Microscopic Exam...

The Microscope...

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Parts of The Microscope...

• Microscope stand
• Specimen stage (where the slide lies)
• Nosepiece (holds the objectives)
• Eyepiece (may be 1 or 2)
• Condenser (concentrates the light on the slide)
• Light source
• Objectives
• 10 X objectives magnify the specimen 100 times
• 20 X objectives magnify the specimen 200 times
• 40 X objectives magnify the specimen 400 times
• 100 X objectives magnify the specimen 1000 times (this objective must be used with immersion oil)

"Parts of The Microscope"...
Types of Microscopes...

• Bright-field Microscope : When you observe a live sample under brightfield, there is very little contrast between the water and the organism, therefore the organisms do not stand out and are not very distinct because the organisms appear as if they are clear. However, when samples are stained, there is more contrast. Brightfield is not good for observing live samples but is ideal for stained slides.
• Phase-Contrast Microscope : Phase-contrast allows you to clearly distinguish organisms and their internal structures. This microscope is more suitable for observing live organism and is the best one to use in water and wastewater laboratories.

Slide Preparation and Staining...

Wet Mount...

1. A drop of activated sludge is placed on a clean grease-free glass slide.
2. A clean cover-glass is placed on the drop.
3. Avoid entrapment of air bubbles as much as possible.
4. The size of the drop should not be so large that the cover-glass floats and it should not be so small that it doesn't fill the entire space under the cover-glass.
5. Use a pipette with a small opening.

Fixed Smears and Staining...

1. Place a drop of activated sludge on a clean, wax-free slide and smear.
2. Let the slide air dry. Do not heat fix !

Mixed Liquor Sample Collection...

The sample should be collected from the discharge end of the aeration basin. Slides should be made from a fresh sample of mixed liquor. If the sample has been collected and can not be examined right away, the sample should be aerated until it can be evaluated.

Nematode...

Microbiology of Activated Sludge...

Activated sludge can be defined as "a mixture of microorganisms which contact and digest bio-degradable materials (food) from wastewater."

• Activated sludge is microorganisms.
• The Activated sludge process is a biological process.
• To properly control the activated sludge process, you must properly control the growth of microorganism. This involves controlling the items which may affect those microorganisms.

Suctoria...

Bacteria...

• Make up about 95% of the activated sludge biomass.
• These single celled organisms grow in the wastewater by consuming (eating) bio-degradable materials such as proteins, carbohydrates, fats and many other compounds.

The Role of Enzymes...

Enzymes are compounds that are made by living organisms. Their purpose is to help biochemical reactions to occur. Almost all biochemical reactions require the presence of enzymes to cause the reaction to occur. Enzymes help bacteria in the process of breaking down nutrients, and in rebuilding broken down nutrients into the new compounds that they require for growth and reproduction. Enzymes only do what they are supposed to when environmental conditions are right. If the conditions are not right the enzymes will not function properly, thus, the bacteria will not function properly, and they will not survive. If conditions are right the bacteria will live and prosper.

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Growth Characteristics...

When there is plenty of food available, bacteria use the food mostly for growth and some for energy.

• A growing bacterium have flagella (hair-like structures on the outside of the cell) which makes it motile, able to move in search of food.
• A bacterium reproduces into two bacteria. The cell splits into two smaller cells and this process occurs over and over again.

When there is very little food available, the bacteria use the limited food to produce energy and to maintain the cell. Very little is available for growth so less reproduction occurs.

• With little food available, and in an attempt to conserve energy, the bacterium loses it flagella and thus, its motility.
• The waste products start to form a thick slime layer outside the cell wall, making the cells stick together.

The growth characteristics of bacteria are better understood by studying the growth curve.

