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Fluorescent Psuedomonads, FLOR-BART™ Quality Control

The pseudomonads are a very important group of Gram negative bacteria that are found in very substantial numbers in soils, waters and many other natural materials. In association with many plants and animals, the pseudomonads can act as agents for disease. In aerobic bioremediation and biodegradation processes, members of the pseudomonads often play critical roles in the biochemical breakdown of critical organic compounds. These various important aspects have led to the development of FLOR-BART which generate conditions favorable to the growth of the pseudomonads.

One critical aspect of this biodetector is the ability to generate soluble fluorescent pigments when some species of the genus Pseudomonas are dominant in the water. These pigments are usually produced after growth has occurred and generally can be detected most easily in the culture medium around the ball (top 20mm of the liquid column). Detection is by the use of an ultraviolet (U.V.) lamp aimed at the top one third of the liquid column in the FLOR-BART. Maximum excitation of these fluorescing molecules is at 400nm. There are two main pigments, pyocyanin and pyoverdins. Pyocyanin is a distinctive pigment that fluoresces with a pale blue to blue color and is most commonly associated with the species, Pseudomonas aeruginosa. This species is commonly associated with clinical specimens (wounds, burns, otitis, sepsis, pneumonia, urinary tract infections), a condition known as "blue pus," and is a hygiene concern in recreational waters. Pyoverdins is the name given to a group of other fluorescent pigments generated by different species of Pseudomonas. Commonly these pigments are referred to as fluorescens and generally have a greenish-yellow glow. The species Ps. fluorescens generates these types of pigments and is commonly associated with the spoilage of foods (eggs, cured meats, fish and milk). The FLOR-BART has been designed to generate these pigments where there is a dominance of fluorescent pseudomonads (hence the prefix, FLOR). If Ps. aeruginosa is detected in a water sample and there is a concern for the potential hygiene risk, it is recommended that confirmatory diagnosis be performed in a recognized diagnostic microbiology laboratory using either the positive FLOR-BART or a fresh sample as the source for the diagnosis.

Other pigments are sometimes produced. These are usually insoluble and non-fluorescent in U.V. light. These are commonly yellow, beige or orange in color and tend to be transitory. One species Ps. stutzeri sometimes generates a reddish-brown pigment later in the growth cycle that is very distinctive. This pigment may concentrate either in the slime ring around the FID ball or in the base of the test vial.

Microorganisms present around the ball in the FLOR-BART can generate these different pigments in the presence of oxygen. Usually these pigments are generated after a cloudy growth has developed in the liquid medium but before there are intense slime-like growths around the ball (as a slime ring). The fluorescent pigments may be difficult to observe with natural and artificial light, but they can be seen using a typical broad spectrum U.V. light whereupon the pigments glow (fluoresce).

There is often a need to test waters for the presence of Pseudomonad bacteria because these bacteria are often dominant in waters which contain oxygen and are rich in a narrow range of organic pollutants (e.g., gasoline, jet fuel, solvents). When these bacteria are present and active, there are two particular events that may need to be considered. First, the presence of Pseudomonad bacteria may indicate that aerobic biodegradation is occurring and biofouling may also be happening within the system being tested. Second, some of the Pseudomonad bacteria that produce the fluorescent (glowing in U.V. light) pigments may be a hygiene risk. The faster that clouding and fluorescing happens, the more aggressive are the Pseudomonad bacteria.

For the FLOR-BART, there are two U.V. fluorescent pigments which can be recognized as:

Pale Bluish Glow that will last for one to four days and then gradually fade. The glow is normally fairly faint and should be viewed against a darkened background since direct light may make viewing more difficult. One major species bearing this pigment (pyocyanin) is Pseudomonas aeruginosa. It is of concern since this species can be associated with a range of opportunistic infections. It is also one of the bacterial species found associated with mastitis in cattle. This species can also be found in a variety of waters.

Greenish-Yellow Glow that may last for two to ten days and then gradually fade away. The glow becomes fairly obvious and is often visible even without using the U.V. light. One major species bearing this pigment (the pyoverdin, fluorescein) is Pseudomonas fluorescens. Generally, this species is not as virulent as Ps. aeruginosa and is often more abundant in waters and can be involved in specialized aerobic degradation of organic pollutants.

