Lesson 1:
Introduction to Microbiology




In this lesson we will learn the following:



Reading Assignment

Along with the online lecture, read Chapter 1 in Microbiological Examination of Water and Wastewater.





Microorganisms are a heterogeneous group of organisms too small to be seen with the naked eye.  They typically exist as single-celled or simple aggregates of cells in varying sizes, generally less than 0.0039 inches.  Microorganisms traditionally include bacteria, protozoa, some algae, fungi and viruses.  They are ubiquitous in nature.
Many people still believe that all microorganisms are harmful.  In truth, the benefits microorganisms provide far outweigh the harms.

Microbiology is more than simply the study of microorganisms.  It is concerned with the roles of microorganisms in disease, changes they make in the environment and products they generate.



Classification of Bacteria

The first classification of living organisms which serves as the basic for the system in use today was developed by Linnaeus in 1700.  This classification system consists of the following:

          Kingdom – a group of related phyla
          Phylum – a group of related classes
          Class – a group of related orders
          Order – a group of related families
          Family – a group of related genera
          Species – organisms of one or the same kind

In water microbiology, the primary focus is on bacteria of the family Enterobacteriaceae.  These include:

**pathogens (all or part of genus)


Linnaeus also developed the binomial naming system, where each living thing is given a generic (genus) and a specific (species) name.

Bacteria are classified based on major observable features.

Cultural classification – the culture media and environmental conditions which support optimal growth (ex. with or without oxygen, incubation temperature)

Microscopic examination – used to examine staining characteristics and cell shape.  The principle stain used is the Gram stain where a cell exhibits either a positive (deep purple) or negative (red) reaction.  Bacterial cell shapes include:
                   Spheres (cocci)
                   Coils (spirochetes)
                   Rods (bacilli)
                   Spirals (spirilla)

These exist as chains, clusters, pairs, tetrads, singles or other configurations.

Characterization of metabolism – when similar gram stain characteristics are noted, physiological characteristics are used, based on the fact that the genetic makeup of a cell reflects its ability to utilize and metabolize nutrients in the environment.  Microorganisms are grown in the presence of a specific nutrient substance, or substrate, and examined to determine what chemical change took place.  For example, the breakdown of starch or the fermentation of glucose to produce acid and gas are often used to separate bacterial species.  The type of metabolism, aerobic, anaerobic, or facultative anaerobic, is also an important classification tool.

Chemical characterization – involves breaking the microorganism apart cell membrane and nuclear structures.

Genetic characterization – DNA determination, PCR technology



Reproduction and Growth

Bacteria reproduce by a process known as transverse fission, an asexual reproduction method (although some species have a sexual mode).  For growth, these microorganisms must have an energy source, typically complex carbohydrates; trace inorganics such as metals, phosphorous and nitrogen compounds; vitamins and water.

A bacterial growth curve consists of three stages – lag, log and stationary – with a generation time of several minutes to hours.  In the initial lag phase, bacteria are increasing in size and adjusting to the environmental conditions.  The log stage is a period of rapid growth (See Bacterial Multiplication Chart), followed by the stationary phase when available nutrients have been depleted and sometimes, toxic products produced.  Without further nutrient sources, most of the bacteria will die off.






Waterborne Bacteria


Most waterborne pathogens can be classified as viruses, bacteria or protozoa, with bacteria recognized as the largest group.  Pathogenic bacteria, protozoa and helminthes (parasitic worms) can be found in a wide range of warm blooded animal hosts, including humans, however, viral pathogens are only shed by infected people.  Several other pathogens, such as Legionella, do not have an animal host and appear to have both pathogenic and non-pathogenic strains ubiquitous in the environment.  The minimum infective dose of these organisms necessary to cause illness can be several hundred to several thousand.  




Pathogenic bacteria are poor competitors and are therefore usually eliminated as a waterborne concern.  The most common bacterial pathogens are the Shigella, Salmonella and E. coli.  Shigella causes dysentery, most commonly in food but isolated in water in underdeveloped countries.  These organisms have a short survival time in sewage and therefore are not prevalent in the United States.  The incidence of illness from Salmonella peaks in the summer months and is carried by humans (1-4% of the population), farm animals (13-17% incidence) and wild animals.  Most Salmonella cause gastrointestinal disease, with one species a strictly human pathogen causing typhoid.  Enteropathogenic E. coli causes gastrointestinal disease and uninary tract infections.

The variety of pathogens in water is high and the incidence is low.  No single method is available to isolate and identify all pathogens, and testing for each individually is expensive and often unreliable due to the lack of acceptable analytical techniques.  Also, the limited methods that are available are laborious, costly, require trained personnel and usually require an extended incubation period.  The result is methods which are unacceptable when a quick public health response is required.  (See following table).