• Lag-phase During this phase bacteria become acclimated to their new surroundings. They are digesting food, developing enzymes and other things required for growth.
• Accelerated Growth-phase The bacteria are growing as fast as they can, since there is an excess of food. The cells are mostly dispersed, not sticking together.
• Declining Growth-phase Reproduction slows down because there is not an excess of food. A lot of food has been eaten and there are now a large number of bacteria to compete for remaining food, so the bacteria do not have enough remaining food to keep the growth rate at a maximum.
• Stationary-phase The number of bacteria is the highest possible, but not much food is left, so the bacteria cannot increase in number. There is some reproduction, but some cells are also dying, so the number of bacteria remain relatively constant. The bacteria have now lost their flagella and have a sticky substance covering the outside of the cell, allowing them to agglomerate into floc. In fact, the floc get big enough that if aeration and mixing were stopped, the floc could settle to the bottom.
• Death-phase The death rate increases with very little if any growth occurring. Therefore, the total number of living bacteria keeps reducing. The bacteria are just trying to keep alive.

"Growth Curve"...

F : M (Food to Microorganism ratio)...

We measure the amount of biodegradable matter the bacteria use for food by measuring the amount of BOD (biochemical oxygen demand) or COD (chemical oxygen demand) in the influent to the aeration basin. We estimate the weight of microorganisms in the mixed liquor by measuring the amount of volatile suspended solids (VSS) in the activated sludge. We use this information to form a relationship called food to microorganism ratio (F/M ratio). The F/M ratio tells us something about growth and cell condition. If the F/M ratio is high, the bugs normally grow quite rapidly (because this means there is a lot of "food" available in comparison to the amount of microorganism); if the F/M ratio is low, the bug normally grow very slowly (because little food is available for growth).

Use of Oxygen...

Microorganisms need oxygen to live. Oxygen use and be used to determine the activity of the organisms. (1) Actively growing organisms are rapidly metabolizing the food, so they are use oxygen at a rapid rate. (2) We measure the rate at which oxygen is used by a test called the Oxygen Uptake Rate (OUR), or the Respiration Rate. It is measured in mg O₂ / g MLSS . hr. (3) Normally a higher uptake rate is associated with high F/M ratios and younger sludges and a lower uptake rate is associated with lower F/M and older sludges. So, if you want a higher uptake rate, more sludge should be wasted. Less should be wasted if you want a lower F/M ratio.

Formation of Floc...

As bacteria begin growing, they generally develop into small chains or clumps. They are very active and motile and it is difficult for them to settle. They have not yet developed the slime layer which aids in their sticking together. So, when mixing occurs, the small chains or clumps are broken up and the bugs are dispersed, and they will not flocculate or settle. As the sludge is allowed to age, the bugs lose their motility and accumulate more slime. Then the clumps and chains are better able to stick together. The clumps grow bigger and bigger until they form a floc. If the organisms are allowed to develop properly, under the right conditions, the floc get large and compact and begin to settle. The mixing in the aeration tank tends to keep the floc small since, even though the bugs are sticky, the bond formed holding the organisms together is not very strong. This is good because it allows the cells, food, and oxygen to contact each other.



Dissolved Oxygen...

Oxygen is required by these bugs to metabolize food for cell maintenance and growth. Although the bugs need oxygen, some bugs can get along with less oxygen than others. Each bug must have a dissolved oxygen of at least from 0.1 - 0.3 mg / L to function properly. So, it is important to maintain about 2 mg / L of DO in the activated sludge so that the bacteria that are contained in the floc can get oxygen. If the DO is less than 2 mg / L, the bugs on the outside of the floc use the DO before it can get to the center of the floc. If this happens, the bugs in the center may die causing the floc to break up.

Effects of Mixing..

Mixing is required to bring organisms, oxygen, and nutrients together, and to remove metabolic waste products. If there is not enough mixing, proper treatment will not take place because of lack of contact between the bugs, their food and oxygen. If too much mixing is provided, it can cause break up of floc or formation of unstable floc particles.

Effects of pH...

