRNA structure:
4 loops
Top loop is Acceptor arm (contains ester bond
left side is Common arm (Guanine-Thymine-pseudouridine-cytosine-guanine)
-Bottom: Anticodon arm
right side: D arm

Question 14

Explain the endosymbiotic theory and recognize the features shared by prokaryotic cells and mitochondria/chloroplasts to support this theory.

Answer 14

The nuclear envelope and ER of eukaryotes may have formed by invagination of the plasma membrane
The Endosymbitoic theory: proposes that eukaryotic cells evolved from prokaryotic cells living inside a host prokaryote
(chloroplasts and mitochondria originated from Bacteria and were engulfed by Eukaryotes)

(according to theory, ancestral eukaryotes engulfed aerobic bacteria that eventually evolved into mitochondria; while the early eukaryotes engulfed cyanobacteria that evolved to become chloroplasts (photosynthetic bacteria)

Question 15

Describe the similarities between prokaryotic cells and eukaryotic mitochondria and chloroplasts that support Endosymbiotic theory

Answer 15

The best comparison is between mitochondria and bacteria (since they are very SIMILAR )
- Prokaryotic Cell and Eukaryotic Organelles (mitochondria and chloroplasts) both have Circular DNA, have 70s Ribosomes and Grow by BINARY FISSION
-Prokaryotic cell Is unique in that it has Histones (in archaea), and the first amino acid in protein synthesis is formylmethionine (bacteria); Methionine (Archaea)
-They also can have one circular DNA; some two circular; some linear DNA
-Eukaryotic cell has LINEAR DNA, and has Histones. Their first Amino acid in protein synthesis is. Methionine and they have 80 s Ribosomes. They grow by Mitosis
-Eukaryotic organelles (mito and chroloplasts) share the same features as bacteria (prokaryotic cell), Except that mitochondria and chloroplasts have NO histones and have Formylmethionine as first amino acid in protein synthesis (like bacteria)
-hence this supports endosymbiotic theory (mito/chlorplasts evolved from bacteria)

Question 16

Discuss the unique characteristics of present-day bacteria Gemmata obscuriglobus and Cyanophora paradoxa

Answer 16

organism that create present day evidence that plasma membrane infolding could produce a nuclear envelope
-Gemmata obscuriglobus: A bacterium (based on rRNA sequence) that has a nuclear envelope surrounding its nucleic
- Cyanophora paradoxa: a protist harboring a photosynthetic endosymbiont (cyanelle) resembling modern day cyanobacteria (Ex; peptidoglycan cell wall and genome organization)
(the cyanelle is closer to a cynaobacteria than a chloroplast)
Both host and endosymbiont require each other survive
Cells also contain mitochondria

Question 17

Explain why scientific names are used, list and apply the naming rules, outline the process for naming newly discovered prokaryote, and renaming based on new information

Answer 17

Scientific Nomenclature is used since
Common Names:
-Vary with geography
2 different organisms are called Spanish Moss (neither actually moss)
-Buttercup (East Texas) name for Oenothera speciosa
whereas Buttercup (elsewhere) misnamed for 250 Ranunculus sp
-Vary with languages
-multiple names for the same organism

Question 18

What are naming rules of organisms using Scientific nomenclature.

Answer 18

Scientific Nomenclature:
-Binomial Nomenclature (genus _ specific epithet)
-Used worldwide
-Names taken from Latin or Greek and may be descriptive or honor a scientist
-Genus name is capitalized and a noun
-Species name is lower case and usually an adjective
-Both names are underlined or italicized
ex: Homo sapiens (man/wise)
providing universal names for organism facilitates research, scholarship and communication

Question 19

Give the scientific names, including their name, Source of genus name, and source of specific epithet for the bacteria: Klebsiella pneumonia, Pfiesteria piscida, Salmonella typhimurium, Streptococcus pyogenes, Penicillum chyrsogenum, and trypanosome Cruz

