Microbiology and Molecular Ecology Techniques in Marine Environments

Marine Microbiology and Biomass Analysis

Alternative methods of direct calculation of bacterial biomass and obtaining clarity through image analysis to study populations. C-ecology is essential for understanding these dynamics.

Genetic and Molecular Techniques

The Green Fluorescent Protein (GFP) is introduced into the genome by genetic recombination for studies in natural media. DAPI binds to nucleic acids of any cell, while fluorescent antibodies are specific to the targeted antigen.

Microbial Ecology and Cultivation

Bacteria require moisture, organic matter (MO), and oxygen (O2), so they must be absent for certain anaerobic types, indicating that oxygen-producing cyanobacteria or sufficient organic compounds limit the system based on light availability.

• Gram-positive bacteria with low G+C content.
• Ultramicrobacteria of the genus Sphingomonas capable of passing through 0.2-micron filters, often dominant in regions like the Pacific with cell counts of 10¹² to 10¹³ cells/mL in polar regions.

Specialized Growth Media and Physical Separation

Using physical parameters as physiological characteristics requires refrigerated incubators. Physical separation helps isolate specific populations from mixed cultures. MPN (Most Probable Number) tests help estimate coliforms in water samples.

Selective and Differential Media

• Thiosulfate-citrate-bile salts sucrose agar (TCBS): A highly selective and differential medium for Vibrio species, where bile salts inhibit other biota and sucrose serves as the carbon source with a pH indicator.
• Complex Media: Marine agar, ferric citrate, bacteriological peptone, and yeast extract as energy and carbon sources for heterotrophs.
• Thermolyzed Media: Altered easily by heat; sterilized through membrane filtration with 0.2-micron pore sizes.

Microcosms and Stratified Communities

An enrichment microcosm is established via a vertical gradient where oxygen decreases upwards and sulfides decrease downwards. Sulfate-reducing bacteria produce hydrogen sulfide in anaerobic conditions, while proteolytic bacteria participate in the nitrogen cycle by producing ammonia from proteins.

Advanced Microscopy and Molecular Profiling

Surface waters are rich in polysaccharides and proteins. Enumeration techniques include:

• Fluorescence microscopy
• Viable cell staining
• Fluorescent antibodies
• Confocal microscopy
• Flow cytometry

PCR and Phylogenetic Analysis

Polymerase Chain Reaction (PCR) uses total cellular DNA extended by universal primers to target conserved regions. Denaturing Gradient Gel Electrophoresis (DGGE), restriction enzymes, and clone libraries allow for sequencing and the creation of phylogenetic trees to identify novel species.

Sampling and Treatment Protocols

Treatments include sonication (50W/20kHz/30s/3x) to break up flocs, followed by the application of sodium pyrophosphate at 0.01% W/V. Specialized techniques are employed to efficiently collect neuston samples from the water's surface layer.