1. Culturing techniques
Microbiology owes it’s roots and much information we know about some organisms from culturing techniques
Culturing generally attempts to develop an isolate (not always of course)
Choose medium, environment (chemical and physical) – innoculate and grow…
Plating or streak culturing – smear a sample so it becomes diluted…

2. Most Probable Number (MPN)
Culturing technique that provides both cell density information and function (grouped into cells that can metabolize a certain way)

3. Environmental Sampling
As soon as you remove organisms from their surroundings, they DO NOT just stop acting – often changing function and ecology quickly
‘Fixing’ is the technique of preserving the cells as close to their actual distribution as possible
Physical (freezing)
Chemical (additives to arrest function)
These techniques kill all, or at least most, cells
Preservation – most also preserve the material you are after until you get to the lab

4. Materials Fixation: Preservation
Freezing – How fast? Dry ice-ethanol, liquid nitrogen
Chemical – Ethanol, Glutaraldehyde, Parafomaldehyde
Preservation
Freezing
Ethanol
RNAlater
Salts, buffers

5. Sterile technique
When should we be MOST concerned with maintaining sterile technique?
SEM samples
PCR samples
FISH samples
MPN samples
Culture samples
Shotgun samples
Discuss the elements of sterile technique

6. Primer Selection
For any process, the primer selected for use in PCR to amplify some piece of DNA is the first point that determines what sort of information you will be working with
Universal for ID, targeted for group selection, gene group targeting (NOT necessarily expressed though)

7. Bias
Bias occurs for any reaction – some organisms will be selected over others in a way that changes their apparent abundance
PCR – selects for organisms in higher abundance (not good to ID organisms at 0.x-5%, roughly) some organisms hybridize to primers faster, conditions through PCR reaction can affect…

8. RFLP Restriction Fragment Length Polymorphism
Cutting a DNA sequence using restriction enzymes into pieces  specific enzymes cut specific places
Starting DNA sequence:
5’-TAATTTCCGTTAGTTCAAGCGTTAGGACC
3’-ATTAAAGGCAATCAAGTTCGCAATAATGG
Enzyme X
5’-TTC-
3”-AAG-
Enzyme X
5’-TTC-
3”-AAG-
5’-CAAGCGTTAGGACC
3’-GTTCGCAATAATGG
5’-TAATTT
3’-ATTAAA
5’-CCGTTAGTT
3’-GGCAATCAA

9. RFLP
DNA can be processed by RFLP either directly (if you can get enough DNA from an environment) or from PCR product
T-RFLP (terminal-RFLP) is in most respects identical except for a marker on the end of the enzyme
Works as fingerprinting technique because different organisms with different DNA sequences will have different lengths of DNA between identical units targeted by the restriction enzymes
specificity can again be manipulated with PCR primers
Liu et al. (1997) Appl Environ Microbiol 63:

10. Electrophoresis
Fragmentation products of differing length are separated – often on an agarose gel bed by electrophoresis, or using a capilarry electrophoretic separation

12. DGGE Denaturing gradient gel electrophoresis
The hydrogen bonds formed between complimentary base pairs, GC rich regions ‘melt’ (melting=strand separation or denaturation) at higher temperatures than regions that are AT rich.
When DNA separated by electrophoresis through a gradient of increasing chemical denaturant (usually formamide and urea), the mobility of the molecule is retarded at the concentration at which the DNA strands of low melt domain dissociate.
The branched structure of the single stranded moiety of the molecule becomes entangled in the gel matrix and no further movement occurs.
Complete strand separation is prevented by the presence of a high melting domain, which is usually artificially created at one end of the molecule by incorporation of a GC clamp. This is accomplished during PCR amplification using a PCR primer with a 5' tail consisting of a sequence of 40 GC.
Run DGGE animation here – from

13. RFLP vs. DGGE RFLP DGGE Advantages Advantages Limitations Limitations
Relatively easy to do
Results can be banked for future comparisons
Limitations
Less sensitive phylogenetic resolution than sequencing
Each fragment length can potentially represent a diversity of microorganisms
Cannot directly sequence restriction fragments,making identification indirect
DGGE
Advantages
Very sensitive to variations in DNA sequence
Can excise and sequence DNA in bands
Limitations
Somewhat difficult
”One band-one species” isn’t always true
Cannot compare bands between gels
Only works well with short fragments (<500 bp), thus limiting phylogenetic characterization

14. RFLP/DGGE info
Grouped into sequences with SIMILARITY, not necessarily identical!
Operational Taxonomic Units (OTU) is the simplest grouping
Can compare these sequences to others prepared in same way (same primers for PCR, same restriction enzyme(s) )

15. FISH Fluorescent in-situ hybridization
Design a probe consisting of an oligonucleotide sequence and a tag
Degree of specificity is variable!
Hybridize that oligonucleotide sequence to the rRNA of an organism – this is temperature and salt content sensitive
Image using epiflourescence, laser excitation confocal microscopy
Technique DIRECTLY images active organisms in a sample

16. Fluorescent in site hybridization

17. B Drift Slime Streamer
10 µm
DAPI
FER656

19. Oligunucleotide design

20. FISH variations
FISH-CARD – instead of a fluorescent probe on oligo sequence, but another molecule that can then bond to many fluorescent probes – better signal-to-noise ratio
FISH-RING – design of oligo sequence to specific genes – image all organisms with DSR gene or nifH for example

21. FISH-MAR Combine FISH to tag specific organisms with autoradiography
FISH ID’s the organism based on oligo hybridization
Isotopically labelled substrates are incorporated into the organism showing metabolism

22. Single cell DNA extraction
Using laser calipers, possible to isolate and move a single cell, lyse, and extract DNA from just that cell for amplification…