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Slime Forming, SLYM-BART™ Quality Control

Slime-forming bacteria (SLYM) is the name given to bacteria that are able to produce copious amounts of slime without necessarily having to accumulate any iron. These slime-like growths are therefore often not dominated by the yellows, reds and browns commonly seen where IRB are present. Some of the IRB also produce slime but it is sometimes denser and has more texture due to the accumulation of various forms of insoluble iron. SLYM bacteria can also function under different reduction-oxidation (redox) conditions but generally produce the thickest slime formations under aerobic (oxidative) conditions. These can develop in the SLYM-BART™ as slime rings growing around the floating ball. Slime growth can also be seen as a cloudy (fluffy or tight plate-like structures) or as gel-like growths which may be localized or occur generally through the body of water medium. Very commonly the gel-like slime growths form from the bottom up in the test vials. One common check for these types of growth is to tilt the BART gently and see that the cloud- or gel- like growths retain their structure and tilt with the tube.

A vast majority of bacteria can produce slime-like growths. The slime is actually formed by a variety of exopolysaccharide polymers that are long thread-like molecules. These extracellular polymeric substances (EPS) literally coat the cells into a common slime-mass within which large volumes of water become clustered and bound. Often 95 to 99% of the volume of slime are actually water. Some bacteria produce an EPS that remains tightly bound to the individual cell. These are called capsules. Other bacteria generate such a copious amount of EPS that it envelops whole masses of cells within a common slime.

The role of the slime appears to be protective. If environmental conditions are harsh (e.g., due to shortage of nutrients), the slime layers tend to get thicker. Not only does the slime act as a protectant to the resident bacteria, but it also acts as a bio-sponge by accumulating many chemicals that could form either a nutrient base or be toxic to the cells. EPS may be produced by enzymatic activity (e.g., dextran sucrase or levan sucrase) on carbohydrates. In addition, EPS may be synthesized within the bacterial cells and released to form an enveloping slime.

Slime-forming bacteria tend to be aerobic and form slimes at redox fronts. In the BART tester, this front may form around the ball causing a slime ring, or deeper down in the liquid medium column to form an observable growth. This growth may be plate-like and appear to float at a specific depth, cloud-like with indefinite edges, form as basal dense slimes in the conical base of the test vial, or be gel-like and maintain its shape even when the vial is tilted. Since slime tends to be formed by bacteria under stress, it is common for the slimes to form after there has been an initial growth that may take the form of a localized or general cloudiness.

Many slime bacteria can produce various pigments that will color the slime. Such growths are usually white, grey, yellow or beige in color. These often darken over time particularly in the presence of daylight. Distinctive colored slimes include red (commonly associated with Serratia marcescens) and violet (associated with either Chromobacterium or Janthinobacterium species). Blackening may also occur particularly after growth. This may be a result of the production of either iron sulfides or carbonates which is commonly associated with the presence of mixed cultures including enteric bacteria in the SLYM-BART.

SLYM-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 SLYM organisms present in the water sample. Different microorganisms utilize various sites along the redox gradient under a ball to grow, and regular careful observations are needed to catch the start of growth so that the time lag can be determined.

Slime-forming bacteria cause very serious engineering problems since the slime formation can compromise the engineered specifications into many systems. Primarily, the effects of the slime growths are to reduce hydraulic or thermal conductivity and reduce water quality (generally, the first symptom is increased turbidity followed by taste, odor or color problems). As the slimes slough into the water later during the infestation, it can be expected to see sudden rises in the total organic carbon, increases in aggressivity and reductions in water quality.

Reaction Patterns

DS - Dense Slime (Gel-Like) (Reactions 1 & 4)
SR - Slime Ring around the Ball (Reaction 4)
CP - Cloudy Plates layering (Reaction 2)
CL - Cloudy Growth (Reaction 5)
BL - Blackened Liquid (Reaction 6)
TH - Thread-Like Strands (Rare Reaction not recognized)
PB - Pale Blue Glow in U.V. Light (Reaction 5-PB)
GY - Greenish-Yellow Glow in U.V. (Reaction 5-GY)

Of the above reactions, it is the CL (cloudy) reaction that is by far the most common. Often the CL will be preceded by a CP which will be transient (lasting commonly less than 24 hours). Descriptions of the various reactions are given below:

DS - Dense Slime
This reaction may not be obvious and may require the observer to rotate gently the BART test at which time slimy deposits swirl up. These deposits may swirl in the form of a twisting slime when the tube is gently rotated. This swirl can reach 40 mm up into the liquid column, or it may rise up as globular gel-like masses that settle fairly quickly. Once the swirl has settled down, the liquid may become clear again. In the latter case, care should be taken to confirm that the artifact is biological (ill-defined edge, mucoid, globular) rather than chemical (defined edge, crystalline, often white or translucent). Generally, these dense slime growths are beige, white or yellowish-orange in color.

