OLEH

SUDRAJATFMIPA UNMUL

2009

Klasifikasi BakteriSomewhat different: a clinical rapid ID is

often important when trying to find causative agent of a disease

Bergey’s manual: Manual is in lab for a reference when doing unknown

Developed on 1940’s for grouping bacteria according to standard diagnostic lab techniques available at the time (such as Grams stain)

Klasifikasi BakteriThe manual divides bacteria into 4 groups or

divisions on the basis of their Cell Wall1. Gram + (stain violet)2. Gram - (destain, and are counterstained

pink or reddish color)3. Bacteria that lack a cell wall (mycoplasma)4. organisms that have a cell wall lacking

“peptidoglycan” (archaeobacteria – Now called “Archaea”

Klasifikasi BakteriMore modern methods now used:1. DNA studies, genomics, gene probes2. using Bacterial viruses “bacteriophages”3. Serology – antibody – antigen reactions

(like blood typing)Examples: Salmonella typhiSalmonella typhimuriumAzotobacter vinelandii 12837E. coli 0157H7 nasty strain of E. coli

Genotypic Characteristics for Identifying Prokaryotesthe use of genotypic testing has increased

with the availability of technologygenotypic testing is particularly useful in

the case of organisms that are difficult to identify

several techniques includegene probesPCRsequencing rRNA

Genotypic Characteristics for Identifying Prokaryotesgene probes

single stranded DNA that has been labeled with a identifiable tag, such as a fluorescent dye

are complementary to target nucleotide sequences unique in DNA of pathogen

Microbe gene probed

Genotypic Characteristics for Identifying Prokaryotes

If there is a suspicion, based on symptoms or other environmental parameters that indicates that the organism to be identified may be “ organism A”, a single strand of “organism A’s” DNA is introduced with a tag attached (such as fluorescent dye). If the introduced DNA binds to the unknown organism, then it is identified as “organism A”. If it does not bind to the unknown organism, then the unknown is not “organism A”.

Genotypic Characteristics for Identifying ProkaryotesPCR: polymerase chain reaction

used to detect small amounts of DNA present in a sample (blood, food, soil)

the PCR chain reaction is used to amplify the amount of DNA present

Genotypic Characteristics for Identifying Prokaryotessequencing ribosomal RNA

of particular use for identifying prokaryotes impossible to grow in a culture

focus is place on the 16S molecules of the RNA because of it’s size approximately 1500 nucleotides

once the 16S molecule is sequenced, it can then be compared to the sequences of known organisms

Machine usedto pick coloniescontaining wantedDNA

Difficulties in Classifying Prokaryoteshistorically prokaryotes have been

grouped according to phenotypic attributesproblems with this approach include

mutation resulting in phenotypic changes “just because they look alike, does not

mean that they are even closely related according the prokaryotics”

new molecular approaches are providing better insight to the relatedness of microorganismsthe more similar the nucleotide

sequence, the more closely related DNAextraction

Genotypic Characteristics used in Classifying Prokaryotescomparison of nucleotide sequences

differences in DNA sequence can assist in determination of divergence of evolutionary path for organisms

DNA hybridizationsingle strands of DNA anneal

16S ribonucleic acidcomparing sequence of ribosomal RNA

relatedness to other organisms can be determined using numerical taxonomydetermined by the percentage of characteristics two

organisms have in commonThe more you have in common phenotypically with

another organism the closer related you are to that organism.

Genotypic Characteristics for Identifying ProkaryotesPCR: polymerase chain reaction

used to detect small amounts of DNA present in a sample (blood, food, soil)

the PCR chain reaction is used to amplify the amount of DNA present

Bacterial TaxonomyCharacteristics used to classify organisms

TraditionalSize, shape, gram reaction, need for O2

Ability to metabolize sugarsMetabolic end products

Supplemented byComparison of 100-300 characteristicsNucleic acid sequence of ribosomal RNA

General Groupings used in Taxonomy

Aerobic (grows in air), obligate if must have O2. Capnophilic if needs CO2.

Facultative anaerobe (grows in air, and can grow without oxygen).

Anaerobe (grows without oxygen, and most species do not grow well in air as O2 is toxic for them).

Microaerophilic (grows in a low concentration of oxygen, but not in its absence or in air).