Don't Abandon Your Microscope! It Still Deserves a Place of Honor

Regular microscopic examination of MLSS can give operators valuable information to help prevent or correct process upsets.
Don't Abandon Your Microscope! It Still Deserves a Place of Honor
FIGURE 1. Phase contrast microscope observation of floc, filaments and surrounding liquid (100x magnification).

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I have seen many changes during my wastewater career, now extending beyond 30 years.

SCADA systems and associated instrumentation have really taken hold in many public and private treatment works, enabling operators to monitor processes, open and close valves, adjust flow rates and chemical dosages, and much more. Some SCADA systems are connected to cameras at key locations to give a real-time view of plant equipment in operation.

Still, SCADA has its limitations. In my opinion, nothing replaces a well-trained operator’s senses and intuition, especially when it comes to daily wastewater or water treatment plant operation. Operators using their eyes and ears and senses of smell and touch during rounds can tell when something doesn’t seem right. Operators of activated sludge facilities run settleability tests, and water plant operators perform jar tests, both using their sense of sight and their instincts in decision-making.

The microscope is one process control tool that doesn’t fit well into the SCADA world. Many utilities use a microscope for process control observation and record analysts’ results in an electronic database, like Excel spreadsheets and Hach Water Information Management Solution (WIMS) software.

However, there seems to be an increasing disconnect between actual plant operation and what the microscopic exam results are telling us. We have become reliant on SCADA systems, yet there are times when that information comes too late — the plant is already upset, and we missed the clues along the way.

I have attended many courses on activated sludge microbiology, and I want to share some of the key points of those sessions. I hope they help you as much as they have helped me.

Four key observations

When viewing a mixed liquor suspended solids (MLSS) sample from an activated sludge plant (Figure 1), there are four significant items I have found very useful.

Floc structure

Pay attention to floc shape and density. Is it uniform in size? Is it small or large? Is it round and granular-shaped, or large and irregularly shaped? Does the floc look like a piece of fluffy cotton or like a BB? Does light pass through the floc easily, or does it seem dark and dense?

Round, granular floc that resemble BBs most commonly point toward a higher sludge age, where the MLSS has become overoxidized (that is, it has been through the aeration process many times). These floc particles usually settle rapidly in the secondary clarifier and settleometer. You might also find the five-minute settleability reading to be very close to the 30-minute reading, and the sludge volume index (SVI) may be low.

Irregularly shaped, fluffy particles normally settle more slowly and compact less in the secondary clarifier and settleometer. The resulting SVI may be higher than normal. Fluffy floc particles could mean a younger sludge age (under-oxidized MLSS), or possibly a filament bulking sludge, discussed further below.

Clarity of the liquid around the floc

When the floc settles in the clarifier or settleability test container, the liquid that rises above the settled sludge blanket (supernate) becomes the secondary effluent. Is the liquid around the floc particles full of tiny particles, or is it relatively clean and clear? Are there very small pieces of round floc dispersed throughout the liquid?

If you have a phase contrast microscope, be sure to use the phase or dark-field settings to get a good look at the liquid surrounding the floc. You may be surprised at what you see. Phase contrast microscopes have special objectives, along with a substage phase contrast condenser that looks like a round turret mounted directly under the stage. Under this objective, the turbidity is much easier to observe, and tiny motile bacteria suddenly appear where they were unseen in bright-field light conditions. Tiny particles suspended in the supernate usually correspond with increased secondary clarifier effluent turbidity.

Filament abundance and identity

This observation can be difficult if you don’t have a quality phase contrast microscope. Filaments are usually colorless; they are found within the floc or extending beyond the floc surface. The term “filament” is more a description of the way the bacteria are growing.

Bacteria come in many shapes and sizes, and some grow in clumps that we call floc. Filaments are chains of bacteria that grow in hair-like strands instead of clumps. Some filaments are very thin and short, making them hard to see with a bright-field microscope. Special stains will help expose them, but this does require stain supplies and training in the proper use of the stains.

The Gram stain (Figure 2) and Neisser stain are used on slides with dried MLSS called smears. They can help an operator get a good visual image of filaments present. Filament abundance is a little easier to determine than the identity of all filament species in a sample.

