Chapter 1

The Microbial World and You

Microbes – minute living things that are too small to be seen with the unaided aid
(micro – small; organism – living)
bacteria, fungi (yeasts and mold), protozoa, microscopic algae, viruses
Virus – noncellular entities on the border between living and nonliving
Microbes are good and bad
Good – bases of food chains, oxygen-generating, waste breakdown, intestine digestion, vitamin synthesis (Vitamin
B and K in human gut), chemical synthesis, food processing, drug synthesis
Bad – pathogenic (minority of microbes)
Found almost everywhere: human skin, intestines, surfaces, marine and fresh water, deserts, forests, mountain
tops, artic circles, deep mines, deep water vents, sulfuric pits

Naming and Classifying Microorganisms

Binomial Nomenclature (bi-two, nom-name clature-process) – method by which we name microbes, developed by
Carolus (Carl) Linnaeus in 1735
Latinized because Latin is traditionally used by scholars
Each organism given two names: genus and species; both used in written text
Genus –capitalized first name, in italics or underlined, general name-equivalent to your last name
Species – lowercase second name, in italics or underlined, specific name-equivalent to your first name
In written text, by custom, name is spelled out completely on first use and every other use following
genus may be abbreviated
Occasionally a third name is introduced after the species epithet, this is called a subspecies, equivalent to
a breed, variety, type, strain, ect.
Example of use:
Scientific names can, among other things, describe an organism, honor a researcher, or identify the habitat of a
species. For example, considerStaphylococcus aureus, a bacterium commonly found on human skin. Staphylo-
describes the clustered arrangement of the cells, coccus indicates the cells are sphere-shaped. The specific
epithet, aureus, is Latin for golden, the color of the bacteria colonies. The genus of the bacterium Escherichia coli is
named for a scientist, Theodor Escherich, whereas the specific epithet, coli, reminds us that E. coli live in the colon,
large intestine. Both S. aureus and E. coli will be studied extensively this semester.

Types of Microbes

A.Bacteria (pl.)/ Bacterium (s.)

unicellular prokaryote (prokaryote (pro=first; kary=nut, nucleus) – genetic material not enclosed in a
special nuclear membrane)
appear in several shapes, most common are:
o bacillus – rod-like
o coccus – spherical or ovoid
o spiral – corkscrew or curved
some can also appear star-shaped or square-shaped
may form in singles, pairs, chains, clusters, or other formations characteristic to a genus or species
enclosed in cell walls composed of carbohydrate and protein complexes called peptidoglycans
reproduce by cell division (binary fission) much like mitosis
nutrition: varies by genus and species
o organic chemicals derived from dead or living organisms
o photoautotroph - photosynthesis

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 o chemoautotroph – inorganic chemical nutrition
movement - some swim by flagella

B.Archea / archeabacteria
unicellular prokaryote
cell walls lack peptidoglycan
usually live in extreme environments
three main groups:
o methanogens –produce methane as a waste production of respiration
o halophiles – live in extremely salty environments (Great Salt Lake or Dead Sea)
o thermophiles – live in hot sulfurous water (hot springs, deep sea vents)
-not known to cause disease in humans

C.Fungi (pl.) / fungus (s.)

eukaryotes, unicellular or multicellular (eu=true) eukaryotes=genetic material is enclosed in a nuclear
membrane
cell walls composed of chitin (carbohydrate-based)
yeast – unicellular form, oval in shape, larger than bacteria
molds – multicellular form, form visible masses called mycelia
o mycelia composed of long filament (hyphae) that branch and intertwine
o appear as a cottony growth on bread and fruit
mushrooms –larger multicellular fungi that resemble plants
reproduction is sexual or asexual (sexual=two parents; asexual = one parent)

nutrition – absorb solutions of organic material from environment (soil, seawater, freshwater, plant host,
animal host)

slime molds- characteristics of both fungi and amoeba (protozoan)

D.Protozoa (pl.) / protozoan (s.)

unicellular eukaryote
classified based on type of locomotion:
o sarcodines (sarco=flesh) move by pseudopodia (false feet), extensions of cytoplasm
and cell membrane
o mastigophorans (mastigo=flagella) flagellated, long whip-like extension of cell
membrane
o ciliophorans – move by cilia, short hair-like extensions of cell membrane
o apicomplexans – no movement by pseudopodia, cilia, or flagella
variety of shapes
free-living or parasitic
reproduction sexual or asexual

E.Algae (pl.) /alga (s.)

- mainly unicellular eukaryotes, few multicellular forms
- photosynthetic
- wide variety of shapes
- reproduction sexual and asexual
- cell walls composed of cellulose

F.Viruses (pl.) / virus (s.)