Coliform Bacteria

In order to rapidly determine if pathogens are likely to be present in a water sample, coliform and E. coli were identified as indicator bacteria which would fulfill the following requirements:


Coliform bacteria are indicators of bacterial pathogens and historically were not considered indicators of protozoa and viruses.  These organisms usually survive longer than E. coli and may also survive disinfection.  However, the EPA recently reviewed data collected through the Information Collection Rule (ICR) and other available studies and concluded that E. coli can be used as a reliable indicator of the potential presence of Cryptosporidium in a source water supply.

The coliform group is typically defined as bacteria in the genera Klebsiella, Enterobacter, Citrobacter, Escherichia.  They are found in large numbers in the microbial flora of warm-blooded animals, with the number of organisms increasing proportionally as the population of animals increase.




Several species of coliform, including Enterobacter, Klebsiella and Citrobacter can be found several miles downstream of a waste discharge point.  These organisms adapt well to the minimal nutrient composition of water and are able to encapsulate in adverse environmental conditions.  As a result, they are often found in distribution system samples.

Domestic animals are often implicated in source water contamination.  In New York City alone, the 500,000 owned dogs contribute 150,000 lbs. of feces and 90,000 gallons of urine daily through storm water runoff.  In rural areas, farm animals and wildlife contribute large quantities of waste material to the potential water source.  Coliforms have also been identified in groundwater from waste sites up to 2 months after burial.

In general, coliform bacteria are aerobic or facultative anaerobic, gram-negative, non-spore forming, rod-shaped bacterium.  Fecal coliforms are a sub-group of coliform bacteria which are present in the gut and feces of warm-blooded animals.  E. coli are one member of fecal coliform bacteria.  Figure 1 explains the relationship of these bacteria groups.




Coliform Testing

Advances in testing techniques have redefined the term “coliform bacteria”, which is now based more specifically on the test method used.  For example, using multiple tube fermentation (MTF) with lauryl tryptose (sulfate) broth, coliforms are defined as those bacteria which will ferment lactose to produce acid and gas within 48 hours at 35°C.  Fecal coliforms are further defined as those bacteria which ferment lactose within 24 hours at 44.5°C.  When using the newer chromogenic/fluorogenic techniques coliforms are defined as those organisms that possess the enzyme ß-galactosidase and E. coli as those coliform organisms that also possess the enzyme ß-glucuronidase.




Bacteria colonize the human environment: homes and businesses, the soil, even the human body. Tiny as they are, bacteria can pose enormous threats to public health if conditions allow them to thrive and multiply. Although all bacteria share certian structural, genetic, and metabolic characteristics, important biochemical differences exist among the many species of bacteria. These differences permit bacteria to live in many different, and sometimes extreme, environments. For example, some bacteria recycle nitrogen and carbon from decaying organic matter, then release these gases into the atmosphere to be reused by other living things. Other bacteria cause diseases in humand and animals, help digest sewage in treatment plants, or produce the alcohol in wine, beer and liquors. Still others are used by humans to break down toxic waste chemicals in the environment, a process called bioremediation.



Bacterial Shapes and Classifications


There are thousands of species of bacteria on earth, many of which have not yet been identified.  When attempting to classify a bacterium, a variety of characteristics are used, including visual characteristics and laboratory tests.  Bacteria are simple, unicellular organisms. Most are free-living organisms, but a few require animal or plant hosts for survival. Bacteria absorb nutrients from their environments, excrete waste products, and secrete various toxins that help them invade tissues. Bacteria have no enclosed nucleus. Their chromosomal material is in the form of a large loop, packed into the cytoplasm of the cell.

Some bacteria can be identified through a simple visual perusal.  First, the operator considers the appearance of the bacterial colony (a group of the same kind of bacteria growing together, often on a petri dish.)  The operator also views individual bacteria under a microscope, considering their shape, groupings, and features such as the number and location of flagella. 

A variety of laboratory techniques can be used to narrow down the identity of a bacterial species if a visual survey is not sufficient.  The operator can stain the bacteria using a gram stain or an acid-fast stain.  The bacteria can be cultured on a specific medium which promotes the growth of certain species, as in the membrane filter method of testing for coliform bacteria.  Other tests can detect bacterial by-products, while yet more advanced tests actually analyze the DNA of the bacteria. 




Bacterial Shapes

The most basic method used for identifying bacteria is based on the bacterium's shape and cell arrangement.  This section will explain the three morphological categories which all bacteria fall into - cocci, bacilli, and spirilla.  You should keep in mind that these categories are merely a way of describing the bacteria and do not necessarily refer to a taxonomic relationship. The most common shapes of bacteria include rod, cocci (round), and spiral forms. Cellular arrangements occur singularly, in chains, and in clusters. Some species have one to numerous projections called flagella enabling the bacteria to swim, making them motile organisms.



Cocci (or coccus for a single cell) are round cells, sometimes slightly flattened when they are adjacent to one another.  Cocci bacteria can exist singly, in pairs (as diplococci ), in groups of four (as tetrads ), in chains (as streptococci ), in clusters (as stapylococci ), or in cubes consisting of eight cells (as sarcinae .) 