The enzymes which regulate many of the biochemical reaction in bacteria are very pH dependent. The optimum pH should be between 7.0 and 7.5 for the proper activated sludge microorganisms to dominate.

Effects of Temperature...

Biochemical reactions are very temperature dependent. Lower temperatures cause such reactions to be much slower. Thus, more bugs are required to do the same job during the winter than in the summer.

Effects of Nutrients...

Microorganisms require certain nutrients for growth. The basic nutrients of abundance in normal raw sewage are carbon (C), nitrogen (N), phosphorus (P), with the ratio of C:N:P ratio approximately equal to 100:10:1. In addition to C,N,and P, trace amounts of sodium (Na), Potassium (K), magnesium (Mg), iron (Fe), and many others are required. In normal municipal sewage, most of these nutrients are provided. Most problems with nutrient deficiency occur when there is a lot of industrial wastes present. When proper nutrients are not available, the metabolism fails and a kind of bacterial fat (slime) will begin to accumulates around the cell. The cell slows down in activity because it cannot produce enough enzymes and because needed nutrients cannot penetrate the slime layer as they should. The sludge will not settle and BOD removal slows down.

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Rotifer...

Protozoa & Rotifers...

The presence of particular types of protozoans is related to effluent quality and plant performance. Protozoan play secondary but important role in purification of aerobic wastewater. The protozoans in the activated sludge treatment process fall into four major classes: amoebae, flagellates, and ciliates (free-swimming, crawling, and stalked).

• Amoebae : Amoebae are the most primitive, single-celled protozoans. They move by false feet. They are frequently present in raw influent, and their presence is short in the aeration basin. Amoebae can only multiply when there is an abundance of nutrients in the aeration tank. They move very slowly and it is difficult for them to compete for food the there is a limited amount available. They are only dominant in the aeration basin for a short time. They feed on small organic particulates. When amoeba are present in large numbers in the aeration basin this usually indicates that there has been some sort of shock loading to the plant (there must be a lot of food available). Their presence may also indicate that there is a low D.O. environment in the aeration basin, because they can tolerate very low amounts of D.O.
• Flagellates : Most flagellates absorb dissolved nutrients. Soon after amoebae begins to disappear and while there is still high concentrations of soluble food. Flagellates and bacteria both feed on organic nutrients in the sewage so as the nutrient level declines they have difficulty out competing the bacteria for soluble food so, their numbers begin to decrease. If large amounts of flagellates are present in the later stages of the activated sludge development this usually indicates that the wastewater still contains a large amount of soluble organic nutrients.
• Ciliates : Ciliates feed on bacteria not on dissolved organics. While bacteria and flagellates compete for dissolved nutrients, ciliates compete with other ciliates and rotifers for bacteria. The presence of ciliates indicate a good sludge, because they dominate after the floc has been formed and after most of the organic nutrients have been removed.
• Free-swimming ciliates - These ciliates appear as flagellates begin to disappear. As the bacterial population increases, a lot of dispersed bacteria is available for feeding and as a lightly dispersed floc appears, free-swimming ciliates begin to dominate and feed on the increased numbers of bacteria.
• Crawling ciliates - As floc particles enlarge and stabilize, crawling ciliates graze on floc particles. Crawling ciliates out compete free-swimming ciliates for food because they can find food within the floc.
• Stalked ciliates - Stalked ciliates appear in the mature sludge. Within the mature sludge the crawling and stalked ciliates compete for dominance.

Ciliates...

Stalked Ciliates...

Factors Influencing Protozoa...

Temperature : Most protozoans can survive and reproduce in a temperature range at which activated sludge processes are carried out. They grow best in ambient temperatures (15 - 25 ^oC).
pH : Protozoans are more sensitive to pH than floc-forming bacteria. They have an optimum pH range of 7.2-7.4 and a tolerance range of 6.0 - 8.0.
Dissolved Oxygen : Like bacteria, protozoan must have oxygen to survive. Thus lack of DO will severely limit both the kind and number of protozoans.
Nutrition : Most municipal wastewater treatment plants, however dilute, contains sufficient nutrients to support most of the protozoan associated with wastewater.

