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MICRO-ALGAE, ALGE-BART™ Quality Control

Micro-Algae (ALGE) is the name given to various plant-like microorganisms that are able to photosynthesize using light as the energy source for growth. The ranges of algae that can grow in this biodetector include grass green Algae (Chlorophyceae), Blue-Green Algae (Cyanobacteria), Desmids, Diatoms and Euglenoids. Such growths may be localized or occur generally throughout the body of the culture medium.

This biodetector is distinctly different from the other products in the series because it is designed to recover and culture phototrophic (photosynthesizing) microorganisms that utilize light and release oxygen as a product. To achieve this, a modified Bold's medium is used which does not contain organics, but does contain the basic nutrients for plant growth (nitrogen, phosphorus, potassium, sulfur etc.). Carbon is presented as bicarbonates and the medium is made slightly alkaline (pH, 8.2) to encourage the micro-algae to utilize this form of carbon. The growth of micro-algae tends to be slower than for the heterotrophic bacteria and does require the presence of light (for photosynthesis). This light does not have to be strong (such as in direct sunlight) and most micro-algae can actually utilize quite low levels of light. The most effective manner for maximizing the probability of photosynthesis is to place the charged ALGE-BARTTM on its side and set it about 60 cms from a single 40 watt daylight fluorescent light or 100 cms away from a 60 watt tungsten light source.

Other differences in the ALGE-BART relate to the lateral diffusing of the nutrient chemicals along the sample and the presentation of a variety of eco-niches for possible growth. These niches are formed by the pores within the woven material layered around a part of the vial and the semi-saturated and saturated nature of this material. Some micro-algae gravitate towards the semi-saturated material above the culturing sample within the test vial while others grow within the saturated material or within the liquid medium itself. In practice, this test takes a minimum of two days and a maximum of 26 days to detect significant populations of micro-algae.

This ALGE-BART can be used as a simple presence/absence (P/A) test capable of indicating to some extent the population size and the types of micro-algae present in the water sample. Different algae utilize different sites in the biodetector because of the two woven materials and the lateral position of the test vial on its side. Twice weekly observations for four weeks should be undertaken to observe the various forms of algal growth. These form into six possible reaction patterns in the test sample (see Reaction Patterns below). Observations can determine:

(1) Level of activity of the micro-algae (aggressivity) through the TL before a reaction is observed; and
(2) Community composition of the micro-algae present and active in the sample.

While a positive test will be recognized by the appearance of the first identifiable reaction, further information beyond the simple P/A result can be achieved by continuing to observe the test vial on a regular basis for subsequent reactions that may appear. The nature of these shifts forms the RPS. Two useful interpretations of the data can therefore be:

(1) Aggressivity and possible population as log. colony forming units per ml; and
(2) Community structure based upon the RPS generated by the chronologically sequenced reaction numbers observed during the test observation period.

By the routine (e.g., monthly) testing of a water or wastewater using this technique, the levels of aggressivity, possible population and community structure can all be determined and the status of any micro-algal induced fouling determined.

To conduct the test, it is necessary to add 15 ml of the water sample to the ALGE-BART biodetector. Once this has been done and the inner test vial returned to the outer test vial that is then capped, the test can begin. To initiate growth, it is recommended that the biodetector be laid on its side under a light source. Continuous light is preferred. Incubation is at room temperature and the biodetector protected from any excessive heating due to any artificial lights that are used. Note that temperatures in excess of 80oF (30oC) may inhibit algal activity. Under no circumstances should the test be severely agitated or shaken during the test period.

If there is a need to determine the micro-algae population in a soil or a slurry, the technique would need to be modified. This is necessary to reduce the potential detrimental effects that could be caused by a high organic nutrient in the soil. Such loadings could stimulate the growth of heterotrophic microorganisms at the expense of the micro-algae. To correct for this, take 1 g of the soil or slurry and suspend it in 9 ml of sterile Ringer's solution. Agitate by vortexing for one minute to disperse the particles evenly into suspension and break up some of the larger structures. Aseptically withdraw a 1ml sample from the midpoint of the suspension and transfer to 14 ml of sterile Ringer's solution. Use this 15ml suspension to charge the ALGE-BART and follow the standard procedures. The possible population would be modified by multiplying the log vu (viable units)/ml by x 2.0 to achieve a possible population in vu/g of soil or slurry.

