1. 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation of medium and incubation conditions Inocula –The sample from which microorganisms will be isolated © 2012 Pearson Education, Inc.

2. Figure 22.1 Mineral salts medium containing mannitol but lacking NH 4 , NO 3 , or organic nitrogen. Soil NH 4  +NH 4  plate  NH 4  plate +NH 4  plate Incubate aerobically © 2012 Pearson Education, Inc.

3. 22.1 Enrichment Enrichment Cultures –Can prove the presence of an organism in a habitat –Cannot prove an organism does not inhabit an environment The ability to isolate an organism from an environment says nothing about its ecological significance © 2012 Pearson Education, Inc. Animation: Enrichment Cultures Animation: Enrichment Cultures

4. The Winogradsky Column –An artificial microbial ecosystem (Figure 22.2) –Serves as a long-term source of bacteria for enrichment cultures –Named for Sergei Winogradsky –First used in late 19th century to study soil microorganisms 22.1 Enrichment © 2012 Pearson Education, Inc.

5. Figure 22.2 GradientsColumn Lake or pond water Mud supplemented with organic nutrients and CaSO 4 O2O2 H2SH2S Foil cap Algae and cyanobacteria Purple nonsulfur bacteria Sulfur chemolithotrophs Patches of purple sulfur or green sulfur bacteria Anoxic decomposition and sulfate reduction © 2012 Pearson Education, Inc.

6. Enrichment bias –Microorganisms cultured in the lab are frequently only minor components of the microbial ecosystem Reason: the nutrients available in the lab culture are typically much higher than in nature Dilution of inoculum is performed to eliminate rapidly growing, but quantitatively insignificant, weed species 22.1 Enrichment © 2012 Pearson Education, Inc.

7. 22.2 Isolation Pure cultures contain a single kind of microorganism –Can be obtained by streak plate, agar shake, or liquid dilution (Figure 22.3) Agar dilution tubes are mixed cultures diluted in molten agar –Useful for purifying anaerobic organisms © 2012 Pearson Education, Inc.

8. Figure 22.3 Colonies Paraffin–mineral oil seal © 2012 Pearson Education, Inc.

9. 22.2 Isolation Most-probable-number technique –Serial 10  dilutions of inocula in a liquid media –Used to estimate number of microorganisms in food, wastewater, and other samples (Figure 22.4) © 2012 Pearson Education, Inc. Animation: Serial Dilutions and a Most Probable Number Analysis Animation: Serial Dilutions and a Most Probable Number Analysis

10. Figure 22.4 1 ml (liquid) or 1 g (solid) Enrichment culture or natural sample Dilution 1 ml 9 ml of broth Growth No growth 1/10 (10  1 ) 10  2 10  3 10  4 10  5 10  6 © 2012 Pearson Education, Inc.

11. 22.2 Isolation Flow cytometry –Uses lasers –Suspended cultures passed through specialized detector –Cells separated based on fluorescence © 2012 Pearson Education, Inc.

12. 22.5 PCR Methods of Microbial Community Analysis Specific genes can be used as a measure of diversity –Techniques used in molecular biodiversity studies (Figure 22.12) DNA isolation and sequencing PCR Restriction enzyme digest Electrophoresis Molecular cloning © 2012 Pearson Education, Inc.

13. Figure 22.12 Microbial community PCR Extract total community DNA Amplify by PCR using fluorescently tagged primers Restriction enzyme digest and run on gel Amplify 16S RNA genes using general primers (for example, Bacteria-specific) or more restrictive primers (to target endospore-forming Bacteria) Excise bands and clone 16S rRNA genes Excise bands Generate tree from results using endospore- specific primers Sample 12 34 Gel All 16S rRNA genes Sample 12 34 T-RFLP gel Different 16S rRNA genes DGGE gel Sample 12 34 Sequence Env 1 Env 2 Env 3 Bacillus subtilis Bacillus megaterium Clostridium histolyticum Bacillus cereus DNA © 2012 Pearson Education, Inc.

14. 22.5 PCR Methods of Microbial Community Analysis DGGE: denaturing gradient gel electrophoresis separates genes of the same size based on differences in base sequence (Figure 22.13) –Denaturant is a mixture of urea and formamide –Strands melt at different denaturant concentrations © 2012 Pearson Education, Inc.