Bacilli (or bacillus for a single cell) are rod-shaped bacteria.  Since the length of a cell varies under the influence of age or environmental conditions, you should not use cell length as a method of classification for bacillus bacteria.  Like coccus bacteria, bacilli can occur singly, in pairs, or in chains.  Examples of bacillus bacteria include coliform bacteria , which are used as an indicator of wastewater pollution in water, as well as the bacteria responsible for typhoid fever.  


Spirilla (or spirillum for a single cell) are curved bacteria which can range from a gently curved shape to a corkscrew-like spiral.  Many spirilla are rigid and capable of movement.  A special group of spirilla known as spirochetes are long, slender, and flexible.




Gram Stain Procedure

The most fundamental technique for classifying bacteria is the gram stain, developed in 1884 by Danish scientist Cristian Gram. It is called a differential stain because it differentiates among bacteria and can be used to distinguish among them, based on differences in their cell wall.

In this procedure, bacteria are first stained with crystal violet, then treated with a mordant - a solution that fixes the stain inside the cell. The bacteria are then washed with a decolorizing agent, such as alcohol, and counterstained with safranin, a light red dye. The walls of gram-positive bacteria (ie. Staphylococcus aureus) have more peptidoglycans (the large molecular network of repeating disaccharides attached to chains of four or five amino acids) than do gram-negative bacteria. Thus, gram-positive bacteria retain the original violet dye and cannot be counterstained.

Gram-negative bacteria (ie. Escherichia coli) have thinner walls, containing an outer layer of lipopolysaccharide, which is disrupted by the alcohol wash. This permits the orignial dye to escape, allowing the cell to take up the second dye, or counterstain. Thus, gram-positive bacteria stain violet, and gram-negative bacteria stain pink. The gram stain works best on young, growing populations of bacteria, and can be inconsistent in older populations maintained in the laboratory.



Although we think of respiration as breathing, respiration is actually the process by which organisms break down organic substances (such as sugars) to produce energy.  All living organisms must perform some kind of respiration.  

In many cases, the chemical process of respiration requires oxygen, although some organisms are able to carry out respiration in the absence of oxygen.  This page will explain the three types of respiration found in microorganisms, as well as how these types of respiration affect the wastewater treatment plant. 



Aerobic Respiration

 Aerobic respiration is respiration in the presence of oxygen.  Most multicellular organisms and many microorganisms produce their energy using aerobic respiration.  In aerobic respiration, sugars are broken down in the presence of oxygen to produce carbon dioxide, water, and energy.  Without oxygen, aerobic microorganisms are unable to produce energy and quickly die.  



Anaerobic Respiration

Other microorganisms are able to survive in environments which lack oxygen by performing anaerobic respiration , sometimes known as fermentation .  Like aerobic respiration, anaerobic respiration breaks down sugars and releases energy.  However, anaerobic respiration is typically slower and less efficient than aerobic respiration.   In addition, anaerobic respiration involves chemicals other than oxygen and carbon dioxide.  

The chemicals used and produced during anaerobic respiration vary from microorganism to microorganism.  Some anaerobic microorganisms use sulfate (SO42-) during respiration and produce hydrogen sulfide (H2S.)  Other microorganisms use nitrate (NO3-), producing nitrite (NO2-), nitrous oxide (NO), or nitrogen gas (N2 ).  Yet other microorganisms are able to use hydrogen gas (H2 ), producing methane (CH4 ) or acetic acid (CH3COOH-) as the byproduct.  

Anaerobic reactions generally lead to more offensive end products than those produced during aerobic respiration.  For example, hydrogen sulfide is very reactive and smells like rotten eggs even at low concentrations.  Hydrogen sulfide can combine with the organic end products of anaerobic respiration to form the dark-colored, odorous substances which are characteristic of anaerobic (also known as septic ) conditions.  



Facultative Anaerobic Respiration

Many microorganisms are either obligate aerobes or obligate anaerobes.  That is, those which perform aerobic respiration will die if the oxygen content of their environment drops too low.  In contrast, those which perform anaerobic respiration will die if they are brought in contact with oxygen.  

The final type of microorganisms - facultative anaerobes - are able to perform either aerobic respiration or anaerobic respiration depending on the oxygen content of their environment.  Since aerobic respiration is more efficient, facultative anaerobes will perform aerobic respiration if there is oxygen present in their environment.  However, in the absence of oxygen, these organisms simply switch over to anaerobic respiration.  Coliform bacteria are a well-known example of facultative anaerobic microorganisms.  



In The Treatment Plant

In most wastewater treatment processes, operators attempt to maintain an environment suitable for aerobic respiration.  By maintaining an aerobic environment the operators prevent the bad smells associated with septic environments and also maintain a higher speed of waste digestion.  Aerobic processes are most common in biological wastewater treatment systems, including the activated sludge process, trickling filters, and many oxidation ponds.  

