Taxonomy
The term taxonomy alone is enough to
cause glazing over of students' eyes in traditional Biology courses. The
significance of taxonomy cannot be overlooked as it is the basis fore categorizing
organisms. It is very important for the student to determine the factor(s), or
characteristic(s) of the organism which cause the specific life form to be included
and excluded by various taxa or categories.
Years ago organisms were classified as plants or animals. The
first scientist known for his work in this arena was Carl von Linne. He
categorized 11,000 organisms with genus annd species epithets. As
more information became available it was clear that these categories did not
cover all life forms. Whittaker's 5-kingdom system was subsequently accepted.
In this system, organisms were categorized in five major divisions as follows:
Ancestral Life Form
Monera Protista
Fungi Plantae Animalia
Increasing technological proficiency has
enabled scientists to establish new relationships between organisms
based on similarities in biochemistry. The most widely accepted method of
classification is the Three Domain System.
Domain Prokarya (bacteria and Blue-green
algae)
Domain Archaea (non-nucleated cells found
in extreme environments)
Domain Eukarya
Kingdoms:
Diplomonadida
(diolomonads, two-nuclei, no miitochondrian.
Parabasala
(Trichomonads, no mitochondria)
Euglenazoa
(Euglena)
Alveolata
(dinoflaggelates, apicomplexans, amoeba, forams, actinopoda, slime molds)
Stramenopila
(diatoms, brown alga, water molds) –
Rhodophyta.
(red algae)
Chlorophyta
(green algae)
Mycetozoa
(slime molds)
Fungi (molds,
cup fungi, mushroom)
Plantae (mosses, ferns,
evergreens, flowering plants)
Animalia (sponges, invertebrates, vertebrates
Domain Bacteria Laboratory
Objectives:
1. Describe
the main. difference between Domain Archaea and Domain Eubacteria.
2.
Describe the main characteristics of methanogens, extreme thermophiles and
halophiles.
3. Describe general
characteristics of domain Eubacteria (domain Bacteria).
4. Describe general characteristics of
Cyanobacteria and their role in ecology
5. Demonstrate the occurrence
of bacteria in nature by culturing microbes from multiple sources.
6. Describe the appearance and function of bacterial endospores.
7. Describe the economic significance
of bacteria.
Prokaryotes
The former kingdom Monera is divided into two Domains under
currently accepted taxonomic classification systems. The first Domain is that
of Bacteria. It is further subdivided
into about a dozen bacterial groups, five of which are:
1.
Spirochetes
2.
Chlamydias
3. Gram-positive bacteria
4. Cyanobacteria, and
5. Proteobacteria.
The second domain is the Domain Archaea which includes
prokaryotes which inhabit the extreme and harsh environments
of the planet.
1. Methanogens
2. extreme halophiles
3. extreme thermophiles.
Prokaryotes
are virtually ubiquitous! ! They are everywhere! Both in respect to numbers and impact, they
dominate the biosphere. Most prokaryotes are very small. Often they are
1/10-1/100 of the size of a eukaryotic cell.
It is estimated that the earliest of prokaryotes developed life forms
about 3.5 billion years ago. Some of them function as decomposers. Life is
possible for all as they assist in recycling inorganic compounds required by plants.
Emphasis
is placed on anti-microbial disinfectants and cleaners today but only a small
number of bacteria are pathogenic (disease-causing.) Many prokaryotes live in
close associations with each other as well as eukaryotes in what are called
symbiotic relationships. Lyn Margulis proposed The Theory of Endosymbiosis. She
suggested that some organelles of the eukaryote (chloroplasts and mitochondria)
existed originally as independent single-celled organisms, which were
prokaryotes.
While
a few thousand prokaryotes are known, it is estimated that perhaps millions
exist in nature. There are many diverse forms capable of unusual metabolic
pathways. It is within these simplest of life forms that more complex
biochemical pathways originated.
The structure of prokaryotes is rather simple when compared with cells of
higher organisms. The cell wall, constructed largely of peptidoglycan, gives
structure to the cell. It is very different from the cell wall of various
eukaryotes. Classification placed them with plants in the antiquated 2 kingdom
system. External to this cell wall are other materials that constitute a type
of extra cellular material, which does differ from species to species.
One of the most well used methods of classification involves
use of the Gram stain. Bacteria can be classified as Gram-positive if they
stain purple via the technique where as Gram-negative bacteria remain pink and
do not capture the Crystal Violet-Iodine Complex because to less peptidoglycan
in the wall. Gram-negative bacteria possess an outer membrane, which consists
of lipopolysaccharides (carbohydrates and lipids.) One of the mechanisms by
which prokaryotes are often limited by antibiotics is by prevention of the
cross-linkage in the cell wall.
Another layer, the capsule, is formed by
many prokaryotes. It is a sticky substance that forms yet another protective
layer. Capsules also allow the bacteria to adhere to substrata as well as to
each other in colonial form. Sometimes surface appendages called pili promote
adherence of these cells to mucous membranes or to each other in exchanging
plasmids (small are loops of DNA that confer advantages such as resistance to antibiotics.)