15. Figure 22.13 PCR amplification DGGE 1 2 3 45 6 78 1 2 3 45 6 78 © 2012 Pearson Education, Inc.

16. 22.5 PCR Methods of Microbial Community Analysis T-RFLP: terminal restriction fragment length polymorphism –Target gene is amplified by PCR –Restriction enzymes are used to cut the PCR products ARISA: automated ribosomal intergenic spacer analysis (Figure 22.14) –Related to T-RFLP –Uses DNA sequencing © 2012 Pearson Education, Inc.

17. Figure 22.14 Position 1 16S rRNA gene 1540 12900 23S rRNA gene 50–1500bp Position 1513 Position 23 Reverse PCR primer ITS region Forward PCR primer containing fluorescent tag ( ) PCR Gel analysis Community DNA 1000 750 500 250 0 400 450500550600650700 750 800 850 900 950 100010501100115012001250 Fluorescence Fragment size (base pairs) © 2012 Pearson Education, Inc.

18. 22.5 PCR Methods of Microbial Community Analysis Results of PCR phylogenetic analyses –Several phylogenetically distinct prokaryotes are present rRNA sequences differ from those of all known laboratory cultures –Molecular methods conclude that less than 0.1% of bacteria have been cultured © 2012 Pearson Education, Inc.

19. 22.6 Microarrays and Microbial Diversity: Phylochips Phylochip: microarray that focuses on phylogenetic members of microbial community (Figure 22.15) –Circumvents time-consuming steps of DGGE and T-RFLP © 2012 Pearson Education, Inc.

20. Figure 22.15 Positive Weak positive Negative © 2012 Pearson Education, Inc.

21. 22.7 Environmental Genomics and Related Methods Environmental genomics (metagenomics) –DNA is cloned from microbial community and sequenced –Detects as many genes as possible –Yields picture of gene pool in environment –Can detect genes that are not amplified by current PCR primers –Powerful tool for assessing the phylogenetic and metabolic diversity of an environment (Figure 22.16) © 2012 Pearson Education, Inc.

22. Figure 22.16 Community sampling approach Environmental genomics approach Outcomes Single-gene phylogenetic tree Total gene pool of the community 1. Identification of all gene categories 2. Discovery of new genes 3. Linking of genes to phylotypes Phylogenetic snapshot of most members of the community 1. Identification of novel phylotypes 2. Amplify single gene, for example, gene encoding 16S rRNA Restriction digest total DNA and then shotgun sequence, OR sequence directly (without cloning) using a high throughput DNA sequencer Extract total community DNA Microbial community Sequence and generate tree Assembly and annotation DNA Partial genomes © 2012 Pearson Education, Inc.

23. III. Measuring Microbial Activities in Nature 22.8 Chemical Assays, Radioisotopic Methods, and Microelectrodes 22.9 Stable Isotopes 22.10 Linking Specific Genes and Functions to Specific Organisms © 2012 Pearson Education, Inc.

24. 22.8 Chemical Assays, Radioisotopes, & Microelectrodes In many studies, direct chemical measurements are sufficient (Figure 22.18) –Higher sensitivity can be achieved with radioisotopes Proper killed cell controls must be used © 2012 Pearson Education, Inc.

25. Figure 22.18 Sulfate reduction Photosynthesis Sulfate reduction 14 C-Glucose respiration Formalin-killed control Light Lactate H2SH2S Lactate or H 2 S 14 CO 2 incorporation 14 CO 2 evolution H 2 35 S H 2 present H 2 absent Killed Dark Killed Time © 2012 Pearson Education, Inc.

26. 22.8 Chemical Assays, Radioisotopes, & Microelectrodes Microelectrodes –Can measure a wide range of activity –pH, oxygen, CO 2, and others can be measured –Small glass electrodes, quite fragile (Figure 22.19) –Electrodes are carefully inserted into the habitat (e.g., microbial mats) Measurements taken every 50–100  m (Figure 22.20) © 2012 Pearson Education, Inc.

27. Figure 22.19 Gold GlassPlatinum 5  m MembranesGlass BacteriaCathode Nutrient solution 50–100  m NO 3  N2ON2O 2e  1 N 2 O  H 2 O N 2  2 OH  © 2012 Pearson Education, Inc.

28. Figure 22.20 Oxygen (O 2 ) concentration (  M) Seawater Oxic sediment Anoxic sediment Nitrate (NO 3  ) concentration (  M) Depth in sediment (mm) 0 100200300 O2O2 NO 3  0 5 10 0 4 8 12 © 2012 Pearson Education, Inc.