Since aerobic microorganisms use up oxygen as they break down waste, it is often necessary to aerate (add air to) the wastewater to maintain an aerobic environment.  Aeration may be achieved by blowing air into the water or (as in the trickling filter) by allowing water to run through the air. 
Despite the advantages of aerobic systems, some wastewater treatment processes are designed to be anaerobic.  Both anaerobic digesters and septic tanks are wholly anaerobic environments.  Since these systems house obligate anaerobic microorganisms, exposing anaerobic digestion systems to oxygen even for a short period of time can seriously affect the systems' ability to function. 

Some systems, through accident or design, can function both aerobically and anaerobically.  Although oxidation ponds are generally aerobic, bottom deposits and stagnant pockets in the ponds often become anaerobic.  On a trickling filter's slime layer, aerobic and anaerobic zones may occur within millimeters of each other, with the surface layers being aerobic and the deeper layers being anaerobic.

Facultative anaerobic microorganism species are very important in many wastewater treatment processes since they area able to perform in both aerobic and anaerobic environments.  However, facultative anaerobic microorganisms can cause problems when  they begin to respire anaerobically, producing unpleasant byproducts.  In general, facultative anaerobic species usually begin performing anaerobic reactions when the dissolved oxygen levels of their environment fall below about 0.5 mg/L for several hours.  This condition is rarely met in aeration tanks unless equipment failure occurs, but keeping sludge in the final clarifiers for an extended period of time can lead to anaerobic conditions.  In addition, conditions of low flow or elevated temperature can result in anaerobic conditions. 




Protozoans are found in all moist habitats within the United States, but we know little about their specific geographic distribution. They are unicellular organisms that range in size from a few microns to several hundred microns. Polluted waters often have a rich and characteristic protozoan fauna. The relative abundance and diversity of protozoa are used as indicators of organic and toxic pollution.

Although protozoa are frequently overlooked, they play an important role in many communities where they occupy a range of trophic levels. As predators upon unicellular or filamentous alge, bacteria, and microfungi, protozoa play a role both as herbivores and as consumers in the decomposer link of the food chain.

Both in organic pollution of the natural environment and in the biological processing of human and domestic animal sewage, the ceaseless activity of the protozoa, particularly the colonial ciliates, in the extraction and digestion of bacteria and other suspended particles is the main element of the natural process by which the water supply is rendered once again fit for consumption by humans and other creatures. Any change in our environment which threatens the life of a balanced community of protozoans threatens also the continuity of a clean water supply for humans. This is particularly relevant in the light of our current over-use of kitchen and lavatory disinfectants and their effect upon the ciliates at sewage processing plants and in the waterways beyond.

Protozoa are members of the Kingdom Protista. There are about 20,000 known species of protozoa that live in water and soil. Some feed on bacteria while others are parasites and feed off their hosts. Most protozoa are asexual and reproduce in one of three ways: fission, budding, and multiple fission. Some protists are sexual and exhange genetic material from one cell to another through conjugation, which is the physical contact between cells.



A protist can survive in an adverse environment by encapsulating itself with a protective coating called a cyst. The cyst defends the protist in extreme temperatures against toxic chemicals and even when there is a lack of oxygen, moisture, and food.



Factors Affecting Growth and Distribution

Most free-living protozoa reproduce by cell division (exchange of genetic material is a separate process and is not involved in reproduction in protozoa). The relative importance for population growth of biotic versuc chemical-physical components of the environment is difficult to ascertain from the existing survey data. Protozoa are found living actively in nutrient-poor to organically rich waters and in fresh water varying between 0°C (32°F) and 50°C (122°F). Nonetheless, it appears that rates of population growth increase when food is not constrained and temperature is increased.




Protists receive nutrients by breaking down organic matter and can grow in both aerobic and anaerobic environments, such as protists that live in the intestine of animals. Some receive nutrients from organic matter and photosynthesis because they contain chlorophyll. These protists are considered both algae and protozoa. Protists obtain food in one of three ways: absorption, ingestion, and engulfing. Food is digested in the vacuole after the food enters the cell. The vacuole is a membrane-bound organelle. Waste products are excreted using a process called exocytosis.



Protozoa in the Wastewater Treatment Process

Most protozoa are free-living in soil and water and enter the activated sludge process through inflow and infiltration. The number of protozoa within the activated sludge process or mixed liquor varies greatly by process and operational conditions, especially hydraulic and organic loadings. The relative abundance of protozoa may be less than 100 per milliliter or greater than 100,000 per milliliter.

Protozoa, especially ciliated protozoa, perform several beneficial roles in wastewater treatment. These roles include cropping action, coating action, and recycling of mineral nutrients.

Bacteria are the primary food source for protozoa, and the consumption of suspended or dispersed bacteria by protozoa is referred to as "cropping" action. Cropping action removes many dispersed bacteria from the bulk solution.

Dispersed growth as well as colloids and particulate materials, collectively known as "fine" solids, also are removed from the bulk solution by the "coating" action of ciliated protozoa. This group releases sticky secretions that cover the surface of fine solids. Through coating action, the surface charge of fine solids is made compactible for adsorption to floc particles in the activated sludge process. The adsorption reduces the quantity of fine solids in the final effluent.