While no organelles exist as in eukaryotic cells, many of the biochemical
pathways still exist in association with infoldings of membrane (mesosomes.)
The DNA tends to be naked and occurs in a snarl of fibers, the nucleoid.
Ribosomes exist and function in a similar manner to those in eukaryotes;
however, they are smaller and less complex. Taxis, movement toward or away from
a stimulus, occurs in response to food, light, magnetic forces or gravity. Some
cells possess a single flagellum or flagella, occasionally under the outer
membrane, as in spirochetes.
Cyanobacteria can exist under very harsh conditions and formerly were
classified with plants not only because of the cell wall but also because many
contained various pigments and were able to carry out photosynthesis with the
net production of oxygen. Some of the earliest fossils of life forms are stromatolites,
layers of cyanobacteria that became fossilized. Some cyanobacteria are able to
carry out nitrogen fixation whereby atmospheric N2 is converted to
NH3, which is then used to synthesize amino acids. The gelatinous
capsule and toxins make them a poor food source for predators. While these
algae are often referred to as the blue-green algae, they are not always that
color. Many possess chlorophyll a, as in plants, but also accessory pigments
that change the absorption spectrum for the specific plant.
Prokaryotes may appear in colonies, in bunches or strings of cell. Monerans
that we will look at today include various forms of bacteria. Some of these may be on prepared slides. Note
that observed internal cell structure is not discernible with the techniques
that you will be using today. Cell walls and capsules will be seen with stains.
Remember that bacteria are very simple life forms but are ubiquitous and
extremely important. Bacteria play a major role in decomposition of organic
material, in disease as well as antibiotic production.
Typical form is in the spherical shape, coccus, rod-shaped bacillus or spiral
shaped spirochete. It is very important to use the oil immersion technique when
examining them microscopically.
bacilli cocci spirilla
Structures present
include cell wall, capsules outside wall, pili, endospores, flagella (simple),
nucleoid, plasmids, ribosomes and mesosomes (infoldings of the plasma membrane
associated with enzymes for specialized reactions like respiration).
Gram positive bacteria
possess more peptidoglycan (polymers of sugars and amino acids) in a simpler
cell wall while gram negative bacteria have a more complex wall with less
peptidoglycan.
Growth
occurs by a process called binary fission. The DNA is replicated and attached
-to the plasma membrane. The membrane slowly pinches: off as the cell wall and
capsule are added in small increments. Replication can occur as often as every
20 minutes with sufficient elimination of waste products and availability of
nutrients.
Endospores are tough capsules that form around the nucleoid yielding
a structure that is resistant to extreme conditions. Only certain bacteria form
endospores but these must be exposed to extreme conditions to destroy the
endospore. Endospores can survive in honey which is generally a great bacteriacidal
agent. It is not recommended that infants be fed honey for this reason. Clostridium
outlines which is responsible for food poisoning be fatal.
Genetic recombination occurs in a
number of ways. Transformation involves taking up genes from the environment. Conjugation
involves transferring genes from bacterium to bacterium. The mode of transduction allows for genes to
be transferred to bacteria and viruses.
All
known nutritional modes evolved in prokaryotic cells. The table below lists the
four modes prokaryotes can be divided into using energy source (phototroph
versus chemotroph) and carbon source (autotroph versus heterotroph) as the
criteria.
Nutrition Mode |
Energy source |
C source |
1. photoautotrophs |
Light |
CO2 |
2. chemoautotrophs |
Inorganic chemicals |
CO2 |
3. Photoheterotrophs |
Light |
Organic compounds |
4. chemoheterotrophs |
Organic chemicals |
Organic compounds |
Relationships with other organisms include bacteria as
saprobes and symbionts (mutualism, commensalism, parasitism.) Nitrogen fixation
is significant as prokaryotes are responsible for using atmospheric nitrogen
and
converting it to ammonia for use by plants. Denitrifying bacteria carry out the opposite
process.
Obligate aerobes include those bacteria
that require oxygen as an electron receiver in cellular respiration.
Facultative anaerobes can operate with oxygen present or harvest energy through
fermentation, which of course does not harvest as much energy as would be
provided through oxidative phosphorylation. Obligate anaerobes are often
poisoned by oxygen and participate in cellular respiration and electron
transport with another molecule acting as an electron acceptor at the end of
the ETC (Electron Transfer Chain).
The first prokaryotes (3.5 billion years
ago) were probably chemoheterotrophs.
ATP might have been the first nutrient. Evolution of glycolysis probably
occurred in a step by step process. The origin of ETC as probably early in the
energy harvesting process. Proton pumps originally were used to expel extra
protons to the environment... Ultimately the cells were able to pair the flow
of protons into cell with the phosphorylation of ADP via chemiosmosis.
Photosynthesis developed a bit
later. The original function of pigments
was probably as a shield from light. Bacteriorhodopsin, a pigment used light to
pump protons out of some cells. Later, other photo systems developed that could
generate reducing power in the form of NADPH by driving electrons from hydrogen
sulfide to NADP+.
The
evolution of specialized bacteria (cyanobacteria), capable of oxygenic
photosynthesis, began the oxygen revolution.
