1. Methods in Microbial Ecology
Chapter 22
Methods in Microbial Ecology

2. I. Culture-Dependent Analyses of Microbial Communities
22.1 Enrichment and Isolation
22.2 Isolation in Pure Culture

3. 22.1 Culture Dependant Microbial Community Analysis
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

4. The Isolation of Azotobacter
Figure 22.1

5. Some Enrichment Culture Methods

6. 22.1 Enrichment and Isolation
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
Animation: Enrichment Cultures

7. 22.1 Enrichment and Isolation
The Winogradsky Column
An artificial microbial ecosystem
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

8. Schematic View of a Typical Winogradsky Column
Figure 22.2a

9. Photo of Winogradsky Column: Remained Anoxic Up to Top
A bloom of different phototrophic bacterium
1: Thiospirillum jenense
2: Chromatium okenii
3: Chlorobium limicola 1 2 3
Figure 22.2b

10. Some Enrichment Culture Methods

11. Some Enrichment Culture Methods

12. 22.1 Enrichment and Isolation
Enrichment bias
Microorganisms cultured in the lab are frequently only minor components of the microbial ecosystem
Because 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

13. 22.2 Isolation in Pure Culture
Pure cultures contain a single kind of microorganism
Can be obtained by streak plate, agar shake, or liquid dilution
Agar dilution tubes are mixed cultures diluted in molten agar
Useful for purifying anaerobic organisms
Most-probable number technique
Serial 10X dilutions of inocula in a liquid media
Used to estimate number of microorganisms in food, wastewater, and other samples

14. 22.2 Isolation in Pure Culture
Animation: Serial Dilutions and a Most Probable Number Analysis

15. Procedure for a Most-Probable Number Analysis
Figure 22.4

16. 22.3 Pure Culture Methods
Figure 22.3

17. 22.2 Isolation in Pure Culture
Axenic culture can be verified by
Microscopy
Observation of colony characteristics
Tests of the culture for growth in other media
Laser tweezers are useful for
Isolating slow-growing bacteria from mixed cultures

18. Principle of the Laser Tweezers
Figure 22.5a

19. The Laser Tweezers for the Isolation of Single Cells
Figure 22.5b

20. II. Culture-Independent Microbial Community Analysis
22.3 General Staining Methods
22.4 FISH
22.5 Linking Specific Genes to Specific Organisms Using PCR
22.6 Environmental Genomics

21. 22.3 General Staining Methods
Fluorescent staining using DAPI or acridine orange (AO)
DAPI (4’-6-diamidino-2-phenylindole) stained cells fluoresce bright blue
AO stained cells fluoresce orange or greenish-orange
DAPI and AO fluoresce under UV light
DAPI and AO are used for the enumeration of microorganisms in samples
DAPI and AO are nonspecific and stain nucleic acids
Cannot differentiate between live and dead cells

22. Nonspecific Fluorescent Stains: Photomicrograph of DAPI
Figure 22.6a

23. Nonspecific Fluorescent Stains: Acridine Orange
Figure 22.6b

24. 22.3 General Staining Methods
Viability stains: differentiate between live and dead cells
Two dyes are used
- Green dye: penetrates all cells
- Red dye: penetrates only dead cells
Based on integrity of cell membrane
Green cells are live
Red cells are dead
Can have issues with nonspecific staining in environmental samples

25. Viability Staining
Figure 22.7

26. 22.3 General Staining Methods
Fluorescent antibodies can be used as a cell tag
Highly specific
Making antibodies is time consuming and expensive
Green fluorescent protein (GFP) can be genetically engineered into cells to make them autofluorescent
Can be used to track bacteria
Can act as a reporter gene

27. Fluorescent Antibodies as a Cell Tag
Sulfolobus acidocaldarius attached to the surface of solfatra soil particles.
Figure 22.8

28. The Green Fluorescent Protein
Pseudomonas fluorescence attached to barley roots.
Figure 22.9

29. 22.4 FISH
Nucleic acid probe is DNA or RNA complimentary to a sequence in a target gene or RNA
FISH (fluorescent in situ hybridization)
Phylogenetics of microbial populations
Used in microbial ecology, food industry, and clinical diagnostics
ISRT-FISH (in situ reverse transcription-FISH)
- Use cDNAs as probes
CARD-FISH (catalyzed reported deposition FISH)
- Peroxidase is attached to the probe
- Treat with tyramide after hybridization
: converted into a very reactive intermediate that binds to adjacent proteins and fluoresces

