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Heterotrophic Aerobic Bacteria, HAB-BART™ Quality Control

Some bacteria are able to degrade organics as their source of energy and carbon. These are known as heterotrophic. By far, the majority of these heterotrophs function most efficiently under aerobic conditions. Much of the biodegradation that occurs under aerobic conditions is due to the activities of these heterotrophic aerobic bacteria (HAB, formerly the total aerobic bacteria or TAB). Since these bacteria play a major role of biodegradation and their presence in oxygen-rich waters can be critical to the efficiency of the engineered operation, the HAB-BART™ was developed to detect these bacteria.

The unique feature of this test is the addition of methylene blue that acts as an indicator of respiratory activity. While there remains free oxygen in the water, the methylene blue dye in the water remains blue. As soon as all of the oxygen has been consumed by bacterial (respiratory) activity, the methylene blue shifts from its observable form to a colorless form. In other words, in the HAB-BART tests, when the liquid medium turns from blue to a colorless (non-blue) form, the heterotrophic aerobic bacteria have been sufficiently aggressive to have "respired off" the oxygen. At this time a methylene blue reductase enzyme becomes activated and this reduces the methylene blue to its colorless form.

Microorganisms present at depths in this test are short of oxygen and "look" for alternatives. The blue dye (methylene blue) in this test forms such an alternate substrate. When the aerobic bacteria use this dye, the color is bleached out. This usually occurs from the bottom (bottom up) or the top (top down) of the tester first. This bleaching action (decolorizing the blue dye) is the indicator of a positive reaction. Note that the dye is added to the test by inverting the charged HAB-BART for 30 seconds to allow the methylene blue chemical dried in the cap time to dissolve into the water. When the HAB-BART is returned to its normal state (cap side up), the ball rolls up through the water sample causing the methylene blue to become mixed into the water to form an even blue solution.

Methylene blue is a basic dye that can bind readily to the negatively charged microbial cells. Traditionally, therefore, this dye has been used to stain microbial cells. A feature of methylene blue is that it changes from a blue color in the oxidized state to a clear form in the reduced state. When methylene blue is added to a medium that is actively converting energy due to microbial respiration, the electrons are transferred to the dye causing it to become reduced and the dye changes from a blue to a clear state (the color disappears). The protocol has been based on the methylene blue reductase test that has been used in the dairy industry for decades to determine the potential for bacterial spoilage of milk. In the HAB-BART the objective is for the user to be able to determine the aerobic bacterial population which may be related to various forms of biofouling and bioremediation. Essentially, the methylene acts as an oxygen substitute and its reduction (bleaching) from the blue to the colorless form can be used an indication of the amount of respiratory function of the bacteria in the sample water. Therefore, this test is an answer to the need to test water and wastewater for the presence of heterotrophic aerobic bacteria as such without trying to determine the particular groups of bacteria that may be present.

The HAB-BART determines the activity of the heterotrophic aerobic bacteria. When these bacteria are present and active, the blue dye in the biodetector becomes bleached (colorless) either from the bottom up or the top down. The faster this happens, the more aggressive are the heterotrophic aerobic bacteria.

Reaction Patterns

UP - Bleaching moves upwards from base (Reaction 1)
DO - Bleaching moves downwards from ball (Reaction 2)

There are only two recognized reactions (UP and DO) and both of these relate to the form with which the bleaching occurs. There are different forms of clouding which follow the bleaching of the methylene blue and these are recognized using the BARTSCAN™ system.

UP – Bleaching moves upwards
Blue solution bleaches from the bottom up. The bleached zone may be clear or clouded. In the latter case, the medium tends to have a light to medium yellow color. Rarely does the bleaching extend beyond the equator of the ball so that a blue ring will remain around the ball with a width of 1 to 5 mm.

