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JoVE Science Education Environmental Microbiology
Algae Enumeration via Culturable Methodology
  • 00:00Overview
  • 01:41Principles of Culturing and Enumerating Algae
  • 04:03Culturing and Enumerating Algae from Soil
  • 05:34Representative Results
  • 07:11Applications
  • 09:02Summary

Enumeración de las algas mediante metodología cultivable

English

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Overview

Fuente: Laboratorios del Dr. Ian Pepper y el Dr. Charles Gerba – Universidad de Arizona
Demostrando autor: Bradley Schmitz

Las algas son un grupo muy heterogéneo de microorganismos que tienen un rasgo común, es decir, la posesión de pigmentos fotosintéticos. En el ambiente, las algas pueden causar problemas para los propietarios de la piscina por el aumento en el agua. Las algas también pueden causar problemas en las aguas superficiales, como lagos y embalses, debido a que liberan toxinas algales. Más recientemente, las algas están siendo evaluadas como nuevas fuentes de energía a través biocombustibles algas. Algas son realmente las bacterias clasificadas como cianobacterias. Cyanobacteria no sólo producir, sino que también tiene la capacidad para fijar nitrógeno de la atmósfera. Otras algas son eucariotas, desde organismos unicelulares hasta organismos multicelulares complejos, como algas. Estos incluyen las algas verdes, los euglenoides, los dinoflagelados, las doradas Algas marrones, diatomeas, las algas marrones y las algas rojas. En los suelos, las poblaciones de algas son frecuentemente 106 por gramo. Estos números son menores que los números correspondientes para bacterias, actinomicetos y hongos, sobre todo porque la luz solar necesaria para la fotosíntesis no puede penetrar bajo la superficie del suelo.

Porque las algas son fototróficas, obtención de energía de la fotosíntesis y el carbono de la biomasa a partir de dióxido de carbono, pueden cultivarse en medios de cultivo que consiste en la totalidad de nutrientes inorgánicos y sin un sustrato de carbono orgánico. La falta de sustrato orgánico impide el crecimiento de bacterias heterotróficas. Utilizando un medio de crecimiento inorgánico, algas originalmente presentan en suelo o agua puede ser cuantificado por el método del número más probable para el (MPN). El método MPN se basa en diluir sucesivamente una muestra, tal que las algas se se diluyen a la extinción. La presencia de algas en cualquier dilución es determinada por un signo positivo de crecimiento en el medio, que suele ser una baba verde de algas que resulta de la fotosíntesis. Uso de tubos de replicar a cada dilución y una evaluación estadística del número de tubos positivos para el crecimiento en cualquier dilución determinada permiten el número de algas presentes en la muestra original para calcular. Tablas MPN se han desarrollado y publicado específico para un diseño particular de la MPN, incluyendo el número de repeticiones en cada dilución.

Procedure

Pesar una muestra de 10 g de suelo que ha sido recogida húmeda del campo, o había agua agregado a él para que permanezca húmedo durante 2-3 días. Tenga en cuenta que el suelo debe ser húmedo pero no saturado. Preparar una serie de diluciones 10 veces mediante la adición de 10 g de suelo en 95 mL de solución de Bristol modificado (figura 1). Para crear solución de Bristol modificado, disolver las siguientes en 1.000 mL de agua: 0.25 g de NaNO3, 0,025 g CaCl2, 0,075 g de MgSO<sup…

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.

p2 and p3 are chosen to be the number of tubes in the next two higher dilutions that show a positive sign of growth. Thus, p2 = 3 and p3 = 1.

The value for p1 can be found by looking down the first column in Table 2. The same is done in the p2 column. Then, the value of p3 (across the top) intersects the two defined by the values of p1 and p2. In this example, the value is 1.1 organisms per mL.

Divide this value by the concentration of soil in the dilution to which you assigned p2. In this example, this is Tube D.

Equation 1

Thus, in this example, there were 1.1 x 104 algae cells per g of soil. This value is fairly typical of the number of algae found in soil.

Figure 2
Figure 2. Hypothetical outcome of an algae enumeration experiment. Shaded tubes indicate the presence of algae. Un-shaded tubes represent the absence of algae.