Answer 19

Scientific Binom Source of genus Source of Epithet
1. Klebsiella Honors Edwin Klebs The disease
pneumonia
2. Pfiesteria Honors Lois Pfiester Disease in fish
piscicida
3. Salmonella Honors Daniel Salmon Stupor (typh-)
typhimurium in mice
4.Streptococcus Chains of cells (strepto) Forms pus
pyogenes (pyo-)
5. Penicillium Tuftlike (penicilli-) produces yellow
chrysogenum (chryso) pigment
6. Trypanosoma Corkscrew-like Honors Oswaldo
cruzi (trypano-, borer; Cruz
soma- body)

Question 20

Describe the process for naming an newly discovered prokaryote

Answer 20

Process for naming a newly discovered prokaryote:
International Committee on Systematics of Prokaryotes
1) **Establish a name based upon Bacteriological Code (rules for naming) **
2) Publish description and evidence of classification in the “International Journal of Systematic and Evolutionary Biology
3) Once published, name is incorporated into the reference book “Bergey’s Manual of Systematics of Archaea and Bacteria, First Edition”

Question 21

What are the 3 common names for Saccharomyces cerevisiae?

Answer 21

Saccharomyces cerevisiae:
1. Baker’s Yeast
2. Brewer’s Yeast
3. Budding Yeast

Question 22

Explain the purpose and distinguish between the two Bergey’s manuals. What are the major groups in Bergey’s 9th edition manual .

Answer 22

Bergey’s Manual and Systematics of Archaea and Bacteria
*First Edition: Published online since April 2015. This online manual replaces and expands upon the first two paper editions of Bergey’s Manual of Systematic Bacteriology (first two editions 1984-2012)
*It is organized by phylum, provides Phylogenetic information on bacteria and archaea, based on rRNA sequencing
Bergey’s Manual of Determinative Bacteriology. 9th Edition
-Book organization is STRICTLY PHENOTYPIC, with not attempt to offer a natural higher classification
-It is utilitarian and is **intended to aid in the identification of bacteria **
-Bacteria are divided into 35 easily recognized phenotypic groups most useful for diagnostic purposes
Group 1: The spirochetes .
Group 4: Gram-negative aerobic/microaerophillic rods and cocci
Group 8: Anaerobic gram-negative cocci
Group 11: Oxygenic photooptic bacteria

An explosion in the isolation and description of new taxa prokaryotes over past 5 years has resulted in description of more than one hundred new genera and six hundred new species each year (first edition)

Question 23

List the major taxa and differentiate between eukaryotic and prokaryotic species. Define strain

Answer 23

Taxonomic Hierarchy:
Domain
Kingdom
Phylum
Class
Order
Family
Genus
Species
(Dear King Phillip Came Over For Good Soup)
Eukaryotic species: Group of closely related organisms that breed among themselves
Prokaryotic species
-A population of cells with similar characteristics

Question 24

What is a strain?

Answer 24

Strain: A variant of a organism.
Strain can be identified by numbers, letters, or name following the species designation (ex: E.coli K12 or S. cerevisiae S288C)

Question 25

Which of the Three Domains do NOT have a Kingdom?

Answer 25

Archaea and Bacteria do NOT have Kingdom in their taxonomy.
They start with Phylum

Question 26

List the major characteristics used to differentiate the kingdoms of Eukarya

Answer 26

Kingdoms in the Domain of Eukarya:
-Animalia: Multicellular; no cell walls; chemoheterotrophic
- Plantae: Multicellular; cellulose cell walls; usually Photoautotrophic
- Fungi; chemoheterotrophic; unicellular or multicellular; cell walls of chitin; can develop from spores or hyphal fragments
-Protista: A catch-all kingdom for eukaryotic organism that do NOT fit other kingdoms
-Grwouped into CLADES based on rRNA
**There is NOT absolute agreement regrind the number of eukaryotic kingdoms

Question 27

Explain how organisms can be renamed based on new information

Answer 27

In 1984, DNA hybridization studies indicated that Streptococcus faecalis and Streptococcus faecium were only distantly related to other streptococcal species. New genus called Enterococcus was formed and species renamed E. faecalis, and E. faecium
-In 2001, based on DNA hybridization and rRNA studies, some Chlamydia species were placed in a new genus Chlamodphila
To avoid confusion when an organism is renamed, the Old name is often written in parenthesis following the new name
ex: Burkholderia (pseudomonas) pseudomalleli

Question 28

Compare and contrast classification and indentification

Answer 28

Classification: Placing organisms in groups of related species. Lists of characteristics known organisms
-estimated that only 1% of prokaryotes have been discovered
Identification: Matching characteristics of an “unknown” organism to lists of known organisms
-clinical lab identification.

