Lesson 5:



In this lesson we will learn the following concepts:
  • The structure of bacteria.
  • Stages of bacterial growth.
  • The different types of respiration.
  • How to identify and classify bacteria.


Reading Assignment

In addition to the online lecture, read chapter 4 in Wastewater Microbiology



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.



Parts of Procaryotic Cells

A procaryotic cell is a cell that does not have a true nucleus. The nuclear structure is called a nucleoid, which contains most of the cell's genetic material and is usually a single circular molecule of DNA. A procaryotic organism, such as bacteria, is a cell that lacks a membrane-bound nucleus or membrane-bound organelles. The exterior of the cell usually has glycocalyx, flagellum, fimbriae, and pili.





Glycocalyx is a sticky, sugary envelope composed of polysaccharides and/or polypeptides that surround the cell. Glycocalyx can be firmly attached to the cell's surface, called capsule, or loosely attached, called slime layer. A slime layer is water-soluble and is used by the procaryotic cell to adhere to surfaces external to the cell.




Flagella are made of protein and appear "whip-like". They are used by the procaryotic cell for mobility. Flagella propel the microorganism away from harm and towards food in a movement known as taxis.

Flagella can exist in the following forms:

  • Monotrichous: One flagellum
  • Lophotrichus: A clump of flagella, called a tuft, at one end of the cell.
  • Amphitrichous: Flagella at two ends of the cell.
  • Peritrichous: Flagella covering the entire cell.
  • Endoflagellum: A type of amphitrichous flagellum that is tightly wrapped around spirochetes. A spirochete is a spiral-shaped bacterium that moves in a corkscrew motion. Borrelia burgdorferi, which is the bacterium that causes lyme disease, exhibits an endoflagellum.



Fimbriae are proteinaceous, sticky, bristle-like projections used by cells to attach to each other and to objects around them. Fimbriae may be responsible for the clinging of cells that leads to biofilms and other thick aggregates of cells on the surface of liquids and for the microbial colonization of inanimate solids such as rocks and glass.




Pili are tubules that are used to transfer DNA from one cell to another cell, similar to tubes used to fuel an aircraft in flight. Some are also used to attach one cell to another cell. The tubules are made of protein and are shorter in length than flagella and longer than fimbriae.



Cell Wall

The procaryotic cell's cell wall is located outside the plasma membrane and gives the cell its shape and provides rigid structural support for the cell. The cell wall also protects the cell from its environment. Pressure within the cell builds as fluid containing nutrients enters the cell. It is the job of the cell wall to resist this pressure the same way that the walls of a balloon resist the build-up of pressure when it is inflated. If pressure inside the cell becomes too great, the cell wall bursts, which is referred to as lysis.

The cell wall of many bacteria is composed of peptidoglycan, which covers the entire surface of the cell. It is made up of a combination of peptide bonds and carbohydrates. The wall of a bacterium is classified in two ways:

  • Gram-positive. A gram-positive cell wall has many layers of peptidoglygan that retain the crystal of violet dye when the cell is stained. This gives the cell a purple color when seen under a microscope.
  • Gram-negative. A gram-negative cell wall is thin. The inside is made of peptidoglycan. The outer membrane is composed of phospholipids and lipopolysaccharides.



The cell wall does not retain the crystal of violet dye when the cell is stained. The cell appears pink when viewed with a microscope.



Cytoplasmic Membrane

The procaryotic cell has a cell membrane called the cytoplasmic membrane that forms the outer structure of the cell and separates the cell's internal structure from the environment. This membrane provides a selective barrier, allowing certain substances and chemicals to move into and out of the cell.



The cytoplasmic membrane regulates the flow of molecules (such as nutrients) into the cell and removes waste from the cell by opening and closing passages called channels. In photosynthetic procaryotes, the membrane functions in energy production by collecting energy in the form of light. This membrane is selectively permeable because it permits the transport of some substances and inhibits the transport of others. Two types of transport mechanisms are used to move substances through the cytoplasmic membrane. These are passive transport and active transport.