Because the micro-algae tend to grow slowly, the generation of a growth may be difficult to determine initially. Stereo-microscopic examination will give an earlier indication of a positive growth. Many micro-algae may initially start to grow generating a green color since the chlorophyll pigments used for photosynthesis are dominant. But as the growth continues, other pigments such as the xanthophylls may become dominant and change the color of the growth. This color shift may involve several different colors dominating over time (e.g., green to yellow to orange to brown).

A number of habitats are provided in this BART test which can encourage the growth of various micro-algae. These habitats include semi-saturated porous, saturated porous, aquatic, liquid: solid and liquid: air surfaces. Nutrients provided are inorganic nutrients commonly used by the micro-algae that, together with constant illumination, provide a preferential habitat for these microbes. Growth is slower because of longer generation times commonly found in the micro-algae.

Reaction Patterns
To observe the test, gently examine the biodetector for the presence of colored patches. If the test is negative, the woven material should remain white and there should be no colored patches or cloudiness in the water medium. A positive reaction may be recognized when there is one of these: a colored cloudiness, a distinctive patch of color or a colored deposit generated in the test device. Low magnification stereo microscopy can be used to directly observe the types of algae growing in the biodetector.

GG Green growth at or above the water level
FG Irregular patches of green growth over the woven material
OB Patches of red, orange or brown growths below water level
YB Yellow patches diffuse over the woven material
GF Green deposits and/or green growth in the woven material
DG Blue-green or black growth commonly at the water level

GG - Grass Green growth (formerly reaction one, ALGE-BART).
A grass-green growth may be seen through the porous textile medium usually concentrated at the water line or below water line. As this reaction matures, flocculent green growth may also be observed in the liquid medium.

FG - Fuzzy Green Patches (formerly reaction two, ALGE-BART)
Much of the reaction seen here may be above the water line in the semi-saturated porous textile medium. The pore structure restricts the extension of the growth and so the growth may be seen as intense grass green zones with ill-defined or radial edges.

OB - Red Orange Brown Patches (formerly reaction three, ALGE-BART)
Red, orange or brown patches usually with clearly defined edges are generated both above and below the water line. These growths may gradually change in color as the growths mature.

YB - Yellow Beige Patches (formerly reaction four, ALGE-BART)
Poorly defined light yellow to beige patches of growth occur on the fabric often at localized sites on the porous fabric, generally these growths are initially difficult to observe.

GF - Green Flocculent (formerly reaction five, ALGE-BART)
Grass-green flocculent deposits are abundant in the liquid medium and on the floor of test vial. Tendency to be dense and lay on the floor of the test vial. The porous fabric may also show some green discoloration particularly on the lower side.

DG - Dark Green to Black Patches (formerly reaction six, ALGE-BART)

Predominantly recognized by dark-green, blue-green or black growths at the water line. Often this is a secondary reaction following reactions GG, FG or GF.

RPS (Reaction Pattern Signatures )
· GG - DG	Cyanobacteria present with possible Nostoc dominate
· FG - DG	Grass-Green algae with cyanobacteria present
· FG - OB Grass-Green algae maturing · YB - OB Diatoms or Desmids may be dominant · GG - GF Grass-Green algae maturing without pigment production · GG-GF-DG Grass-Green algae dominant but cyanobacteria dominate

The RPS displays the reaction patterns in the order that they were observed. For example, GG-GF-DG signature indicates the order for the reactions observed were firstly, GG; secondly, GF; and thirdly, DG. The signature obtained from an individual sample will provide an initial understanding of the type of algal community present in the sample.

Population counts are based on direct microscopic examination of samples with the range being set at two standard deviations. This population projection is based on the number of viable units (vu) per ml of original sample volume. In this event, each vu is considered to be a single cell rather than some form of multicellular structure.