Question 29

What are the morphological characteristics that can be used to identify bacteria ?

Answer 29

Morphological Characteristics:
Bacteria:
Hundreds of bacterial species are small rods or cocci
Although, shape tells little about phylogenetic relationship, it can still be useful for identification
-Endospores, inclusions, pigments or flagella differences can also be helpful
Eukaryotes
-larger size and intracellular structures may help classifications

Question 30

Explain how biochemical tests are used to identify bacteria

Answer 30

Biochemical tests:
widely used to identify/differentiate even closely related bacteria
-Can also be used to provide insight into an organism’s niche in the ecosystem (ex; Nitrogen fixation/plant association)
-Tests previously discussed include:
catalase, urea utilization, acid production via sugar fermentation of sugars, CO2 production, H2S production, hemolysin, antibiotic resistance, O2 tolerance

Question 31

Describe how morphological characteristics, biochemical tests and differential staining are used to identify bacteria

Answer 31

Early identification methods:
Morphological Characteristics: limited Usefulness
Differential staining: Gram staining, acid-fast staining
Biochemical Tests: Determines the presence of bacterial enzymes

Question 32

Explain the use of a clinical microbiology lab report form

Answer 32

Bacterial identification facilities physician diagnosis
Clinical Microbiology Lab report form (used to identify an unknown bacterium using microbiology tools and methods)
-A genitourinary sample is examined for sexually transmitted disease
-The physician swabs the appropriate surface, then inserts the swab into a tube of transport media (Not nutritive)- PREVENT overgrowth and prolong viability of fastidious pathogens
This report form contains gram stain type, source of specimen, tests requested and patients information, lab and date/time test occured.

Question 33

What is the information obtained from microbes used for? know how to use a dichotomous key to identify an unknown bacterium

Answer 33

Information obtained about microbes is used to identify and classify them.
Methods of using this information include:
Dichotomous keys- used for identification
Cladograms: show evolutionary relationships

Question 34

Explain how the Enterotubue II is used

Answer 34

Numerical/Rapid identification is only used for ENTERICS
-it is a rapid, multi step test used to identify unknown bacteria of family Enterbacteriaceae
-Enterotube II from Beckton Dickinson
Process:
1. one tube contains media for 15 biochemical tests is inoculated with an unknown enteric bacterium
2. After incubation, the tube is observed for results
3. The value for each POSITIVE test is Circled, and the numbers from each group of tests are added to give the ID value
4. Comparing the resultant ID value with a computerized listing shows that the organism in the tube is proteus mirabilis
(Id value: 21007; atypical results ornithine- )
-Numbers are assigned to avoid ambiguity
-Mutations and plasmids can produce strains with different characteristics

Question 35

What are the NEWER methods used to identify bacteria?

Answer 35

Newer Methods to identify bacteria
-Serology
-Phage Typing
-Fatty acid profiles
-Flow cytometry
-DNA base composition
-DNA fingerprinting
-Nucleic Acid Amplification Tests (NAATS)
-Nucleic acid hybridization
-Ribotyping and rRNA sequencing
-FISH

Question 36

Explain how Serology can be used to identify an unknown bacterium.
REVIEW

Answer 36

Serology- combines a known antiserum with an unknown bacteria .
antiserum: antibodies in blood serum
antiserums are produced by injecting whole bacteria or specific part of protein of bacteria (like E.coli) into an animal (blood then drawn and centrifuged? )
Can also differentiate among strains within species

Question 37

What are strains that express different antigens called? What is the rationale for using serological testing to determine the relatedness of two microbes?