Passive Transport

Passive transport moves substances into and out of the cell down a gradient similar to how a rock rolls downhill, following the gradient. There are three types of passive transport:

  • Simple diffusion: Simple diffusion is the movement of substances form a higher-concentration region to a lower-concentration region. Large molecules are too large to enter the cell.
  • Facilitated diffusion: Facilitated diffusion is the movement of substances from a higher-concentration region to a lower-concentration region with the assistance of an integral protein across a selectively permeable membrane.
  • Osmosis: Osmosis is the net movement (diffusion) of a solvent (water in living organisms) from a region of higher water concentration to a region of lower concentration as in the image below.



Active Transport

Active transport is the movement of a substance across the cytoplasmic membrane against the gradient by using energy provided by the cell. This is similar to pumping water against gravity through a pipe. Energy must be spent in order for the pump to work. A cell makes energy available by removing a phosphate (P) from adenosine triphosphate (ATP). ATP contains chemical potential energy that is released by a chemical reaction within the cell. It is this energy that is used to change the shape of the integral membrane protein-enabling substances inside the cell to be pumped through the membrane. For example, active transport is used to pump sodium from a cell.




Procaryotic cells reproduce asexually. Asexual reproduction is a process through which one parent gives rise to genetically identical offspring - in other words, two clones of the parent. 

Binary fission animation.

Asexual reproduction in microorganisms is often known as binary fission since it consists of a cell splitting in half.  As you can see in the animation above, the microorganism first makes a second copy of its DNA in a process known as replication .  Next, the cell begins to constrict in the middle, leaving one set of DNA and organelles on each side of the constriction.  Eventually, the cell splits apart into two identical daughter cells.  Once these daughter cells enlarge to adult size, each one is ready to split into two more daughter cells.  The physical and chemical requirements for growth can vary widely among different species of bacteria, but in general, the physical requirements include proper temperatures, pH and osmotic pressure. Most bacteria thrive only within narrow ranges of these conditions, however extreme those ranges may be.



Stages of Bacterial Growth

The term "bacterial growth" generally refers to growth of a population of bacteria, rather than of an individual cell. Under optimal conditions, a bacterium can divide into two daughter cells every 15 to 30 minutes.  After another 15 to 30 minutes, the two daughter cells can each divide into two more cells.  These four cells then divide into eight cells, and so on.  As you can see, microorganism populations have the potential to grow tremendously within a very short period of time.

Population growth curve.

You might expect for a microorganism population to continue to grow indefinitely.  However, in a closed system (an environment such as a test tube or a batch of sewage which is separate from the outside world), microorganism populations usually follow a predictable pattern of growth and death shown in the diagram above. 

When microorganisms are first introduced to a new environment, they go through a lag phase.  The lag phase is a time when the microorganisms do not reproduce, so the number of microorganisms in the population remains constant.  During the lag period, microorganisms are adjusting to their new environment. 

After a short time, the microorganisms begin to reproduce.  At first, they reproduce relatively slowly, but the reproduction rate quickly speeds up as they pass out of  the accelerated growth phase and into the logarithmic growth phase . 

As the microorganisms grow, they begin to use up the food and oxygen in their environment.  They also excrete wastes which pollute their environment.  Eventually, the environmental conditions degrade to a point where the bacterial growth begins to slow - the decelerated growth phase .  Then the number of organisms found in the population levels off as the death rate equals the birth rate in the stationary phase . 

The environment continues to be degraded, and soon the microorganism death rate exceeds the birth rate and the population size begins to fall in the accelerated death phase .  At the maximum death rate, the population is in the logarithmic death phase in which the population shrinks very quickly until nearly all of the cells are dead.  At this point - the survival phase - the population will level off at a relatively small size. 

The wastewater operator needs to be familiar with this population growth curve since it will influence the functioning of an activated sludge system.  During conventional treatment, the activated sludge is held for a sufficient time for the microorganism population to enter the logarithmic death phase. 



Bacterial Shapes and Classification


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. 

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.


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. 



Types of Bacteria


A large variety of bacteria can cause waterborne diseases.  On this page, we will consider a few of the most widespread species - Campylobacter, Escherichia coli, Salmonella, Shigella, and Vibrio cholerae



Campylobacter is a spiral-shaped bacterium which can infect humans as well as several other animals.  When humans are infected with Campylobacter, they experience gastroenteritis for about a week, then usually recover on their own without treatment.  However, in a few cases, Campylobacter can lead to a more serious disease called Guillain-Barre syndrome. 

