1. OLEH SUDRAJAT FMIPA UNMUL 2009

2. Klasifikasi Bakteri Somewhat 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)

3. Klasifikasi Bakteri The manual divides bacteria into 4 groups or divisions on the basis of their Cell Wall 1. 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”

4. Klasifikasi Bakteri More modern methods now used: 1. DNA studies, genomics, gene probes 2. using Bacterial viruses “bacteriophages” 3. Serology – antibody – antigen reactions (like blood typing) Examples: Salmonella typhi Salmonella typhimurium Azotobacter vinelandii 12837 E. coli 0157H7 nasty strain of E. coli

5. Genotypic Characteristics for Identifying Prokaryotes the use of genotypic testing has increased with the availability of technology genotypic testing is particularly useful in the case of organisms that are difficult to identify several techniques include gene probes PCR sequencing rRNA

6. Genotypic Characteristics for Identifying Prokaryotes gene 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

7. 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”.

8. Genotypic Characteristics for Identifying Prokaryotes PCR: 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

9. Genotypic Characteristics for Identifying Prokaryotes sequencing 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 used to pick colonies containing wanted DNA

10. Difficulties in Classifying Prokaryotes historically prokaryotes have been grouped according to phenotypic attributes problems 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 microorganisms the more similar the nucleotide sequence, the more closely related DNA extraction

11. Genotypic Characteristics used in Classifying Prokaryotes comparison of nucleotide sequences differences in DNA sequence can assist in determination of divergence of evolutionary path for organisms DNA hybridization single strands of DNA anneal 16S ribonucleic acid comparing sequence of ribosomal RNA relatedness to other organisms can be determined using numerical taxonomy determined by the percentage of characteristics two organisms have in common The more you have in common phenotypically with another organism the closer related you are to that organism.

12. Genotypic Characteristics for Identifying Prokaryotes PCR: 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

13. Bacterial Taxonomy Characteristics used to classify organisms Traditional Size, shape, gram reaction, need for O 2 Ability to metabolize sugars Metabolic end products Supplemented by Comparison of 100-300 characteristics Nucleic acid sequence of ribosomal RNA

14. General Groupings used in Taxonomy Aerobic (grows in air), obligate if must have O 2. Capnophilic if needs CO 2. Facultative anaerobe (grows in air, and can grow without oxygen). Anaerobe (grows without oxygen, and most species do not grow well in air as O 2 is toxic for them). Microaerophilic (grows in a low concentration of oxygen, but not in its absence or in air).