Protozoa also release excretions to the bulk solution. These excretions contain many mineral nutrients, including nitrogen and phosphorus, and help to recycle mineral nutrients in the activated sludge process. These nutrients then are available for bacterial activity in degrading wastes, the biochemical oxygen demand (BOD).

Protozoa in the activated sludge process commonly are placed in one of five groups: amoebae, flagellates, free-swimming ciliates, crawling ciliates and stalked ciliates. Amoebae and flagellates are considered "lower" life forms, while crawling ciliates and stalk ciliates are considered "higher" life forms. Free-swimming ciliates are considered "intermediate" life forms. Some treatment plant operators often perform routine microscopic examinations of the protozoa in the activated sludge to determine the health of the activated sludge process.

Typically, operators base the health of the process on the protozoan groups that are dominant as revealed by microscopic examination (Table below). For example, if lower life forms are dominant, the activated sludge is considered unhealthy and unacceptable. An unhealthy activated sludge produces an aeration tank effluent having a BOD greater than 30 mg/L. If the higher life forms are dominant, the activated sludge is considered healthy and acceptable. A healthy activated sludge produces an aeration tank effluent having a BOD less than 30 mg/L.


Protozoan Groups and Bioindicator Values



Rarely predominant except for start-up conditions and conditions that mimic start-up such as over-wasting, recovery from toxicity, washout, and organic overloading

Flagellates, plant-like

Dominant under high organic loading, dispersion of floc particles, such as through chlorination, and start-up conditions or conditions that mimic start-up. Also may dominate in the presence of excess soluble phosphorus.

Flagellates, animal-like

Except for the presence of excess soluble phosphorus, these are dominant for operational conditions listed for plant-like flagellates and usually follow plant-like flagellates as the dominant group.

Free-swimming ciliates

Transition group that dominates between healthy and unhealthy conditions and proliferates when large numbers of free-swimming bacteria are present.

Crawling ciliates

Dominant in the presence of mature floc particles and low BOD in the bulk solution. Alternate with stalked ciliates as the dominant group.

Stalked ciliates

Dominant in the presence of mature floc particles and low BOD in the bulk solution. Alternate with crawling ciliates as the dominant group.


Operators should exercise caution when correlating final effluent quality with the health of activated sludge as indicated by the dominant protozoan groups. For example, loss of secondary solids from the clarifier due to a bulking condition would increase the BOD above that achieved in the aeration tank.




Diseases Caused by Protozoa

Several protozoa can cause waterborne diseases.  The most common of these pathogenic protozoa are Cryptosporidium , Entamoeba , Giardia , and Toxoplasma .  These species are all able to form cysts , or protective coatings, which allow them to survive outside a host for extended periods of time.  Protozoan cysts also protect the organism from chlorine, so these species are not effectively controlled by chlorination.  Instead, protozoa are usually removed from water by filtration.  






Cryptosporidium is a sporozoa which is one of the most common pathogens responsible for waterborne diseases in the United States.  Scientists estimate that between 1% and 5% of Americans are infected with Cryptosporidium at any one time.  Cryptosporidiosis, the disease caused by Cryptosporidium infection, results in diarrhea, abdominal cramps, and fever.  Healthy adults usually do not require treatment for cryptosporidiosis since the body will heal itself naturally within a couple of weeks. 

Cryptosporidium , like most other waterborne pathogens, is spread to new hosts through the ingestion of contaminated food and drinking water.  In addition, several outbreaks have been found to result from fecal accidents in swimming pools.  Water treatment plants find it difficult to prevent the spread of Cryptosporidium since the cysts are very resistant to chlorination and can sometimes pass through filters.  Instead, the cysts can be killed by ozonation or by boiling the water for at least a minute.  




Entamoeba histolytica


Entamoeba histolytica is an amoeboid protozoan which lives in anaerobic environments.  Like the other pathogenic protozoa, Entamoeba is capable of forming cysts which can remain dormant for extended periods of time in the water, in soil, or in food.  These cysts spread to new hosts when we ingest contaminated food or water. 

In humans, Entamoeba causes amoebic dysentery, which is usually treated with antibiotics.  Entamoeba outbreaks are rare in the United States but are quite common in developing countries when raw sewage contaminates drinking water supplies or when the soil is fertilized with untreated wastes.  In the water treatment plant, Entamoeba can be removed from water using a sand filter.  




Giardia lamblia


Giardia lamblia is a flagellate protozoan which is capable of forming cysts.  The species is the most important cause of waterborne disease outbreaks in the United States.  

Infection with Giardia lamblia results in a disease called giardiasis, which is also known as traveler's diarrhea or Montezuma's Revenge.  Symptoms of giardiasis include diarrhea, abdominal cramps, fatigue, and weight loss.  Although most people who contract giardiasis heal naturally within a week or two, the illness sometimes lingers for up to a year, in which case antibiotics may be prescribed.  