30. Morphology and Genetic Diversity
Phase contrast
Phylogenetic FISH
Figure 22.10a

31. FISH Analysis of Sewage Sludge: Nitrifying Bacteria
Red: ammonia-oxidizing bacteria
Green: nitrite-oxidizing bacteria
Figure 22.11a

32. FISH Analysis of Sewage Sludge
Figure 22.11b

33. In-situ Reverse Transcription
Stained with DAPI
Stained with ISRT probe
Figure 22.12b

34. 22.5 Linking Genes to Specific Organisms Using PCR
Specific genes can be used as a measure of diversity
PCR, DGGE, molecular cloning, and DNA sequencing and analysis are tools used to look at community diversity
DGGE (denaturing gradient gel electrophoresis) separates genes of the same size based on differences in base sequence
Denaturant is a mixture of urea and formamide
Strands melt at different denaturant concentrations
- Use gels with different gradient of the denaturant

35. Steps in Single Gene Biodiversity Analysis
Figure 22.13

36. PCR and DGGE Gels
Figure 22.14a

37. PCR and DGGE Gels
Figure 22.14b

38. 22.5 Linking Genes to Specific Organisms Using PCR
T-RFLP (Terminal Restriction Fragment Length Polymorphism)
Target gene is amplified by PCR using a primer set in which one of the primers is end-labeled with a fluorescent dye
Restriction enzymes are used to cut the PCR products
Molecular methods demonstrate that less than 0.5% of bacteria have been cultured
Phylochip: microarrays that focus on phylogenetic members of microbial community
Circumvents time-consuming steps of DGGE and T-RFLP

39. Phylochip Analysis of Sulfate-Reducing Bacteria Diversity
Figure 22.15

40. 22.6 Environmental Genomics
Environmental Genomics (metagenomics)
DNA is cloned from microbial community and sequenced
Idea is to detect as many genes as possible
All genes in a sample can be detected
Yields picture of gene pool in environment
Can detect genes that would not be amplified by current PCR primers
Powerful tool for assessing the phylogenetic and metabolic diversity of an environment

41. Single Gene Versus Environmental Genomics
Figure 22.16

42. III. Measuring Microbial Activities in Nature
22.7 Chemical Assays, Radioisotopic Methods, and Microelectrodes
22.8 Stable Isotopes

43. 22.7 Chemical Assays, Radioisotopes, & Microelectrodes
In many studies direct chemical measurements are sufficient
Higher sensitivity can be achieved with radioisotopes
Proper killed cell controls must be used
Radioisotopes can also be used with FISH
FISH microautoradiography (FISH-MAR)
Combines phylogeny with activity of cells

44. Microbial Activity Measurements
Figure 22.17

45. An autotroph using 14CO2 as a carbon source.
FISH-MAR
An autotroph using 14CO2 as a carbon source.
Figure 22.18a

46. FISH-MAR
FISH
MAR with 14C-glucose
Figure 22.18b

47. 22.7 Chemical Assays, Radioisotopes, & Microelectrodes
Can measure a wide range of activity
pH, oxygen, CO2, and others can be measured
Small glass electrodes, quite fragile
Electrodes are carefully inserted into the habitat (e.g., microbial mats)

48. Schematic Drawing of an Oxygen Microelectrode
Figure 22.19a

49. Microelectrodes Being Used in a Hot Spring Microbial Mat
Figure 22.19b

50. Microbial Mats and the Use of Microelectrodes
Figure 22.20a

51. Oxygen, Sulfide, and pH Profiles in Hot Spring Microbial Mat
Figure 22.20b

52. 22.8 Stable Isotopes
Stable isotopes: non-radioactive isotopes of an element
Can be used to study microbial transformations in nature
Isotope fractionation
Carbon and sulfur are commonly used
Lighter isotope is incorporated preferentially over heavy isotope
Indicative of biotic processes
Isotopic composition of a material reveals its past biology (e.g., carbon in plants and petroleum)

53. Mechanism of Isotopic Fractionation Using Carbon
Figure 22.21

54. Isotopic Geochemistry of 13C and 12C

55. Isotopic Geochemistry of 34S and 32S

56. 22.8 Stable Isotopes
Stable isotopes probing (SIP): links specific metabolic activity to diversity using a stable isotope
Microorganisms metabolizing stable isotope (e.g., 13C) incorporate it into their DNA
DNA with 13C can then be used to identify the organisms that metabolized the 13C-labelled substrates
SIP of RNA can be done instead of DNA

57. Stable Isotope Probing