Most probable number for indicated values of p3
p1 p2 0 1 2 3 4 5
0
0
0
0
0
0
0
1
2
3
4
5

0.018
0.037
0.056
0.075
0.094
0.018
0.036
0.055
0.074
0.094
0.11
0.036
0.055
0.074
0.093
0.11
0.13
0.054
0.073
0.092
0.11
0.13
0.15
0.072
0.091
0.11
0.13
0.15
0.17
0.090
0.11
0.13
0.15
0.17
0.19
1
1
1
1
1
1
0
1
2
3
4
5
0.020
0.040
0.061
0.083
0.11
0.13
0.040
0.061
0.082
0.1
0.13
0.16
0.060
0.081
0.10
0.13
0.15
0.17
0.080
0.10
0.12
0.15
0.17
0.19
0.10
0.12
0.15
0.17
0.19
0.22
0.12
0.14
0.17
0.19
0.22
0.24
2
2
2
2
2
2
0
1
2
3
4
5
0.045
0.068
0.093
0.12
0.15
0.17
0.068
0.092
0.12
0.14
0.17
0.20
0.091
0.12
0.14
0.17
0.20
0.23
0.12
0.14
0.17
0.20
0.23
0.26
0.14
0.17
0.19
0.22
0.25
0.29
0.16
0.19
0.22
0.25
0.28
0.32
3
3
3
3
3
3
0
1
2
3
4
5
0.078
0.11
0.14
0.17
0.21
0.25
0.11
0.14
0.17
0.21
0.24
0.29
0.13
0.17
0.20
0.24
0.28
0.32
0.16
0.20
0.24
0.28
0.32
0.37
0.20
0.23
0.27
0.31
0.36
0.41
0.23
0.27
0.31
0.35
0.40
0.45
4
4
4
4
4
4
0
1
2
3
4
5
0.13
0.17
0.22

0.34
0.41
0.17
0.21
0.26
0.33
0.40
0.48
0.21
0.26
0.32
0.39
0.47
0.56
0.25
0.31
0.38
0.45
0.54
0.64
0.30
0.36
0.44
0.52
0.62
0.72
0.36
0.42
0.5
0.59
0.69
0.81
5
5
5
5
5
5
0
1
2
3
4
5
0.23
0.33
0.49
0.79
1.3
2.4
0.31
0.46
0.7
1.1
1.7
3.5
0.43
0.64
0.95
1.4
2.2
5.4
0.58
0.84
1.2
1.8
2.8
9.2
0.76
1.1
1.5
2.1
3.5
16
0.95
1.3
1.8
2.5
4.3

Table 2. Most probable numbers for use with the experimental design in this exercise.

Applications 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 to malachite green.

A further application is the estimation of mycorrhizal fungi by inoculating soil dilutions onto a plant host and looking for root colonization by the fungi.

Transcript

Algae are photosynthetic organisms that live in a variety of environments. Soil dwelling algae can be cultured in the laboratory, and their concentration enumerated using simple calculations.

Algae are a highly heterogeneous group of organisms that have one common trait, namely the possession of photosynthetic pigments, commonly chlorophyll. The vast majority of algae are microscopic, however, the exact definition of the group is controversial, and also includes seaweeds, which are typically macroscopic.

In the environment, algae can cause problems in surface waters such as lakes or reservoirs, forming algal blooms that deplete the water nutrients, blocking light passing beyond the water surface, and releasing toxins. The ability to enumerate algae in samples allows scientists to evaluate the health of an ecosystem, and the potential risk of algal overgrowth. 

Algal populations in soils frequently occur at around ten thousand cells per gram. These numbers are typically lower than corresponding concentrations of bacteria, fungi, or actinomycetes, as algae require sunlight for photosynthesis, which cannot penetrate far below the soil surface.

This video will illustrate how to culture algae from soil in the laboratory, and how to enumerate the concentration of algae in the starting soil sample.

Algae have beneficial effects on ecosystems. Blue-green algae, or cyanobacteria, have the ability to fix nitrogen gas from the atmosphere, making them useful in increasing soil nitrogen in semi-arid environments and also as a potential tool for biofuel production.

Other algae are eukaryotic, and range from single-celled to complex multicellular organisms, like seaweeds. These include green algae, euglenoids, dinoflagellates and diatoms, brown algae, and red algae.

Algae are phototrophic, obtaining energy from photosynthesis and carbon for biomass from carbon dioxide. As a result, they can be grown in media consisting entirely of inorganic nutrients, without an added organic carbon substrate. This lack of organic substrate prevents the growth of heterotrophic bacteria, which are dependent on external organic carbon for growth.