Giardia lamblia is spread when people ingest contaminated food or water.  In the water treatment plant, chlorine is somewhat effective at inactivating the cysts at a dosage of 1.5 mg/L chlorine with a contact time of 10 minutes.  Filtration and boiling are more effective at killing Giardia. 




Toxoplasma gondii f

Toxoplasma gondii is a sporozoa which causes a disease known as toxoplasmosis.  In most cases, toxoplasmosis has mild flu-like symptoms which are often unnoticed or undiagnosed and do not require treatment.  However, a pregnant woman may pass on the infection to her unborn child, in which case the child will have much more serious symptoms.  

Toxoplasma gondii has a complex life cycle involving at least two different hosts.  The protozoan reproduces sexually within its primary host, the cat, and releases cysts in the cat's feces.  The cysts are then ingested by intermediate hosts, which include a large number of vertebrate species such as pigs, cows, and humans.  In the intermediate host, the cysts develop into an active form of the protozoan which reproduces asexually.  The offspring produced in the intermediate host then infect a cat, completing the cycle. 

In most cases, humans contract toxoplasmosis by eating contaminated meat or by handling cats or changing cats' litter boxes.  However, a recent outbreak of toxoplasmosis linked to drinking water in Canada has alerted us to the possibility of Toxoplasma gondii being spread in water.  




Viruses are non-living organisms which can only reproduce in a living host cell.  As a result, all viruses are obligate parasites and all cause some sort of disease.  Infectious hepatitis, polio, influenza, smallpox, AIDS, and a variety of intestinal disturbances are all caused by viruses.  

Viruses can attack many different kinds of organisms ranging from bacteria through plants and animals, though each type of virus is specific in its type of host.  For example, a plant virus will not attack an animal and a dog virus is unlikely to attack a human.  

Viruses are too small to be to be seen with a light microscope, so their presence is usually recognized only by the harm they cause.  They often enter water in animal feces and are thus expected to be present in domestic wastes.  In addition, viruses can often survive for long periods of time in natural waters.  Viruses are a public health concern in water and wastewater treatment since many are not removed by conventional treatment methods such as disinfection. 






Viruses are very simple organisms consisting primarily of genetic material (which can be either DNA or RNA) enclosed within a protein coat called a capsid.  The genetic material can have a variety of forms, being either double-stranded or single-stranded and either circular or linear.  The capsid coat can have several shapes, including spherical and icosahedral (20-sided), and may further be surrounded by an envelope.  The envelope is made up of lipids and is usually imbedded with envelope proteins which help the virus recognize its host cell. 

As you can see in the picture above, there are several different kinds of viruses.  The virus on the left is a typical bacteriophage, which is a virus which infects bacteria.  Bacteriophages have complex tails which are used to attach to and inject DNA or RNA into the host cell.  The cell on the right is a typical animal virus and has a much simpler structure.  




Animation of viral reproduction.


The animation above shows how a virus reproduces.  First, the virus attaches to receptors on the host cell using its envelope proteins.  Then the virus inserts its DNA or RNA into the host cell.  This second step can be achieved through fusion (as shown in the animation), through endocytosis (which is a process in which the host cell's membrane engulfs the virus), or through direct penetration (a common process in bacteriophages in which the virus's DNA or RNA is injected into the host cell through the virus's tail.)
If the virus's genetic material is RNA, then the RNA must be turned into DNA in the host cell's cytoplasm.  The viral DNA then makes its way into the host cell's nucleus.  There, the viral genetic material is spliced into the host cell's DNA.  

A cell's DNA tells the cell how to perform cell processes, such as making proteins and new DNA.  So, once the virus's DNA has been inserted into the host's DNA, the viral DNA can tell the cell to produce new viral parts - DNA, proteins, and lipids.  After the cell has produced the viral parts, the virus's DNA tells the cell to assemble the viral parts into new viruses, known as virions.  

Once the cell is full of virions, the virions are released into the environment.  The animation shows one method of virion release common in simple host cells, in which the host cell lyses (or breaks apart).  In other types of host cells, the virions may simply bud out of the cell.  In either case, the new virions float through their environment until they find a new host and can repeat the reproductive cycle.  


Lysogenic cycle



In some cases, the virus's reproductive cycle also has a lysogenic phase in which the virus is dormant within the host cell for a time.  The viral DNA enters the host cell and splices into the host DNA, but does not immediately begin producing new viruses.  Instead, the host cell is able to grow and reproduce normally.  As the host cell reproduces, however, it copies the virus's DNA as well as its own DNA.  After the host cell has reproduced several times, all of the daughter cells begin to produce new viruses.  Many bacteriophages go through a lysogenic phase in their life cycle.  

As you read through the description of viral reproduction above, you may have noticed that the virus was not active in much of the reproduction process.  After the virus inserted its genetic material into the host cell, the host cell did all of the work of making new viruses.  This dependence on a living organism for reproduction is one of the reasons that many scientists consider viruses to be non-living.  