DO – Bleaching moves downwards
Blue solution bleaches from the top down. The bleached zone is more commonly cloudy. The bleached liquid medium tends to have a light to medium yellow color. Commonly the bleaching does extend up beyond the equator of the ball and any blue ring remaining around the ball is relatively thin with a width of 0.5 to 2 mm.
Note: that there is almost always a blue ring remaining around the ball and that the DO reaction will usually leave this ring intact. Furthermore, the test reaction can only be one or the other and so interpretation is restricted to one or other of these two reactions:

UP: Strictly aerobic bacteria may be dominant with some facultative anaerobes often present DO: Facultatively anaerobic heterotrophs dominate along with some anaerobic bacteria

The relationship between the time lag (days of delay) to the bacterial population is given in Table Eleven.

Table Eleven The Relationship Between Time Lag and the Population for Heterotrophic Aerobic Bacteria
Time Lag (days) Population cfu/ml 1 7,000,000 2 500,000 3 50,000 4 7000

The heterotrophic aerobic bacteria, like the slime formers, grow very quickly and are readily detectable because of the reduction of the methylene blue from the blue (oxidative) to the colorless (reductive) state. Essentially, the methylene blue acts as a redox indicator and rapidly shows when respiratory activity is occurring because the test liquids become reductive and the methylene blue decolorizes. This test is one of the fastest of the BART tests as well as being the easiest to read. It functions most effectively when the bacterial consortia in the sample are dominated by heterotrophic aerobes.

Risk Potential Assessment

The heterotrophic aerobic bacteria are subdivided into two major consortial groups in the HAB-BART. These are dominated by either: the strictly aerobic (UP), or the facultatively anaerobic (DO) heterotrophic bacteria. The risk potential for the severity of a detected HAB event can be expressed through the shortness of the time lag (in days) as follows:

1. Very aggressive (treatment should be started as early as convenient)
2. Aggressive (treatment should be considered in the near future before the condition degenerates further)
3. Moderately Aggressive (treatment may not be required but vigilance through ongoing testing should be practiced)
4. Normal Background Levels (routine testing is recommended).

Twelve Relationship Between the Time Lag to the CG Reaction in a HAB-BART and the Aggressivity of the Anaerobic Bacteria
Aggressivity Very Sign. Moderate Not UP -Bleach Up <0.5 1-2 3-4 >4 DO-Bleach Down <1 2-3 4-6 >6

There is a wide variety of bacteria that are aerobic and they share a common feature of the ability to respire using oxygen. One substitute for oxygen that is used in this BART test is methylene blue. This is utilized by bacteria instead of oxygen and it becomes bleached from blue (oxidized form) to a colorless (reduced) form. This test incorporates methylene blue and the activity of the aerobic bacteria is reflected in the speed and form in which the methylene blue is bleached. Occasionally, bacteria will bioaccumulate the methylene blue before it begins to be used for respiration. As a result, the medium may tend to darken where there is a dominance of bacterially initiated bioaccumulation. The major feature of this test is, however, the color shift from blue to colorless (bleaching). The other features are secondary.

HAB-BART Medium

A brown hardened gel-like mass with a darker inner circle extends fully up the cone to the walls of the test vial. On the sidewall of the inner test vial above the basal cone there is a transparent film that extends approximately 3mm up the walls. This film is also brown in color and contains gel-like deposits. The edge is ill defined and there is normally a thin ring of salt deposit visible around the wall at 6 to 8 mm above the basal cone. The characterization of the medium is given in Table Thirty-One.

HAB-BART CAP

On the inside of the cap, there are blue crystalline deposits around the outer edge in a complete circle, or in an incomplete arc. Flakes of the blue crystalline material may cover 20 to 50% of the inside area of the cap itself. These flakes are firmly held to the cap surface but excessive vibration is shipping may cause some of the smaller crystals to detach. If this happens, the water sample, when added to the inner test vial, may show some small areas of blue coloration. Occasionally, also, these flakes may be seen on the ball or attached to the walls of the test vial. Their presence does not interfere with the test procedure since they dissolve rapidly once the sample is added to the test vial.