Keeping track of the relative abundance and the trend of filament presence should be a part of the routine microscopic exam of the MLSS. More than 20 types of filaments could exist in your mixed liquor, many showing up in pairs. Training in filament identification is critical if you really want to uncover the cause of their growth and ways of reducing or eliminating them.

Remember, filaments are essential to good, strong floc formation and structure. Mixed liquor that contains no filaments will usually settle very rapidly, leaving a turbid appearance in the supernate. Secondary effluent quality can be reduced as the small, suspended particles flow over the weirs.

Floc is more durable when it contains some filaments, since sticky bacteria can attach to and grow along the filaments. Filaments are a nuisance when they become excessive, causing inter-floc bridging or slowing the rate of settling and sludge compaction. This condition, known as bulking, can be detrimental, especially if solids wash out of the secondary clarifiers.

If this occurs, the filaments should be identified and their root cause uncovered. This information can help operators eliminate or reduce the cause and improve settling and effluent quality.

Protozoa abundance, identity and activity

Most operators are familiar with the diverse population of protozoa and metazoa that can be found in the MLSS; many have a favorite they enjoy watching when using the microscope. Protozoa are single-celled organisms with organelles like contractile vacuoles, a nucleus and hair-like projections used for locomotion (cilia).

Protozoa observed in MLSS include amoeba, flagellates, free-swimming and stalked ciliates. Rotifers, nematodes (roundworms), gasterotrichs and waterbears (tardigrades) are multicelled organisms called metazoa.

When observing slides of MLSS, use several magnifications and illumination settings to get a complete picture of the protozoa and metazoa present. It is easy to overlook the almost transparent, blob-like amoebae if you do not adjust the light, contrast and magnification. Some organisms are much easier to see than others; don’t get into the practice of only looking for your favorite active protozoa (free-swimmers) and metazoa (rotifers) and overlook the slower-moving organisms like flagellates and amoebae.

When sludge age is relatively young and there is a large population of free-living, dispersed bacteria in the liquid around the flocs, bulk-liquid free-swimming ciliates may be common. These can be quite large, move quickly through the liquid and have large oral grooves. Bulk-liquid free-swimmers expend energy while moving about, gulping bacteria as they swim.

They are common due to the abundance of bacteria available as food. This abundance of bacteria also contributes to elevated BOD results in the clarifier effluent or final plant effluent.
As the abundance of free-living bacteria declines with rising sludge age and bacteria begin to form durable flocs, the population of bulk-liquid free-swimmers declines, giving rise to crawling free-swimming ciliates. These creatures have specialized cilia (cirri) that they use as scrapers to dislodge bacteria from the floc particle they are crawling on.

As they crawl on the floc, they graze on surface-level bacteria. The numbers of free-living bacteria in the bulk liquid decline or vanish, and bulk-liquid free-swimmers slowly disappear. During this process, the effluent quality is improving, and BOD and turbidity are decreasing.

Carnivorous free-swimmers are just as the name implies: they capture and consume the innards of other protozoa. Still other protozoa, like the large Spirostomum, are considered by some to be omnivorous, consuming algae, bacteria, fungi and small protozoa.

As the numbers of free-living bacteria in the liquid around the floc continue to decline and the floc continues to build, the available food value in the water also declines. Ciliates become more specialized in how they obtain their nourishment and energy. Stalked ciliates attach themselves to floc particles and use a ‘stalk’ to extend themselves into the open liquid. Once there, they use their cilia to move the water about in a circular motion and toward their gullet, consuming bacteria caught in the current. They expend less energy overall to get their food as compared to motile free-swimmers.

Getting clues

With this information, operators can use population observations to get an idea of what is occurring in the activated sludge process. Watching for trends or shifts in populations of protozoa and metazoa can be a clue to increases or decreases in plant effluent quality.

You don’t have to be an academic microbiologist to make the observations listed above. There are many reference manuals available on activated sludge microbiology, and quality training can be found almost everywhere. The topics explored in this article are only a few of the valuable lessons I’ve learned during courses I have attended. I continue to take microbiology courses when I can.

In summary, electronic monitoring instruments found in today’s modern treatment plants are valuable tools. Coupling SCADA information with timely, hands-on microscopic observation of the MLSS a few times per week can greatly enhance any process control regimen and help operators prevent or recover from plant upsets.

About the author

Ron Trygar, CET, is a senior training specialist at the University of Florida TREEO Center. He can be reached at


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