Answer 37

Serotypes, servers or biovars
Serological testing is used to determine relatedness of two microbes because if both reacted with the same serum, they must be sharing proteins
REVIEW

Question 38

What are the different forms of Serology?

Answer 38

Serology
1. Slide Agglutination test
2. ELISA
3. The Western Blot

Question 39

How is the Slide agglutination test performed?

Answer 39

Serology 1: Slide Agglutination test
-Unknown bacteria are placed in a drop of saline on several slides
-Different antibodies are added to each sample
-bacteria will agglutinate (clump) when mixed with antibodies produced against them.
(**Drop will appear to contain many clumps)

Question 40

What occurs in ELISA? What are the Advantages of ELISA. Explain how an ELISA test is done

Answer 40

Serology 2: ELISA
-Enzyme linked immunosorbent assay to identify bacteria
-Unknown bacteria + known antibodies
-Antibodies are linked to enzyme
Enzyme substrate
Advantages: Fast and readable with a computer scanner
other Elisa variations can be used.
Virtually all microbrial species have one antigen that is unique

Question 41

Explain how an ELISA test is done. Differentiate between Direct Assay and Indirect Assay for ELISA

Answer 41

Elisa Test
-A technician uses a micropipette to add sample to a microplane for ELISA
-ELISA results are then read using spectrophotometer
other Elisa variations can be used.
Direct Assay: uses 1 antibody the primary antibody is conjugated to fluorphore and enzyme
Indirect assay uses 2 antibodies: the unconjugated primary antibody will be bound to antigen, while second antibody is conjugated to flurophore and is directed against primary antibody.

Question 42

What occurs in the process of Western Blotting? What can be revealed with this method.

Answer 42

Serology 3: Western Blot
used to detect specific protein in blood sample or tissue
Lyme disease is often diagnosed by Western blotting
HIV infections are often confirmed by Western blotting
Capillary action for western blots is rarely used in practice

Question 43

Explain the process of The Western Blot

Answer 43

Process:
1. if Lyme dies is suspected in patient: Electrophoresis is sued to separate Borrelia burgoderfi proteins. Proteins move at different rates based on their charge an size when the gel is exposed to electric current
2. The bands are transferred to a nitrocellulose filter by blotting. each band consists of many molecules of a particular protein (antigen). The bands are not visible at this point
3. The proteins (antigens) are positioned on the filter exactly as they were on gel. The filter is then washed with patient’s serum followed by antihuman antibodies tagged with an enzyme. The patient antibodies that combine with their specific antigen are visible (shown in red) when the enzyme’s substrate is added
4. The test is read. If the tagged antibodies stick to the filter, evidence of the presence of the microorganism in question (B. burgodrferi in this case) has been found in patient’s serum.

Question 44

How does Electroblotting work?

Answer 44

Electroblotting: technique used to transfer proteins or nucleic acids from polyacrimide onto a membrane from using PVDF or nitrocellulose. membrane
-**if proteins are hydrophobic, use PVDF membrane

Question 45

What is Phage typing? How does this process work?

Answer 45

Phage typing: measures a bacterium’s sensitivity to different phages
Phage are highly specialized and usually only infect members of a particular species, or even specific strains within a species
Phage sensitivity is shown by PLAQUE formation on a lawn of bacterial cells.
-Surgical and food. associated infections can be traced by this method
-There are at least 2,523 serovars of Salmonella that can be subdivided into 49 phage types

Question 46

What is the process of phage typing?

Answer 46

process:
1. An inoculum of Staphylococcus aureus is spread over the surface of agar medium
2. Different bacteriophage suspensions are deposited in a fixed pattern
3. After incubation, different patterns of lysis are seen with different strains of S. aureus

Question 47

Explain what fatty acid profiles are and why they are important

Answer 47

Bacteria synthesize a wide variety of fatty acids that are constant for particular species
-Gas chromatographs can separate cellular fatty acids
-The fatty acid profile is matched to a library of 700+ bacterial species representing over 180 genera
-These profiles, called FAME (fatty acid methyl ester) are widely used in clinical and public health labs
(triglyceride + methanol use NAOH as base to form FAME and glycerol)

can see differences in FAME profile in cell membrane lysates (by looking at length of peaks in graph; bordetella pertussis have higher peaks than bordetella bronchiseptica)

Question 48

What is Flow cytometry? How does it work?