In essence, this test selectively allows the detection of pseudomonad bacteria in the water with the separation of the fluorescent species. Pseudomonad bacteria can cause a range of problems in waters. Problems range through slime formations, turbidity, taste and odor, corrosion and biodegradation through to greater hygiene risks. In recreational waters (such as swimming pools, hot tubs, restricted natural bathing sites), the presence of aggressive fluorescent pseudomonads should be taken as a potential cause for concern since these bacteria may cause a range of skin, eye, ear and urinary tract infections. Occasionally the pseudomonad bacteria will cause skin infections particularly under tight fitting bathing apparel. This is particularly a potential problem in warmer waters and hot tubs where the bathers remain relatively inactive in the waters for prolonged periods.

The pseudomonad bacteria often dominate aerobic biodegradation of organic pollutants and determining the aggressivity and possible population size can often monitor the rates of degradation. If the organic pollutant is being degraded aerobically or in a situation where there is a significant quantity of nitrates to support respiration, there is a potential for the degradation to be dominated by the Pseudomonad bacteria. Monitoring the aggressivity of these bacteria using the FLOR-BART enables the user to monitor the amount of biodegradation occurring.

Pseudomonad bacteria are also sometimes associated with taste and odor problems in water since many of the species produce distinctive odors such as "fishy" or "kerosene-like" which can become very dominant in the water.

Reaction Pattern
PB - Pale Blue Glow in U.V. Light
GY - Greenish-Yellow Glow in U.V. Light

These reactions are described in more detail below. Care should be taken to follow manufacturers cautionary notices when using a U.V. light source to observe glowing in the BART tests.

PB – Pale Blue Glow
Solution very cloudy (passes through reaction one) and then generates a glowing around FID when ultraviolet light is shone onto the side-walls of the inner test vial. This glowing fluorescence occurs usually in the top 15 to 20 mm around the ball and gives a pale blue glow. This glowing commonly lasts 2 to 3 days.

GY – Greenish Yellow Glow
Solution very cloudy (passes through reaction one) and then generates a glowing around FID when ultraviolet light is shone onto the side-walls of the inner test vial. This glowing fluorescence occurs usually in the top 15 to 20 mm around the ball and gives a greenish-yellow glow. This glowing lasts commonly for 4 to 8 days (latter case).

RPS Reaction Signatures

  • PB Pseudomonas aeruginosa likely to be present
  • GY Pseudomonas fluorescens species group likely to be present

    Table Thirteen The Relationship Between Time Lag and the Population For Fluorescing Pseudomonad Bacteria
    Time Lag (days) Population cfu/ml 1 1,000,000 2 100,000 3 10,000 4 5,000 5 1,000 6 100 7 100 8 100

    The fluorescing pseudomonads are only a part of typical slime-forming or heterotrophic aerobic bacterial consortia. As such, they have to be in high populations and very aggressive in order to begin to produce the ultraviolet (U.V.) fluorescence that is typical for the species of Pseudomonas that are capable of doing this. A time lag of longer than five days may have a small population but only if the U.V. glow produced is pale blue. This would still be a concern if the nosocomial pathogenic bacterial species Pseudomonas aeruginosa was present in the sample and confirmatory tests using the traditional microbiological procedures may need to be undertaken as a precaution.

    Population Assessment of Fluorescing Pseudomonads using a BART Extinction Dilution Technique
    To measure the population of fluorescing pseudomonad bacteria, four dilutions of the original water sample should be used. These dilutions can be achieved using the following semi-quantitative technique:

    1. Dispense 14 ml of sterile water into each of four FLOR-BART™ tests. Label these tubes: "1", "2", ""3", and "4."
    2. Charge a FLOR-BART™ with the water sample (15 ml) and label "0."
    3. Withdraw 1 ml of water from tube "0" and transfer into tube "1." Invert and gently shake tube for 10 seconds. Allow to settle (5 seconds).
    4. Withdraw 1 ml of water from tube "1" and transfer into tube "2." Invert and gently shake tube for 10 seconds. Allow to settle (5 seconds).
    5. Withdraw 1 ml of water from tube "2" and transfer into tube "3." Invert and gently shake tube for 10 seconds. Allow to settle (5 seconds).
    6. Withdraw 1 ml of water from tube "3" and transfer into tube "4." Invert and gently shake tube for 10 seconds. Allow to settle (5 seconds).
    7. Observe the tubes for PB or GY fluorescence after three days of incubation at room temperature. Note that this day may be changed if an alternate day is found to display maximum fluorescence.
    8. Refer to table below to semi-quantitatively determine population based upon the tests that exhibit fluorescence in a U.V. light.

