1. MODERN SYSTEMS OF BACTERIAL TAXONOMY
DNA ANALYSIS
NUCLEIC ACID HYBRIDIZATION

2. Traditional vs Modern
Binomial taxonomy
DNA sequences

3. Modern systems
The routine laboratory identification are the microscopic observation, culturation biochemical test and serology test
Latest and the most modern era of identification are molecular biological test
Most of the methods relates to the content of genetic materials

4. Serology Studies of serum and immune responses thru serum
m/o is antigenic, normally response as antibodies
The reagent used in test kit is antiserum
Present of m/o usually causing the clotting effects towards antiserum (+ve result)
Oppositely, the absent of m/o will be diluted effects (-ve result)

5. Types of serology test Slide agglutination test
ELISA (Enzyme-linked Immunosorbent Agglutination)
Western blotting (same approach but not categorize as serological test)

6. Phage typing To determine which phages a bacterium susceptible to
Eg. Bacteriophage (the bacterial virus) only attacking the particular strain or types of bacteria
Receptor sites for bacteriophages are highly specific.
Clear sites(plaques)are left when phages have killed bacterial cells.
On the basis of which phages attacked a bacteria culture ,can determine which strain of that bacterial species present.

7. Fatty acid analysis To compare the fatty acid profiles among m/o
Also known as FAME (fatty acid methyl ester)
Widely used in clinical and public health laboratories

8. Flow cytometry
To identify bacteria in sample without culturing the bacteria
Cell and m/o go through a small portion
Pass thru a laser and show scattering graph determining size, shape, density and surface
Also consist of fluorescent detection

9. DNA base composition
Can be group by comparing the relative oercentage of bases present in their DNA cell.
Show the relatedness via % of (Guanine G and cytosine C)
G complement with C, A complement with T
Pair s of GC resulting pairs of AT; (GC + AT = 100%)
Studies shown G-C content varies in 23-75% in bacteria

10. Example:
Staph aureus and Clostridium tetani have similar DNA composition while Pseudomonas aeruginosa has a different DNA composition.
But similar percentage of G-C content does not prove that the organisms are closely related.Because the sequence of bases may be quite different.
Example: Human & B.subtilis nearly identical G-C content.

11. DNA Fingerprinting
To determine the source of hospital-acquired infections
Spread via finger tips
Relate to hospital personnel or patients from the same ward
Short-time period required, instead of routine process
Should have the control sequences
Similar sequences shows >% of relationship

12. Polymerase Chain Reaction (PCR)
To make multiple copies of a piece of DNA enzymatically
Used to
Clone DNA for recombination
Amplify DNA to detectable levels
Sequence DNA
Diagnose genetic disease
Detect pathogens
ANIMATION PCR: Overview
ANIMATION PCR: Components

13. Polymerase Chain Reaction
m/o cannot be cultured through conventional methods
This method can be used to increase the amount of microbial DNA to levels that can be tested by gel electrophoresis
Eg. From the amber of ancient period
If there are primer of particular m/o in the sample, there will be amplified DNA indicates that m/o, obviously shows in the gel

14. Nucleic acid hybridization
Southern Blotting
DNA chips
Ribotyping and Ribosomal RNA sequencing
Flourescent In Situ Hybridization (FISH)

15. Introduction Each complimentary DNA containing complementary bases
When heat, the hydrogen bonds separated
When cool, it reunite almost back to normal
Basic purpose is to identify unknown m/o
Can hybridize any single-stranded nucleic acid chain; DNA-DNA, RNA – RNA, DNA-RNA

16. DNA BASE COMPOSITION

17. Nucleic acid hybridization

18. Nucleic acid hybridization

19. Southern Blotting To identify unknown m/o
Using DNA probe for rapid identification
Method involves breaking enzyme, then selecting a specific fragment as a probe , then the probe able to hybridize with the DNA of all particular m/o strains, but not with the DNA of closely related group m/o

20. DNA probe used to identify bacteria

21. Southern Blotting
Arrangement of papers in Blot tray.

22. Southern blot

23. DNA chips
Possible to quickly detect a pathogen in a host by identifying a gene that is unique to that pathogen
The chips composed of DNA probe
Unknown DNA label by fluorescent dye and placed in a chips
Hybridization between DNA probe and DNA detect by fluorescent

24. DNA chip technology

26. Fluorescent In Situ Hybridization
Fluorescent dye-labeled RNA or DNA probe are used to specifically m/o in place or in situ
Cell will be treated then the probe will enter the cell easily and react to the target ribosome in the cell (in situ)
To determine the identity, abundance and relative activity of m/o in an environment
Also to detect bacteria that have not yet been cultured (tiny quantity)

27. FISH

28. Ribotyping and Ribosomal RNA Sequencing
To determine the phylogenic relationships among organisms
3 advantages:
All cells contain ribosomes
RNA genes undergo <changes over time – each sequences looks ‘same’ to each other
Cell do not have to be cultured in laboratory

29. Ribosomal sequencing

30. Task of the week
Compare between Western Blotting and Southern Blotting. Differentiate in table form.

31. The end
SCORE A!!!