Answer 48

Flow Cytometry: measure the characteristics of single cells suspended in a flowing saline stream
**A focused laser scatters light from these cells and can trigger fluorescence
-Scattered light provides information about cell size, shape, density, and surface
-Natural fluorescence of some species can be used for detection
-Cells can be selectively stained with antibody coupled to fluorescent dye
- Can be used to identify bacteria in a sample without culturing (ex: Listeria in milk)
**Also can tell you cell number in sample **
(laser light source, shine red: fluorescence is emitted from stain cells; shine green: forward and side scattered light from all cells are detected)

Question 49

What is DNA composition? What does a local area of abnormal GC content in chromosome sometimes indicate?

Answer 49

DNA BAse composition: observe Guanine + cytosine moles % (GC%)
- Greater than 10% difference in GC ration between organisms Indicates they are likely NOT related
-Identical GC ratios do NOT necessarily indicate relatedness
-GC % ranges from 10-90%
-S. cerevisiae (chromomsomes 98%; GC; mitochondria 18%). E. coli (51%), Frankia alni (73%)
-it suggests it comes from another source

Question 50

What is DNA fingerprinting? Why is it useful?

Answer 50

DNA fingerprinting: Identification method based the analysis of DNA restriction fragments resolved by gel electrophoresis
-a comparison of the number and size of restriction fragments produced provide information about their similarities and differences
Can be used to determine an infection source
(Short tandem repeats give differences in restriction fragments for closely related strains)
-Although determining the entire sequence of an organism’s DNA is possible, it is NOT practical
(**DNA from several different bacteria is digested with same restriction enzyme)

(can be used in forensics to identify an individual by analyzing differences in DNA by individuals )

Question 51

What is NAATS? How does this process work?

Answer 51

NAAT (Nucleic acid Amplification Test)
-PCR test used to detect microbes (easy way to ID microbes)
-Includes PCR, RT-PCR and real-time PCR
-Allow direct identification with species-specific primers
-also employed to increase DNA amounts to levels that can be tested by gel electrophoresis
Rapid identification that does NOT require a pure culture of the organism.
-1992, Tropheryma whipplei, causative agent of the gastrointestinal and nervous system disorder called Whippie’s disease (cannot be cultured)
-PCR is used to diagnose Zika virus in pregnant thought to have been exposed to the virus
-Strain comparisons don’t require culture
Ex: Compare E. coli from contaminated juice and sick patient

Question 52

Describe what occurs in nucleic acid hybridization

Answer 52

Nucleic Acid Hybridization is used to determine relatedness (between two organisms)
process:
1. heat to separate strands
2. Combine single strands of DNA (both organisms A and Organism B DNA)
3. cool to allow renaturation of double-stranded DNA
4. Determine the degree of hybridization
-If there is complete hybridization: organism are IDENTICAL (if strands completely stuck together)
-if their is partial hybridization: organism are RELATED (if some parts open, some stuck together)
if there is NO hybridization (strands separate); organisms are UNRELATED
**RNA-RNA and RNA-DNA hybridization are also possible

Question 53

What is FISH? How does this process work?

Answer 53

FISH: Fluorescence in situ Hybridization uses fluorescent DNA or RNA probe to detect and locate specific DNA sequences on a chromosome
- can be used to determine the identity, abundance and relative activity of microbes in environment
-microbes do NOT need to be cultured
-Future uses include detecting bacteria in drinking water or patients without the 24 hour plus wait required for culturing.

Question 54

Describe process of FlSH?