CP - Cloudy Plates Layering
When there are populations of aerobic bacteria, the initial growth may be at the Redox front that commonly forms above the yellowish-brown diffusion front. This growth usually takes the form of lateral or "puffy" clouding which is most commonly grey in color. Often the lateral clouds may be disc-like in shape (plates) and relatively thin (1 to 2 mm). It should be noted that if the observer tips the BART slightly, the clouds or plates often move to maintain position within the tube. The edges of the plates are distinct while the edges of the "puffy" forms of layering are indistinct. These formations are most commonly observed 15 to 30 mm beneath the fill line. While cloud formations will tend to extend to cause an overall cloudiness of the liquid medium (CL). These plates sometimes appear to divide (multiple plating) before coalescing into a cloudy liquid medium.

SR - Slime Ring
A slime ring, usually 2 to 5 mm in width forms on the upper side of the ball. The appearance is commonly mucoid and may be a white, beige, yellow, orange or violet color that commonly becomes more intense over time on the upper edge.

CL - Cloudy Growth
Solution is very cloudy and there may sometimes be a poorly defined slime growth around the ball. Sometimes a glowing may be noticed in at least a part of the top 18mm of the liquid medium. This glowing is due to the generation of U.V. fluorescent pigments by some species of Pseudomonas. The common pigments doing this are a pale blue (PB) or a yellowish green (YG) color. Note that this glowing may not be readily observable unless a U.V. light is used. The occurrence of the glowing in a U.V. light means that there is a probability of potentially pathogenic species of Pseudomonas and confirmatory testing is recommended.

BL - Blackened Liquid
This is commonly a secondary or tertiary reaction rather than an initial reaction. It is recognized as a clear, often colorless, solution that is surrounded by large blackened zones in the basal cone and up the walls of the test vial. The BL often parallels the BL reaction in the IRB when the two BARTs are used together to test the same water sample.

TH - Thread-Like Strands
On some occasions, the slime forms into threads that form web-like patterns in the liquid medium. Sometimes these threads which interconnect from the ball to the floor of the inner test vial.

RPS (Reaction Pattern Signatures )

  • DS - CL Dense slime-forming bacteria producing copious EPS, facultative anaerobes dominate
  • SR - CL Aerobic slime-forming bacteria (such as Micrococcus) dominating with some facultative anaerobes
  • CP - CL Motile facultatively anaerobic bacteria dominate (e.g., Proteus)
  • CL - SR Mixed bacterial flora including some aerobic slime-formers
  • CL - BL Slime formers dominated by Pseudomonads and Enteric bacteria
  • CL - PB Pseudomonas aeruginosa dominant member of the bacterial flora
  • CL - GY Pseudomonas fluorescens species group present in the flora
  • TH - CL Aerobic bacteria dominant which are able to generate slime threads (e.g., Zoogloea)

    The slime-forming bacteria are amongst the fastest growing aggressive consortia and the medium used in this BART is very enriching and causes a wide variety of bacteria to grow rapidly. However, when the bacteria do not grow quickly, this indicates a very low population of aggressive bacteria. As a result of this, the time lags of between 3 and 6 days show a rapid decline in populations when compared to the IRB- or SRB-BART tests.

    Table Nine The Relationship Between Time Lag and the Population For Slime-forming Bacteria
    Time Lag (days) Population cfu/ml 1 1,800,000 2 350,000 3 66,500 4 12,500 5 2500 6 500

    Risk Potential Assessment
    The slime-forming bacteria are complex consortia involving many bacteria. These consortia inhabit a common "growth" of slime that acts as a communal chamber. Within these slimes, the bacterial cells are commonly dispersed and occupy only a small part of the total volume (<0.1%). Most of the slime is water bound to the organic polymers that bind the slime together. The SLYM-BART reflects the activities of bacteria that are present in the water as a result of the sloughing from the slime. As a result of this, the test may exhibit a complex set of reactions depending upon precisely which bacterial species are present in the water sample. Like the other BART tests, the shorter the time lag to the SLYM-BART displaying a reaction, then the greater becomes the aggressivity and the more urgent the need to treat. Not all reactions are equally important in determining the aggressivity of the slime-forming bacteria (and therefore the need to treat). Below is a list of the reactions described above and their relative importance in relation to the need to treat. Concern can be expressed through the shortness of the time lag (in days) as:

    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)
    Table Ten Relationship Between the Time Lag to the Reactions in a SLYM-BART and the Aggressivity of the Slime-Forming Bacteria
    Agressivity Very Sign. Moderate Not DS -Dense Gel Slime <1 2 3-7 >7 SR -Slime Ring <1 2-3 4-6 >6 CP -Cloudy Plates <0.5 1-2 3-6 >6 CL -Cloudy Growth <1 2 3-6 >6 BL -Blackened Liquid <1 2-4 5-8 >8 TH -Threads <2 3-4 5 >6 PB -Pale Blue Glow <1 2-4 5-8 >8 GY -Green-Yellow Glow <1 2-3 4-8 >8

    Some remedial treatments should be considered urgently where the time lag (in days) shows aggressivity to be at the very aggressive or aggressive (1 or 2) levels. Where there has been an RPS (sequence of reactions to form a signature), the aggressivity should be considered to be equivalent to the most aggressive using the above table.

    Hygiene Risk
    The most significant hygiene risk generated by this test is the BL reaction that indicates that Pseudomonads and enteric bacteria are present. If this reaction occurs within eight days, then a fecal coliform test should be performed on that water to determine the hygiene risk directly. Where PB or GY reactions are observed, this should be confirmed using the FLOR-BART™.

    SLYM-BART™
    Some bacteria that are not IRB also do form copious slimes. They can cause many of the problems commonly associated with IRB but tend to generate white, grey, beige or black forms of slime rather than the browns. Under the right cultural conditions, many aerobic bacteria will generate slimes. The selective medium used in the SLYM-BART specifically encourages the formation of slimes. These are commonly seen as condensed cloudy/plate-like growths suspended in the liquid medium, gel-like rings (around the ball) or globular/swirl forms of slime in the basal cone. This group is known as the slime-forming bacteria.

    SLYM-BART™ Medium
    A brown hardened gel-like mass with a darker inner circle that 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 3 mm 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 deposits visible around the wall at 6 to 8 mm above the basal cone. The characterization of the medium is given in Table Twenty-Seven.

    Table Twenty-Seven Medium Diffusion in a Sterile SLYM-BART Inner Test Vial to Confirm a Negative Reaction
    Time (days) Color Basal Lower column Upper column 0.25 Brown-yellow Clear Clear 0.5 Dark brown Light brown Clear 1.0 Light brown Light brown Light brown 2.0 - 10.0* Very light brown Very light brown Very light brown

    *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 crystalline deposits forming 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 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 to ensure any surplus saturation of the water with oxygen to have adjusted by the venting off of the surplus oxygen.

    Table Twenty-Eight QC Characterization of Medium Diffusion in a Sterile SLYM-BART Inner Test Vial
    Time (hrs) Color Contamination Basal Column* 0.5 Brown-yellow Clear Clear 12.0 Dark brown Light Brown Clouding 48-240 Very light brown Very light brown Clouding/slimes

    *See note for Table Twenty-Eight.

    Generally, where contamination does occur in the SLYM-BART™, it will initially be seen as a clouded zone floating above the diffusion front. These clouds may be plate-like or diffuse in form. After a period of time, these growths may condense into a basal gel-like mass that will either settle in the basal cone of the test vial, or form a slime-like ring around the FID. Colors will generally lighten except around the FID where any slime growths may cause colors such as beige, yellow, red, or violet to be generated. For the QC to be acceptable, there must be no evidence of contamination in 240 hours and the color generation should follow the pattern given in the above table.

    Confirmation of Selective Media Composition in the SLYM-BART
    In order to confirm the suitability of the selective medium for the biodetection of the various slime-forming 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 SLYM-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 uses a suspension of 0.1 ml of the broth culture in 15 ml of the sterile Ringer's solution. This innoculum should be taken from the midpoint of the broth culture immediately after the culture had been gently agitated. This inoculated solution is 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 Twenty-Nine.

    Table Twenty-Nine Cultural Characterization of the SLYM-BART
    A.T.C.C. Genus/species Characterization 8090 Citrobacter freundii CL Reaction 5 13048 Enterobacter aerogenes CL-BL Reaction 5 & 6 27853 Pseudomonas aeruginosa CL-PB Reaction 5 to PB 12228 Staphylococcus epidermidis DS Reaction 1 23355 Enterobacter cloacae CP-CL Reaction 2 to 5 13315 Proteus vulgaris CP-CL Reaction 2 to 5 13883 Klebsiella pneumoniae SR-CL Reaction 4 to 5 25922 Escherichia coli CL-BL Reaction 5 to 6

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

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