    Hygiene Risk
    If a PB reaction is observed, there is a risk that Pseudomonas aeruginosa may be present in the water sample and could cause an infection in humans. These infections can range from pneumonia to skin, eye and ear infections. Where a population is detected and confirmed using the extinction dilution technique described above, the tube "0" FLOR-BART should be submitted to a suitable microbiology laboratory to confirm the diagnosis. If the fluorescence is of the GY type, then a similar precaution should be taken if the population is >2.0 log fluorescing pseudomonads/ml.

    Table Fourteen The Relationship of Positive Detection of Fluorescence to The Population
    Tube # Population Assessment "0" F F F F F "1" F F F F ---- "2" F F F ---- ---- "3" F F ---- ---- ---- "4" F ---- ---- ---- ---- ____ ____ ____ ____ ____ Possible Population: >5.0 >4.0 >3.0 >2.0 >1.0 (log . fluorescing pseudomonads/ml)

    The fluorescent pseudomonad bacteria are a group of bacteria that have created some level of hygiene risk particularly in hospital and recreational environments. This BART utilizes a selective medium that encourages the growth of species of Pseudomonas which are able to generate fluorescent pigments particularly in the upper part of the inner test vial around the ball.

    FLOR-BART Medium
    A yellowish semi-transparent hardened film covers the basal cone and may extend as a more transparent film up the sidewalls of the test vial to a height not exceeding 3mm. The texture of this deposit is roughened granular and some lighter linear crystalline deposits may be seen radiating out from the central peg in the basal cone. Diffusion characteristics of the FLOR-BART using sterile water is given in Table Thirty-Four.

    Table Thirty-Four Medium Diffusion in a Sterile FLOR-BART Inner Test Vial to Confirm a Negative Reaction
    Time (days) Color Basal Lower column Upper column 0.25 Light yellow Clear Clear 0.5 Light yellow Clear Clear 1.0 Very light yellow Very light yellow Clear 7.0* Very light yellow Very light yellow Clear

    *Note that the liquid medium is crystal clear and has been generated using sterile distilled or deionized water. Natural water samples can cause minor chemical reactions that may be seen through an intensification of the color in the diffusion front and sometimes crystalline deposits may form in the base of the test vial. These crystalline deposits can be differentiated from a basal slime since the crystalline deposits swirl up and have a sharply defined edge, do not have a gel-like appearance, and settle rapidly to the base after shaking. 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. Therefore do not use water taken directly from a refrigerated or cold source but allow the water to rise to room temperature before beginning the test. This will ensure that any surplus saturation of the water with oxygen will be vented off and not interfere with the test.

    Table Thirty-Five QC Characterization of Medium Diffusion in a Sterile FLOR-BART Inner Test Vial
    Time (days) Color Contamination Basal Mid- column 1.0 Very light yellow Clear Clouding 7.0 Very light yellow Clear Turbid or slimes

    The FLOR-BART should remain very clear with only a very light yellow coloration near the base of the test vial. If there is contamination, this is usually caused by pseudomonads or aerobic Gram positive bacteria. These growths usually develop as a general cloudiness that then intensifies to make the medium turbid. Slimes sometimes are formed.

    Confirmation of the Selective Media Composition in the FLOR-BART
    In order to confirm the suitability of the selective medium for the biodetection of the various pseudomonad bacteria recognized by this test method, it is recommended that the following A.T.C.C. (American Type Culture Collection) strains be applied to the FLOR-BART to determine the standard reaction patterns. Each culture should be prepared as a 48 hour broth culture incubated at 30oC to reach the stationary growth phase using Brain Heart Infusion broth. Inoculation of the inner test vial should be using a 0.1 ml 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 inoculated solution should be applied directly over the ball as the test vial is filled. Do not shake the vial. Incubate at 22 to 24oC for five days and observe for activities and reactions. Typical results are listed below for the recommended A.T.C.C. strains in Table Thirty-Six.

    Table Thirty-Six Cultural Characterization of the FLOR-BART
    A.T.C.C. Genus/species Characterization 13048 Enterobacter aerogenes CL Reaction 1 27853 Pseudomonas aeruginosa CL-PB Reaction 1 with PB 12228 Staphylococcus epidermidis No growth 19606 Acinetobacter calcoaceticus CL Reaction 1

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