Answer 54

Process of Fluorescence in situ Hybridization
1. FIXATION of cells in mixed population (put holes in membrane to allow DNA probe enter cell)
2. Attach cells to glass slide and permeabilized (for hybridization)
3. expose cells to probe, which is a piece of purified DNA that is tagged with fluorescent dye
4. The fluorescently labeled probe enters and reacts with target ribosome (16s RNA) and then finds and binds to its matching sequence within the set of chromosomes
(hybridized cells)
5. Use FISH Analysis to locate the DNA sequence that the probe is attached to, using Fluorescence microscopy and Flow cytometry

Question 55

Explain what diochotomous keys are and how they are useful

Answer 55

Dichotomous keys/Identification schemies
(scheme for medically important ENTERIC genera)
-Used to IDENTIFY bacteria based on successive or Series of questions where each question has two possible answers (YES or NO)
-After each Yes, or NO question, the investigator is directed to another question util the organism is identified
-Dichotomous: means cut in two.
(useful for limited bacteria; enteric organism)

selected species of human pathogens can be isolated from marine animals

Question 56

Explain how ribotyping and rRNA sequencing can be used to to identify an unknown bacterium

Answer 56

Already answered
(ribotyping and rRNA sequencing, FISH, southern blotting and DNA chip all use Nucleic acid hybridization to identify bacteria )

Question 57

Compare and contrast between Serology, phage typing, fatty acid profiles, flow cytometry and other methods listed above and recognize their advantages and limitations

Answer 57

A

Question 58

Distinguish between slide agglutination tests, ELISA tests, and Western blots

Answer 58

A

Question 59

What is Southern blotting? How is it achieved?

Answer 59

Southern blotting; a method routinely used in molecules biology for detection of specific DNA sequence in DNA samples
Process:
1. DNA is isolated from bacterial cells and cut into fragments by restriction enzymes
2. The fragments are separated according to size by gel electrophoresis. Each band consists of many copies of particular DNA fragment. (band are invisible and can be made visible by staining)
-at this step, double-stranded pieces of DNA are denatured or separated into single strands within gel
3. The DNA bands are transferred to a nitrocellulose filter by blotting. The solution passes through the gel and filter to paper towels.
4. This produces a nitrocellulose filter with DNA fragments positioned exactly as on the gel (Capillary transfer”)
5. The filter is exposed to a radioactively labeled probe for a specific gene. the probe will base pair (hybridize) with a short sequence present on the gene.
6.The filter then exposed to a X-ray film The fragment is containing gene of interest is identified by band on developed film.

Question 60

What are DNA probes? Why are they necessary?

Answer 60

DNA Probes: small segments of labeled DNA used to find a specific sequence of nucleotides in a DNA molecule
-can be sued to rapidly identify microbes
-pathogenic-specific probes can reveal pathogen locations in body tissues or food
-other species-specific probes can locate and identify microbes in soil
-Probes can also be tagged with other molecular markers

Question 61

Distinguish between standard Southern Blotting, colony hybridization and DNA Chip Technology

Answer 61

Standard southern blotting can be used to identify bacteria, but Colony hybridization is a more RAPID modification
Standard southern blotting: uses combo of transferring electrophoresis separated DNA fragments and transferring them onto filter membrane, followed by fragment detection by probe hybridization
Colony hybridization: selecting bacterial colonies with desired genes through straightforward cloning and transfer process.

Question 62

What is the process of Colony Hybridization?

Answer 62

Colony hybridization:
1. salmonella DNA fragment is cloned in E.coli
2. cloned DNA fragments are marked with fluorescent dye and separated into single strands, forming DNA probes
3. Unknown bacteria are collected on a filter
4. The cells are lysed and the DNA is released
5. The DNA is separated on single strands
6. DNA probes are added to dNA from unknown bacteria
7. DNA probes hybridize with salmonella DNA from sample. Then excess probe is washed off. Fluorescence indicates presence of Salmonella

Question 63

-What is DNA Chip/microarray Technology? How does it work?