Oxygen was released and the atmosphere became more oxidizing. This
resulted in great changes in the ecosystems.
Many eubacteria are responsible for disease in humans. About 50%of all human disease is caused by
bacteria. Our immune system is generally able to protect us from these
pathogenic bacteria. Periodically, however, the bacterium evades the body's
defenses and illness occurs. Other pathogens are opportunistic. These bacteria
are normal residents of the human body but only cause illness when the body's
defenses are weakened.
Robert Koch,
a German doctor, was the first to make the connection .between a disease and
the specific bacterium that caused it. Koch established four criteria, now
called Koch’s postulates for establishing a pathogen as the specific cause of a
disease. These guidelines are still applied to associate microbes and resulting
diseases today. It is necessary to
1.
find the same pathogen in each case of
the disease,
2.
isolate and culture the microbe,
3.
re-infect a population and
4.
isolate and culture it once again.
Some pathogenic bacteria cause illness by
disrupting the health of the host. However, most bacteria cause illness by
producing poisons called exotoxins and endotoxins.
Exotoxins
are typically soluble proteins secreted by living bacteria d exponential growth.
The production of the toxin is generally specific to a particular bacterial
species that produces the disease associated with the toxin (e.g. only Clostridium
tetani-produces tetanus toxin;
only Corynebacterium diphtheriae produces the diphtheria toxin).
Usually, virulent strains of the bacterium produce the toxin while nonvirulent
strains do not, and the toxin is the major determinant of virulence (e.g.
tetanus and diphtheria). At one time it was thought that exotoxin production
was limited mainly to Gram-positive bacteria, but both Gram-positive and Gram-negative
bacteria produce soluble protein toxins. Bacterial protein toxins are the most
powerful human poisons known and retain high activity at very high
dilutions.Endotoxins are part of the outer membrane of the cell wall of
Gram-negative bacteria.
Endotoxins are invariably associated with Gram-negative
bacteria whether the organisms are pathogens or not. The term
"endotoxin" is occasionally used to refer to any cell-associated
bacterial toxin. However, it more properly refers to lipopolysaccharide toxins
associated with the outer membrane of some Gram-negative bacteria (for example,
E. coli, Salmonella, Shigella, Pseudomonas, Neisseria, and Haemophilus).
Using the Oil Immersion Objective
Filling the space between the slide and the lens with oil
increases the resolution of the image. Oil has a much higher index of
refraction than air and contributes to the magnification of the image. The
following directions must be followed carefully to avoid damage to the
microscope or to the slides.
1.
Locate the object and center it with scanning lens.
2. Move to low, and then to high power, refocus while using
the fine adjustment knob only.
3.
Swing the high power lens away from the slide. Place a drop of immersion oil on
the center of the cover slip and move the oil immersion lens into position.
Refocus using the fine adjustment knob only. 4. When you are finished,
swing the lens away. Do not move any other lens into position.
5.
Remove the slide and wipe most of the oil off with paper, then clean with soap
and water.
6. Clean the lens with xylene and then dry lens paper.
7. Students MUST
show the microscope to professor before placing in the cabinet
The
bacteria that you will examine are found in yogurt with live cultures of
bacteria. The bacteria commonly used are Streptococcus
thermophilus, which
ferments the sugar lactose, and Lactobacillus bulgaricus, which produces
the flavors and aroma of yogurt.
Prepare a slide of yogurt culture:
1. Obtain a slid and cover
slip. With a bacterial loop or a toothpick transfer a small amount of yogurt to
the center of the slide.
2. Smear the yogurt in an
area slightly smaller than the cover slip. Allow it to dry
3.
Place two drops of crystal-violet or carbolfuchsin stain on the air-dried yogurt
smear.
4. Place a coverslip on
the stained smear and examine under the microscope.
Draw the observed
bacterial types in the space below.
Cyanobacteria are blue- green
algae. Blue-green algae is and in aquatic environments as well in damp,
terrestrial environment. They exist largely as colonies and filaments and
produce spores that resist desiccation. They generally have gelatinous capsules
that are often toxic and are not a normal food source for heterotrophs.
You
may be using prepared slides or using fresh cultures of these organisms. A few
cyanobacteria are Anabeana, Gloeocapsa, Oscillatoria and Merismopedia. Your
instructor may provide a survey culture of various organisms to key out and to
exam.
Draw and label several
representatives of cyanobacteria in the space below.
Anabana GloeCocapsa Merismopedia Oscillatoria
Often the source of a
specific disease or type of contamination can be determined by culturing microbes,
growing them under rather specific conditions (media, temperature, and gasses)
and using indicators to demonstrate specific reactions that are being carried
out.
Your
instructor may give you nutrient agar plates. The instructor will give
directions as to the manner in which they shall be used. Students will use
sterile swabs to inoculate the Petri dishes. Various surfaces may be swabbed to
check for presence of various types of prokaryotes. Definitive techniques will
not be used at this level to attempt to identify specific genera. Plates should
be sealed with tape and placed in a safe place for 2-3 days. Each round colony
that grows is likely to be the result of a single bacterium's growth.
Lab
Questions to be answered