Rotifers...

Rotifers are rarely found in large numbers in wastewater treatment processes. The principal role of rotifers is the removal of bacteria and the development of floc. Rotifers contribute to the removal of effluent turbidity by removing non-flocculated bacteria. Mucous secreted by rotifers at either the mouth opening or the foot aids in floc formation. Rotifers require a longer time to become established in the treatment process. Rotifers indicate increasing stabilization of organic wastes.

Indicator Microorganisms...

The indicator microorganisms that feed on bacteria can be observed by using inexpensive microscopes at lower powers of magnification. Some forms may need to be stained for viewing. Process status can then be interpreted from these observations. For operational purposes, the relative types of microorganisms present in an activated sludge sample, the predominance of each of the various types and the mobility of the microorganisms are the primary concerns. The microorganisms that appear most frequently and the activity of these microorganisms enables the operator to interpret occurrences in the process. The more common microorganisms encountered and their relative numbers are shown below.

"Relative Predominance of Microorganisms"...

One important operational philosophy that merits a constant reminder is that ; (1) any specific microorganism predominates in a given environment because it thrives in the existing conditions, (2) normally the operator has created the conditions favorable to the microorganism and (3) to decrease the microorganism predominance, the operator need only change its environment (that is control the process) to provide less favorable conditions. If the environment is changed drastically, a different microorganism will quickly predominate (unless all microorganisms are destroyed). A normal transition results in the gradual disappearance of the former, during routine observation, with the latter gradually taking over.

"Wanted Dead"...
"Wanted Alive"...

Filamentous bacteria...             Nocardia...

Introduction to Filament Identification...

Filament Identification...

In order to identify many of the following filament characteristics, the mixed liquor must be examined under 100X using immersion oil. It is difficult to see many of these characteristics under lower magnifications.

Filament Shape and Length...

Filaments may be long, short, smoothly curved, coiled, irregularly bent, straight, or bundled.

Individual Cell Shape...

Filamentous bacteria are made up of a chain of cells. The shape of the individual cells is a characteristic that can help us to identify the different filamentous bacterial types. Cell shape may be round, square, rectangular, oval, or discoid.

Cell Septa...

The cell septa is the "line" which separates each individual cell which makes up the bacterial filament. The septa are clearly seen in some filaments an is very difficult to see in others. Some septa are "indented" and some are not. Indentations and the ability to clearly see the cell septa are other characteristics which can help us to identify the different filamentous bacteria.

Motility...

Motility is the ability of an organism to produce motion or to move. Beggiatoa spp is only one filamentous bacterium found in activated sludge that is motile.

Intercellular Granules...

Some filaments store by-products as intercellular granules (mostly sulfur granules). Sulfur granules can be seen very clearly under phase contrast and are found usually in septic wastes. Sulfur granules are commonly found in Beggiatoa, Thiothrix and Type 021N.


Filamentous bacteria...
Branching...

Branching may be "true" or "false". If a filament has true branching the intercellular fluids will flow freely throughout all the branches of the filament. Intercellular fluids cannot flow through false branches. In false branching the filament are simply attached to each other simulating a branch. There are only two filaments which exhibit branching; one has true branching and the other false. Nocardia spp has true branching and Sphaerotilus natans exhibits false branching.

Sheath...

The cells of some filamentous organisms are contained in a tight fitting sheath. The easiest way to detect a sheath is to look for "missing spaces" between the cells. Some filaments which have a sheath are Haliscomenobactor hydrosis, Sphaerotilus natans, Type 1701, Type 0041, and Type 0675.

Attached Growth...

Some filaments have bacterial cells attached along the side, perpendicular to the filament. There are three filaments on which this commonly occurs. Type 0041, Type 0675, and Type 1701.