The bacteria are spread when we eat contaminated food or drink contaminated water.  Campylobacter infections are usually sporadically distributed through the population, although outbreaks may occur.  Scientists estimate that between 1 and 4 million Americans are infected with Campylobacter every year. 

Escherichia coli

E. coli

Escherichia coli are gram-negative, facultative anaerobic, rod-shaped bacteria which live in the intestines of warm-blooded animals.  Although most E. coli bacteria are not harmful, a few strains can cause gastroenteritis.  Pathogenic E. coli bacteria are usually transmitted through contaminated food and water.  


Salmonella are rod-shaped, gram-negative bacteria which are related to E. coli.  Different species of Salmonella bacteria can cause gastroenteritis, typhoid, and paratyphoid.  Gastroenteritis resulting from Salmonella usually lasts for only a few days and seldom needs to be treated, but typhoid and paratyphoid are more serious illnesses, lasting up to three weeks and requiring antibiotics. 

Salmonella infections are usually spread through contaminated food and water.  Like Campylobacter infections, Salmonella infections are very common, with up to four million infections being reported each year.


Shigella are gram-negative, rod-shaped bacteria related to E. coli and Salmonella.  When infected with Shigella, people develop bacillary dysentery, an illness which is usually overcome by the body in a few days without treatment.  However, bacillary dysentery can be fatal to the young and old and, in severe cases, even healthy adults become so dehydrated by the dysentery that they require hospitalization. 

Shigella is unique in that only a few bacterial cells are required for infection.  These cells can be transmitted by direct contact from person to person, or they can be transmitted through contaminated food and water.  A few outbreaks of bacillary dysentery have been reported, but in most cases the bacteria do not survive for long enough in contaminated water to cause widespread problems. 

Vibrio cholerae

Vibrio cholerae

Vibrio cholerae is a gram-negative, curved rod bacterium.  Cholera, the disease caused by this bacterium, is a serious illness which can be fatal over a short period of time if left untreated.  The bacteria is transmitted primarily through contaminated food and water.  Although the disease is rare in the United States and Europe, cholera outbreaks commonly occur in Asia and Africa as well as in Central and South America. 

Antibiotic Resistance

Diseases caused by bacteria can be treated with antibiotics, which are drugs able to kill microorganisms.  In every population of bacteria, however, there are often resistant bacteria which are able to survive the antibiotic treatment.  These resistant bacteria are able to survive and reproduce in the presence of antibiotics while more susceptible bacteria in the population die.  The result is a population of antibiotic resistant bacteria which are unaffected by antibiotics.  Antibiotic resistance can also be spread through a bacterial population when bacteria exchange plasmids. 

Recent studies have shown that antibiotic resistant bacteria are quite widespread in America's rivers, probably due to antibiotics making their way through wastewater and into natural waters.  In some rivers, the antibiotic resistant bacteria made up as little as 2% of the population while in other rivers all of the bacteria were found to be antibiotic resistant.  Antibiotic resistant bacteria can quickly spread from location to location through rivers and can contaminate drinking water supplies, infecting humans and causing diseases which are very difficult to treat. 




A procaryotic organism, such as bacteria, is a cell that lacks a membrane-bound nucleus or membrane-bound organelles. The exterior of the cell usually has glycocalyx, flagellum, fimbriae, and pili. The procaryotic cell's cell wall is located outside the plasma membrane and gives the cell its shape and provides rigid structural support for the cell. The cell wall also protects the cell from its environment. The wall of a bacterium is classified in two ways, gram-positive or gram-negative. The procaryotic cell has a cell membrane called the cytoplasmic membrane that forms the outer structure of the cell and separates the cell's internal structure from the environment. This membrane provides a selective barrier, allowing certain substances and chemicals to move into and out of the cell. Two types of transport mechanisms are used to move substances through the cytoplasmic membrane, passive and active. The general shapes of bacteria are cocci, bacilli and spirilla. The Gram Stain Procedure is the most fundamental technique for identifying bacteria. There are three types of respiration that can occur: aerobic, anaerobic and facultative.




Betsy, Dr. Tom and Keogh, Jim. 2005. Microbiology Demystified . McGraw-Hill Publishing



Complete Assignment 5 on Bacteria Identification . You may do the Assignment online to get credit or print it out and send it to the instructor.

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




Please read the Total Coliform Bacteria lab procedure. No work needs to be done in regards to this lab.




Answer the questions in the Lesson 5 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.