Identification
What is the classical approach to identifying DNA - 3
morphological characteristics differential staining biochemical tests
what do each group look like on EMB agar
-aerogenes-coli: black or dark centered colonies thyphoid - clear
Why is simple streaking on a complex culture not sufficient for getting a pure culture from nature - 2
1. many microgorganisms are present 2. the microorganism amy be present in a small porportionn to other organsims
two drawbacks to dichotomous keys
1. time consuming 2. genomic DNA or plasmid may change the characteristics of the cell and give false results
EMB agar - distinguish between what bacteria?
Aerogenes-coli group and thyphoid
Nomenclature system used
Binomial system - genus, speceis
How are bacterial species defined and how much of it percentage wise must be the same?
DNA- DNA hybridization 70% hybridization 16s rRNA seqeunces 97% similarity
3 Real world applications of identification
Diagnosis a disease cause of food contamination water treatment plant basic research
What are the two choices called in a dichotomous key?
Dichotomy
Steps of DNA fingerprinting- 4
Digest DNA into smaller fragments via restriction enzymes seperate the fragments based on size visualize the bands unique patter of bands identifies the Donor DNA
What is a good first choice to use in a dichotomous key?
Distinguish between broad categories - split the options into two, reliable test - gram stain, morphology, catalase
Order of classificaiton
Domain phylum class order family genus species
Common Biochemical tool used in the best fit method
Enterotube
What are 7 common things we exammine in identifcaiton procedures
Metabolic processes morphological characteristics serology phage typing amino acid sequencing fatty acid analysis by gas composition base composition of fatty acids
After using selective or differential media what do you do?
Move it to a complex agar (such as LB) to insure pure growth
What do you need for identifcaiton before you even begin?
PURE CULTURE
Why PCR is useful in idneifcation
Primers hybridize to complementary sequences to amplify the region of DNA these can then be sequenced
Nucleic Acid Hybridization
Southern blotting test if DNA from one organisms can bind with the organism of interest if so they may be similar
Steps to obtain a pure culture to be used for biochemical tests and morphological identification
Streak on a plate isolated is restreaked to test purity transferred to a slant of be used as a master culture
What cant we just use morphological characteristics?
There are a limited number of visible characteristics
Assumption made by dichotomous key
a set of characteristics unique to a species in at least one respsect
What is a dichotomous key
a tool used for the identification of an organisms based on a series of choices between alternative characteristics
What is serology
aggulination technqiues
What is an alternative to a dichotomous key?
best fit method
MacConkey Agar - what does it have and what do colones look like
bile salts, lactose, methyl red dye coliform bacteria are brick red with precipitated salts salmonella are clear gram positve cannot grow
What are common morphological characteristics? - 5
cell shape size arrangement presence of spores flagella
What 4 identifcaiton methods can we use when analyzing base composition of nucleic anlaysis
detmeine the entire seqeunce of bases in an organisms DNA DNA fingerprinting Hybridization PCR
What do we do to prevent the problems in using a complex culutre
differential and selective meida
What is differential media
differentiation between the bacteria
Mueller Tellurite agar distinguish between what bacteria? what does it contain
diphtheria bacteria tellurium
what do we look at in metabolic processes
enzymatic activities, utilization of carbon and nitrogen sources, waste prodcuts
Use of a personal computer - biolog, how does it work?
identify many bacteria use a microtiter plate, and detemines the oxidation of 95 different energy carbon sources that the bacteria is on. The test results are red by the computer and it determines which bacteria is present
What are we interested in finding in the biochemical tests
not the specific enzymes but some net end result
what is the best fit method and why is it better
use several tests at once and score the tests. The identifcaiton is made based on what organism the results show that it is closest to. Therefore even if one test gave a false result it can still be given the proper identifcaiton because it is still the most similar to a normal organism. it is also much more quick - use of primary tests to narrow it down from a large number of possibilites and secondary tests to identify it
What is phage typing
use to identify which phage (virus) a bacterium is susceptible to