Answer 63

DNA Chip/microarray Technology; used to measure a large number of genes all at once
(ex: Salmonella antimicrobial resistance array to detect antibiotic resistant bacteria from farm animals)
-DNA is attached to a solid surface of glass, plastic or silicon. In this example, the DNA chip contains probes for different antibiotic resistance genes
-up to 400,000 DNA spots on single array
DNA from either a S. typhimirium or S.typhi is separated into single strands, enzymatically cut, and labeled with a fluorescent dye
process:
Unknown DNA is inserted into the chip and allowed to hybridize with the DNA on the chip
-Tagged DNA will only bind complementary DNA on the chip. The bound DNA will be detected by its fluorescence.
In this microarray, S. typhimurium DNA probes are GREEN while s. type DNA probes are RED Resistance genes found in both serovars (combo of both bacterial probes) appear yellow. Black/gray indicates ABSENCE of gene

Question 64

Distinguish between Ribotyping and rRNA sequencing

Answer 64

Ribotyping: way of identifying and characterizing bacteria by using information from rRNA-based phylogenetic analyses
(determine if two strains are the same)
16S rRNA gene (from small subunit, 1500 bases) used most often (large subunit 23S rRNA gene also used)
Advantages to using rRNA for phylogeny:
-all cells contain rRNA
-rRNA genes have undergone few changes overtime so all members of a domain, phylum, and in some cases genus, have the same “signature” sequences in the rRNA
-cells do NOT have to be cultured

Question 65

How does ribotyping differ from rRNA sequencing? Why is PCR necessary?

Answer 65

Ribotyping takes DNA sequence, cuts with restriction enzyme
rRNA sequencing: determine nucleotide sequence that have different patterns
PCR is necessary to amplify gene from chromosomes, take DNA cut it. (get gene away from chromosome)
-

Question 66

Build and interpret a cladogram

Answer 66

Cladograms: maps that show evolutionary relationships
(tell you about relationships between bacteria )
Process of buliding Cladogram:
1. Determine the sequence of bases in an rRNA molecule for reach organism. Only a short sequences sequence if bases (shown in example)
2. Calculate the percentage of similarity in the nucleotide bases between pairs of species. (for example, there is a 70% similarity between sequences for L. breves and L. acidophilus
3. Construct a cladogram. The length of the horizontal lines correspond to percent similarity values.
Each brand point, or node, in the cladogram, represents an ancestor common to all bond that node; Each node is defined by similarity in rRNA present in all species beyond that branch point.
**The longer the length of similarity, the less repeated they are*

Question 67

What is the taxonomy hierarchy for Eukarya, Archaea, and Bacteria?

Answer 67

The Taxonomic Hiearchy
Doman: Eukarya Archaea Bacteria
Kingdom: Fungi NONE NONE
Phylum: Ascomycota Euryarcheaota Proteobacteria Class: Hemiascomycetes Methanococci Gammaproteo-
bacteria
Order: Saccharmycetales Methanococales Enterobactier
-iales
Family: Sacharomycetaceae Methanococcae Enterbacte
-riaceae
Genus: Saccharomyces Methanothermococcus Escheric
-chia
Species: S. cerevisiae M. okinawensis E. coli

Question 68

How does Electroblotting work? What are its components?

Answer 68

Electroblotting: technique used to transfer proteins or nucleic acids from polyacrimide onto a membrane from using PVDF or nitrocellulose. membrane
-**if proteins are hydrophobic, use PVDF membrane
Components:
uses transfer buffer solution and current to drive proteins onto membrane
after gel electrophoresis occur, the dry blotting system is set up: containing stacks of filter paper and ordered fro CATHODE to ANODE and has filter sheets soaked in transfer buffer solution. The membrane must be between the gel and anode (since current moves in that direction). Stack is placed in transfer system and current is run.

Question 69

Distinguish between Western blotting and Southern blotting

Answer 69

Western blotting: detects RNA sequences
-uses primary and secondary antibodies
-used in diagnosing diseases (Lyme disease; HIV infections) ; uses SDS PAGE
Southern blotting: detects DNA sequences
-uses a radioactive PROBE; uses agarose gel electrophoresis
both process used electrophoresis gel, transfer to a membrane and use hybridization

Question 70

What are the 5 mains steps of Western blotting?

Answer 70

Western Blotting
1. SDS poylacrimde Gel electrophoresis (separate proteins)
2. Protein Blot on Nitrocellulose (transfer proteins on Nitro membrane)
3. Label with specific antibody
4. Detect antibody ( reveals protein of interest).