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Algae Enumeration via Culturable Methodology

Overview

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Bradley Schmitz

Algae are a highly heterogeneous group of microorganisms that have one common trait, namely the possession of photosynthetic pigments. In the environment, algae can cause problems for swimming pool owners by growing in the water. Algae can also cause problems in surface waters, such as lakes and reservoirs, due to algal blooms that release toxins. More recently, algae are being evaluated as novel sources of energy via algal biofuels. Blue-green algae are actually bacteria classified as cyanobacteria. Cyanobacteria not only photosynthesize, but also have the ability to fix nitrogen gas from the atmosphere. Other algae are eukaryotic, ranging from single-celled organisms to complex multicellular organisms, like seaweeds. These include the green algae, the euglenoids, the dinoflagellates, the golden brown algae, diatoms, the brown algae, and the red algae. In soils, algal populations are frequently 106 per gram. These numbers are lower than corresponding numbers for bacteria, actinomycetes, and fungi, mostly because the sunlight required for photosynthesis cannot penetrate far beneath the soil surface.

Because algae are phototrophic, obtaining energy from photosynthesis and carbon for biomass from carbon dioxide, they can be grown in growth media consisting entirely of inorganic nutrients and without an organic carbon substrate. The lack of organic substrate precludes the growth of heterotrophic bacteria. Using an inorganic growth medium, algae originally present in soil or water can be quantitated by the most probable number (MPN) method. The MPN method relies on successively diluting a sample, such that the algae themselves are diluted to extinction. The presence of algae in any dilution is determined by a positive sign of growth in the medium, which is typically a green slime of algae that results from photosynthesis. Use of replicate tubes at each dilution and a statistical evaluation of the number of tubes positive for growth at any given dilution allows for the number of algae present in the original sample to be calculated. MPN tables have been developed and published specific to a particular MPN design, including the number of replicates used at each dilution.

Procedure
  1. Weigh out a 10 g sample of soil that has either been collected moist from the field, or had water added to it so that it remains moist for 2-3 days. Note that the soil should be moist but not saturated.
  2. Prepare a 10-fold dilution series by adding the 10 g of soil into 95 mL of Modified Bristol’s Solution (Figure 1). To create Modified Bristol’s Solution, dissolve the following in 1,000 mL of water: 0.25 g NaNO3, 0.025 g CaCl2, 0.075 g MgSO-4 · 7H

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Results

Figure 2 is an example of representative results.

p1 is chosen to be the number of replicate tubes of the highest dilution (least concentrated in soil) that has the highest number of positive tubes. Here, the replicates from Tube B do not count, because those of Tube C are from a higher dilution. In contrast, the number of tubes from Tube D that show a positive sign of growth is less than those from Tube C. So, p1 = 5.<

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Application and Summary

The MPN methodology is useful, because it allows estimation of a functional population based on a process-related attribution. In the example, the functional process was photosynthesis undertaken by algae, which allowed for growth in the absence of organic carbon. This allowed for total algal populations in soil to be enumerated.

MPN is also used to estimate the number of a particular type of microbial pathogens in water, such as Salmonella, utilizing the resistance of Salmonella<

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Tags
Algae EnumerationCulturable MethodologyPhotosynthetic OrganismsLaboratory CultureConcentration CalculationPhotosynthetic PigmentsAlgal BloomsSurface WatersAlgal OvergrowthEcosystem HealthRisk AssessmentSoil Algae PopulationSunlight RequirementSoil Surface PenetrationAlgae Culturing From SoilAlgae Concentration EnumerationBeneficial Effects Of Algae

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0:00

Overview

1:41

Principles of Culturing and Enumerating Algae

4:03

Culturing and Enumerating Algae from Soil

5:34

Representative Results

7:11

Applications

9:02

Summary

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