Taxonomy is the arrangement of organisms into related groups based on natural relationships. Taxonomy has three components: classification, nomenclature, and identification. The most commonly used rank to identify organisms, in order from most general to most specific is Domain, Kingdom, Phyla, Class, Order, Family, Genus, Species. The diagram below shows how microorganisms are classified.  In addition to the microorganisms listed on the tree, worms and rotifers are very small, multicellular animals.  

Tree of microorganisms classification. 



Nester, E.W., C.E. Roberts, and M.T. Nester.  1995.  Microbiology: A Human Perspective.  Wm. C. Brown Publishers, Dubuque.

Sterrit, R.M., and J.N. Lester.  1988.  Microbiology for Environmental and Public Health Engineers.  E. & F.N. Spon, New York.  

Tree of Life.  2004.  University of Arizona College of Agriculture and Life Sciences and University of Arizona Library, Tucson.  

Van Egmond, Wim.  1998.  The Smallest Page on the Web.  

Wikipedia.  2005.  



Wastewater Treatment


Wastewater treatment refers to the process of removing pollutants from water previously employed for industrial, agricultural, or municipal uses. The techniques used to remove the pollutants present in wastewater can be broken into biological, chemical, physical and energetic. These different techniques are applied through the many stages of wastewater treatment.


Primary treatment usually includes the removal of large solids from the wastewater via physical settling or filtration. The first step in primary treatment is screening.

Secondary treatment typically removes the smaller solids and particles remaining in the wastewater through fine filtration aided by the use of membranes or through the use of microbes, which utilize organics as an energy source. Energetic techniques may also be employed in tandem with biological techniques in the secondary phase to break up the size of particles thus increasing their surface area and rate of consumption by the microbes present. A common first step in the secondary treatment process is to send the waste to an aeration tank.

Tertiary treatment involves the disinfection of the wastewater through chemical or energetic means. Increasing the number of steps in a wastewater treatment process may insure higher quality of effluent; however employing additional technologies may incur increased costs of construction, operation, and maintenance.



Primary Treatment

Screening is the first technique employed in primary treatment, which is the first step in the wastewater treatment process.

This step removes all sorts of refuse that has arrived with the wastewater such as plastic, branches, rags, and metals. The screening process is used primarily to present the clogging and interference of the following wastewater treatment processes.

Screens are considered coarse if their opening are larger than 6mm, fine if their openings are between 1.5 and 6mm, and very fine if their openings are between 0.2 and 1.5mm.

This type of screen, called a bar screen, removes debris from wastewater.


Screens are cleaned manually if the object caught is larger and mechanically if finer particles are caught. The angle of the screen may also be varied to affect the efficiency of filtration.


In order to remove coarse solids, numerous types of detritus tanks, grinders, and cyclonic inertial separation are utilized, including a comminutor and a grit chamber. The type of grit removal separation depends upon the characteristics of the grit itself.

A comminutor, also known as the grinding pump, houses a rotating cutting screen. This cutting screen shreds any large chunks of organic matter in the wastewater into smaller pieces. This makes it easier for the microorganisms to use the organic matter as food and prevents the large chunks from harming the internal workings of the treatment plant.


A grit chamber allows pieces of rock, metal, bone, and even egg shells, which are denser than organic materials, to settle out of the waste stream. Removal of grit prevents damage to machinery through abrasion or clogging.



The last step in primary treatment is sedimentation, which occurs in the primary clarifier.


Sedimentation simply entails the physical settling of matter, due to its density, buoyancy, and the force of gravity. Certain chemicals known as coagulants and flocculants are often used to expedite this process by encouraging aggregation of particles. Through sedimentation, the larger solids are removed in order to facilitate the efficiency of the following procedures and also to reduce the biological oxygen demand of the water.

The biochemical oxygen demand (BOD) refers to the amount of oxygen required by the microbes within the wastewater to digest the matter that they are using for food. By removing these solids early on, the efficiency of the microbial digestion at later stages in increased.



Secondary Treatment

Once the wastewater leaves the primary treatment steps, it enters secondary treatment. The first step in the secondary treatment process is the aeration tank.

Bacteria are single celled organisms, which have basic requirements for existence and reproduce rapidly. Many occupy unique niches and consume only certain types of food. Many types of bacteria have been utilized in wastewater processing. If certain bacterium is supplied with an environment in which the proper pH, temperature, micro and macronutrients, and oxygen levels are present, it can quickly and effectively break pollutants present in wastewater down into less harmful components.

The types of bacteria utilized in wastewater processing can be categorized based upon their necessary or intolerance of oxygen to survive. Those bacteria that require oxygen to convert food into energy are called aerobic, those that will perish in the presence of oxygen are anaerobic, and finally facultative anaerobes may thrive in either the presence or absence of oxygen. Typically aerobes, which can degrade pollutants 10-100 times faster than anaerobes, are utilized most frequently. Increases in temperature and pollutant food source have shown to increase the rate of degradation, but if all elements necessary for conversion of food to energy are not in balance, the microbial degradation will be thwarted.


The wastewater is then passed through a secondary clarifier, which performs sedimentation again, which is described earlier and occurs in primary treatment as well.