Table Thirty Medium Diffusion in a Sterile HAB-BART Inner Test Vial to Confirm a Negative Reaction
Time (days) Color Basal Lower column Upper column 0.25 Greenish-blue Blue Blue 0.5 Greenish-blue Greenish-blue Blue 1.0 Greenish-brown Greenish-blue Blue

Note:The blue color is crystal clear and has been generated using sterile distilled or deionized water. Natural water samples can cause secondary chemical reactions that may be seen through an intensification of color at the diffusion front. Water saturated with oxygen stored at low temperatures can, when used in this test, cause bubbles to form as oxygen comes out of solution as the temperature rises to room temperature. Water samples with higher salt concentrations may cause the color of the methylene blue to shift from a blue to very greenish shade of blue. This color shift does not, however, affect the sensitivity of the test.

Table Thirty-One QC Characterization of Medium Diffusion in a Sterile HAB-BART Inner Test Vial
Time (days) Color Contamination*** Mid-point color 1.0 Blue Bleached* 4.0 Greenish-blue Bleached** 8.0 Greenish-brown Bleached***

A satisfactory QC would involve the left hand column of events with no signs of cloudiness, or localized concentrations of the blue color. Generally, contamination would be first observed by a bleaching in which the blue color turns to a colorless form. Commonly where this has happened in less than one day (*) there is only a slight clouding. If the bleaching occurs after four days (**), then the liquid will usually also be cloudy. In the event that bleaching did not occur until eight days (***) this would indicate either a very low contamination with aerobic microbes or that the contamination was dominated by anaerobes. Where anaerobic microbes dominate, the blue color may become locally concentrated (e.g., near the basal cone of the test vial).

Confirmation of the Selective Media Composition in the HAB-BART

In order to confirm the suitability of the selective medium for the detection of the various HAB recognized by this test method, it is recommended that the following A.T.C.C. (American Type Culture Collection) strains be applied to the HAB-BART to determine the standard reaction patterns. Each culture should be prepared as a 48 hour Brain Heart Infusion broth culture incubated at 30oC to reach the stationary growth phase. Inoculation of the inner test vial should be using a 1.0ml suspension of the broth culture in 15 ml of the sterile Ringer's solution. This inoculum should be taken from the midpoint of the broth culture immediately after the culture had been gently agitated. This inoculum should be applied directly over the ball as the test vial is being filled. Do not shake the vial. Incubate at 22 to 24oC for seven days and observe for activities and reactions. Typical results are listed below for the recommended A.T.C.C. strains in Table Thirty-Two.

Table Thirty-Two Cultural Characterization of the HAB-BART
A.T.C.C. Genus/species Characterization 27853 Pseudomonas aeruginosa DO Reaction 2 25922 Escherichia coli UP Reaction 1

The HAB (or heterotrophic aerobic bacteria) BART test is very specifically designed for the determination of aerobic bacteria that are heterotrophic. This means that this test is designed to determine the activity of aerobic bacteria that will possess the methylene blue reductase enzyme system and not function well anaerobically. If the biofouling problems are thought to be generated by aerobic microbial activities, the HAB-BART™ performs a role as the "scout" for aerobically generated problems. Commonly, the HAB-BART can therefore be used as the initial test that is then followed by tests using other BART testers. Some of these patterns of potential usage are given in HAB Table Thirty-Three:

Table Thirty-Three Possible Further Testing of Waters Giving an Aggressive Diagnosis Using the "Scout" HAB-BART Test Method
Problem Recommended Testing Strategy using BART IRB- SRB- SLYM- FLOR- ALGE- White slime 4 4 4 Grey slime 4 4 4 Black slime 4 4 4 Brown slime 4 4 4 Green slime 4 4 4 Turbidity 4 4 Taste 4 4 4 Odor 4 4 4 4 Color 4 4 4 Corrosion 4 4 4 Hygiene risk* 4 4 4

* Waters that have a possible hygiene risk should be tested for the presence of coliform bacteria.

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