To culture algae for enumeration, soil samples are serially diluted tenfold to 10-6 g soil per mL, and cultured in growth media. Several replicates are made for each dilution. They are then incubated in a well-lit area for up to 4 weeks to allow algal growth.

The presence of algae in any dilution is determined by a positive sign of growth in the medium, which will typically appear as a green slime. Finally, empirically developed MPN tables designed for algal growth are consulted, enabling the user to determine the original algal concentration based on growth in dilution replicates. The MPN method relies on the serial dilution of samples such that the algae are diluted to extinction, meaning that at some dilution, no algal growth ensues.

Now that we are familiar with the concepts behind growing and enumerating algae from samples, let’s take a look at how this is carried out in the laboratory.

To begin the experiment, first weight out 10 grams of moist soil that has either been collected moist from the field, or been rehydrated and remained moist for 2 to 3 days. The soil should but not saturated.

Next, prepare a ten-fold dilution series by adding the 10 grams of soil first to 95 mL of Modified Bristol’s solution, or MBS. Label this as suspension A.

After shaking vigorously, continue the dilution series by adding 1 mL of suspension A to 9 mL of MBS in a test tube. Continue this ten-fold dilution series another 4 times to give dilutions up to 10-6 g per mL.

Next, inoculate 5 replicate tubes, each containing 9 mL of MBS with 1 mL of each of the dilutions 10-1 to 10-5. This results in 5 replicates tubes for each dilution from 10-2 to 10-6. Cap the tubes loosely.

Finally, incubate the tubes for a full 4 weeks in an area exposed to sunlight. Observe the tubes for algal growth once every 7 days. Tubes exhibiting algal growth will appear green.

Most Probable Number, or MPN, analysis is a commonly used mathematical method to enumerate microorganisms grown from dilution of a concentrated initial substrate. By taking into account the dilution factors of the solutions, and the number of tubes which show positive signs of growth at each dilution, the most probable number of organisms per gram of original soil sample can be calculated using an MPN table and simple formula.

To calculate MPN, the highest dilution with the highest number of positive replicate tubes is assigned the label of p1, in this case, the replicates of tube C. In contrast, some of the tubes from D & E are negative with no signs of algal growth.

The number of tubes in the next two higher dilutions that show positive growth are labeled as p2 and p3. Here, p2 = D and p3 = E.

The value for p1 can be found by looking down the first column in the MPN table. The same should be done with the p2 column. Finally, the value of p3, across the top, is used to intersect the two defined by p1 and p2, to give a value of the most probable number of organisms per mL.

Next, to calculate the concentration of organisms per gram in the original soil sample, this value is divided by the concentration of soil in the dilution to which p2 was assigned. The following equation is used to define the actual number of organisms per gram of soil.

Algal enumeration and MPN analysis have a wide range of applications, some of which are explored here.

This culturing method of algal enumeration can be used in a variety of settings. It can be applied to rivers or lakes to determine algal levels, and assess the risks of harmful algal blooms. Alternatively, it can be used to assess the cleanliness and safety of waters more directly used by humans, including swimming pools, water fountains, or other drinking water sources. Ideally, in potable water samples and swimming pools, there are no algae present.

The MPN analysis for enumeration can also be applied to other non-algal microorganisms. For example, water quality can be assessed using indicator organisms such as coliforms or E. coli. Here, samples can be cultured with media containing chemicals that are altered to produce color or fluorescence in the presence of the indicator organisms. By performing multiple small replicates of this experiment in individual cells, with samples diluted to a known concentration, the ratio of positive cells can be referenced to an MPN table for the specific indicator organism, and the starting concentration in the samples determined.

Algae may also be cultured for commercial applications. For example, some types of biofertilizer utilize blue-green algae, which can act as symbionts with plants, aiding their fixture and take-up of nitrogen, which is particularly useful in aiding crop growth in areas with poor soil. Similarly, algae can be grown for biofuels, or as a source of nutrient rich food for livestock.

You’ve just watched JoVE’s introduction to algal culture and enumeration. You should now understand how to dilute soil samples for algal growth, how to culture algae in the laboratory, and how to enumerate the algal concentration of your starting samples. Thanks for watching!

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JoVE Science Education Database. JoVE Science Education. Algae Enumeration via Culturable Methodology. JoVE, Cambridge, MA, (2023).

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