The disinfection of wastewater through the use of chemicals such as chlorine typically acts as the final step in wastewater treatment. Disinfection seeks to remove harmful organics and pathogens causing cholera, polio, typhoid, hepatitis, and a number of other bacterial, viral, and parasitic diseases from the water.

Due to security concerns, some wastewater treatment facilities are using sodium hypochlorite to eliminate the need for chlorine. Sodium hypochlorite is more expensive than liquid chlorine, but is also safer. Although chlorine is considered the tried and true solution to reducing pathogens in contaminated water, the method of disinfection, such as UV disinfection, must fit the type of pathogen the wastewater harbors, to be truly effective.

Through disinfection a significant portion of the pathogens are inactivated, however, it is difficult to identify individual pathogens within wastewater, and therefore indicator pathogens are used. In wastewater, fecal coliform acts as the indicator pathogen, but there has been discussion of using E. coli or total coliform, the indicator for potable water, to check wastewater.




Coagulation and Flocculation

Coagulants and flocculants are chemicals used to precipitate insoluble substances. The purpose of coagulation and flocculation is to cause small pollutant particles such as metals to aggregate and form large enough floc so that they can be separated from the wastewater through sedimentation.

There are three main types of coagulants that are used to overcome the repulsive forces of particles, thus causing them to aggregate. Electrolytes, organic polymers, and synthetic polyelectrolites are added to wastewater and then flocculation tanks mix the water to promote flocs and subsequent physical separation.

Rate of flocculation is dependent upon many factors including concentration of particles, particle contact, and range of particle sizes. Coagulation targets dissolved ions such as metal and radionuclides. Some difficulties with this technology include the frequent need to adjust pH levels, the creation of toxic sludge that must be eventually mitigated, and the difficulty that results in trying to address the chemical nature of multiple compounds. This technology has been used consistently in the electronics and electroplating industry as well as for applications in groundwater treatment.



Membrane Filtration

The three main types of membrane-based filtration technologies include reverse osmosis, nanofiltration, and ultrafiltration. Although categorized as different technologies, the three types of membrane filtration have a great deal in common. All three act as membranes created by coating a thin layer of a very porous polymer, or plastic, onto a backing material. The end result is the finest form of filtration presently known, with reverse osmosis being the smallest, nanofiltration being a slight step larger and ultrafiltration being a bit larger again.


The pore sizes are typically measured in angstroms (one billionth of a meter) and thus are extremely tiny. These membrane technologies offer a host of advantages over traditional filtration. Due to the fine pore space and indiscrimination of influents of these membrane filtration systems, a very high quality effluent emerges. Additionally, membrane technologies take up only a fraction of the space needed for other tertiary treatment systems. The disadvantage of having extremely fine pores means that clogging is a frequent and costly problem with membrane filtration technologies.



Constructed Wetlands

Scientists have long recognized the abilities of wetlands to purify water. Through the correct sequencing of base media, plant species, and microbe species, constructed wetlands can successfully reduce nitrogen content, filter out solids, and reduce the presence of heavy metals.

The type and amount of pollutant removed depends upon the species and oxygen affinity of the organisms present in the wetland. Wetlands utilize physical and chemical processes to clean wastewater and typically serve as the secondary and tertiary steps.

Although constructed wetlands tend to take up a great deal of space, they require less investment of time and money than traditional waste treatment procedures. Ultimately, constructed wetlands area cost-effective and environmentally-benign method of wastewater processing.




In this lesson we learned the relationship between microbes, wastewater, and wastewater treatment. One goal of biological treatment is nitrification/denitrification. Nitrification is an aerobic process in which bacteria oxidize reduced forms of nitrogen. Denitrification is an anaerobic process by which oxidized forms of nitrogen are reduced to gaseous forms, which can then escape into the atmosphere. If water with a large amount of BOD is discharged into the environment, it can deplete the natural oxygen resources. Eutrophication is the process by which bodies of water become rich in mineral and organic nutrients causing plant life, especially algae, to proliferate, then die and decompose thereby reducing the dissolved oxygen content and often killing off other organisms. Wastewater treatment in the plants involve primary, secondary and tertiary treatment. Primary treatment deals with removing large solids while secondary treatment removes the smaller solids and tertiary treatment involves the disinfection of the wastewater through chemical means.




The Fundamental Microbiology of Sewage

"Wastewater Treatment Technology Tutorial"  2006. Earthspace



Complete the interactive assignment on Wastewater Treatment .

This assignment will give you practice with the topics covered in Lesson 1.  You should print the assignment and become familiar with the exercises before doing them online. You may do the Assignment online to get credit or print it out and send it to the instructor.  It will require the Flash player to view, which should already be installed on your machine.

In addition, the first Project paper is due this week.  Once you have completed the project either mail, fax, or email it to your instructor.





Answer the questions in the Lesson 1 quiz .  When you have gotten all the answers correct, print the page and either mail or fax it to the instructor. You may also take the quiz online and directly submit it into the database for a grade.