Time Lag (days of delay) to ALGE-BART Population Table Seventeen The Relationship Between Time Lag and the Population For Micro-Algae
Time Lag Aggressivity Population (days) (cfu/ml) 4 Extremely High 100,000 8 Very High 20,000 12 Very High 7500 16 High 600 20 High 100 24 Moderate 20 25 Very Low Not Aggressive

g) ALGE-BART™
This biodetector is distinctly different from the other products in the series because it is designed to recover and culture phototrophic (photosynthesizing) microorganisms that utilize light and release oxygen as a product. To achieve this, a modified Bold's medium is used which does not contain organics, but does contain the basic nutrients for plant growth (nitrogen, phosphorus, potassium, sulfur etc.). Carbon is presented as bicarbonates and the medium is made slightly alkaline (pH, 8.2) to encourage the micro-algae to utilize this form of carbon. The growth of micro-algae tends to be slower than for the heterotrophic bacteria and does require the presence of light (for photosynthesis). This light does not have to be strong (such as in direct sunlight) and most micro-algae can actually utilize quite low levels of light. The most effective manner for maximizing the probability of photosynthesis is to place the charged ALGE-BART on its side and set it about 60 cms from a single 40 watt daylight fluorescent light or 100 cms away from a 60 watt tungsten light source.

ALGE-BART Medium
White opaque crystalline deposits extend from the central peg towards the walls. Commonly, the deposits may not reach out to the side-wall of the inner test vial and exhibit an irregular form but being more granular towards the center. A transparent crystalline film may extend for 2 mm up the sidewall but this is obscured by the textile walls of the test vial. In the normal event of the confirmation of sterility, there is a change in the characteristics of the liquid medium forming in the test vial. These are detailed in Table Forty-Four.

Table Forty-Four Medium Diffusion in a Sterile ALGE-BART Inner Test Vial to Confirm a Negative Reaction
Time (days) Color Basal Above weave 0.25 Clear Clear 28* Clear Clear

*Note that the culture medium should remain crystal clear and have been generated using sterile distilled or deionized water. Natural water samples can cause secondary chemical reactions that may be seen through crystalline deposits forming on the floor of the test vial.

Table QC Forty-Five QC Characterization of Medium Diffusion in a Sterile ALGE-BART Inner Test Vial
Time (days) Color Contamination Fabric Solution 7 Off white Clear Cloudy 28 Discolored Cloudy Cloudy or slime deposits

The ALGE-BART has a very low organic nutrient load in this QC test and so any contaminant will take a period of time to grow. This test should be done under constant illumination at 22 to 24OC and bad contamination may occur, and be visible, in seven days. The woven material may discolor slightly under these circumstances. If this discoloration includes the formation of black or grey specks, then it is possible that the contaminants are molds particularly if this occurs above the water line.

Confirmation of Selective Media Composition in the ALGE-BART
There is a need to confirm the suitability of the selective medium for the biodetection of the various algae. This need is recognized by this test method (Table Forty-Six). It is recommended that the following Culture Collection of Algae and Protozoa (CCAP) strains be applied to the BART to determine the standard reaction patterns. The CCAP 5th edition was issued as ISBN 1 871105 01 3 in 1988. Each culture should be prepared according to the protocols described in the 5th edition CCAP catalog. The cultures should be used when they have reached the stationary growth phase. Inoculation of the inner test vial should be using a suspension of 0.5 ml of the culture in 15 ml of the sterile isotonic salt solution. This inoculum should be taken from the midpoint of the micro-algal culture immediately after the culture had been very gently agitated. Do not shake the vial. Incubate under continuous light at 22 to 24oC for twenty-eight days and observe twice a week for activities and growth reactions. Typical results are listed below for the recommended CCAP strains in Table Forty-Six.

Table Forty-Six Characterization of the ALGE-BART
CCAP Genus/species Characterization 211/62 Chlorella sp. GF 11/77 Chlamydomonas baca GG to GF 276/21 Scenedesmus quadricauda GF to YB 678/4 Spirogyra sp. GF to DG

Note that some of these cultural tests will shift from one reaction type to another as the growth in the ALGE-BART matures.

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