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 acellular (not quite a true cell)
very small, only seen with electron microscope
consists of a core made of only one type of nucleic acid (DNA or RNA)
core surrounded by a protein coat
sometimes the coat is encased by an additional layer, lipid membrane called envelope
can only reproduce in a host cell using the host’s cellular machinery
meaning they are parasitic and cannot survive without a host
on the border between living and nonliving

G.Multicellular animal parasites

not microbe called the helminthes (helmin = worm)
divided into two groups:
o flatworms – dorsoventrally flattened, nonsegmented
o roundworms – cylindrical in shape, nonsegmented
are microscopic during some life stages

Classification of Microorganisms

three domains of organisms (developed by Carl Woese, 1978)

o bacteria (cell walls contain peptidoglycan, no true nucleus)

o archae (cell walls, if present, do not have peptidoglycan, no true nucleus)
o eukarya (true membrane bound nucleus)
Protists/ protozoan – slide molds, protozoa, algae
Fungi – yeasts, molds, mushrooms
Plants – mosses, ferns, conifers, flowering plants
Animals – sponges, worms, insects, vertebrates
 

HISTORY OF MICROBIOLOGY
First Observations
Robert Hooke (1665) first defined life’s smallest structural unit as a cell, marked the beginning of cell
theory
Cell theory – all living things are composed of cells
Antoni van Leeuwenhoek (1673 thru 1723) describe "animalcules" in water-based substrates (we call
these animalcules bacteria and protozoa)

Debate over Spontaneous Generation

after discovery of these small creatures, scientists become interested in where these organisms originate
until the 1850’s scientists believe in spontaneous generation, the formation of new life from non-living
substances
in spontaneous generation, mice, toads, snakes could arise from moist soil, flies emerge from manure,
maggots arise from decaying corpses
even created recipe books on how to create all kinds of living organisms

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Evidence for and against spontaneous generation

- Francesco Redi (1668) demonstrated that maggots do not simply arise from dead meat
- maggots appeared only when flies where allowed to leave their eggs on meat
- results were a serious blow to the spontaneous generation idea
John Needham (1745) demonstrated that nutrient broth boiled to destroy life and then poured into
cooled beakers would spontaneously generate microorganisms
He forgot to cover the beakers and air-borne microbes contaminated the broth
Lazzaro Spallanzani (1765) debunked Needham’s work by suggesting that that air-borne microbes could
have landed in the boiled broth and begun growing
he proved this by sealing the broth in a beaker first and then boiling the broth
Sealed, boiled broth did not grow microbes
Needham responded by saying the "vital force" necessary for spontaneous generation had been
destroyed by the heat and kept out of the flask by the seals
"vital force" was given more credence by the discovery of importance of oxygen to life, indicating there
was not enough oxygen in the sealed flasks to support life
The issue remained unresolved until 1858-1861 when the concept of biogenesis was theorized and Louis
Pasteur got involved.

Theory of Biogenesis
living cells can arise only from preexisting living cells Rudolf Virchow, 1858
1861, Pasteur demonstrated that microbes are present in the air and can contaminate sterile solutions,
but the air itself does not create microbes
Pasteur filled several short-necked flasks with beef broth and boiled the broth.
Some flasks were left open for several days and were found to be contaminated
Other flasks were sealed after boiling and were free of microbes (remember before the flasks were sealed
before boiling)
Results allow for reasoning that microbes in the air contaminated the flasks
to further prove that air-borne microbes do not spontaneously generate, Pasteur performed a second
experiment
Pasteur placed broth in a long-necked flask and bent the neck of the flask into a "s" curve
he then boiled the broth and allowed it to cool
broth in the flasks did not decay or become contaminated for long periods
air was allowed to freely move in or out of the flask but air-borne microbe were trapped in the curve of
the neck
some of the flasks still show no contamination even now over 140 years later
Later experiments of Pasteur’s proved that microbes can be present in nonliving matter, solids, liquids, in
air
Also demonstrated that microbes can be destroyed by heat
And methods can be devised to block the access of airborne microbe to nutrient environments
These discoveries form the basis of aseptic techniques, techniques that prevent contaminations by
unwanted microbes standard practice in laboratory and medical procedures.
FIRST AND MOST IMPORTANT THING YOU WILL LEARN IN LAB

Golden Age of Microbiology

Starting with Pasteur and continuing for about 50 years, there was an explosion of discoveries in
microbiology (1857-1914)
Fermentation and Pasteurization
commercialization of anti-microbe technique
French merchants asked Pasteur why wine and beer soured
If they could prevent the beverages from souring, they could ship longer distances and make more money
Pasteur discovered that there were two different processes occurring in these alcoholic drinks

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 Yeasts converted the sugars from the plants used in the drinks into ethanol (Good thing) This process was
called fermentation and is used very commonly to make wine and beer and well as several other food
products including bread dough (ethanol evaporates during baking)
Other microbes (mainly bacteria) cause the souring and spoilage. These bacteria change the alcohol into
vinegar (acetic acid) in the presence of air
solution to prevent spoilage: heat the beverage just enough to kill the bacteria that cause spoilage
process is called pasteurization and is now used for milk as well as alcoholic beverages

Germ Theory of Disease

fermentation led to the thought that some microbes might also cause diseases
if yeast could interact with the sugars in dough and wine and beer, other microbes could have similar
relationships with plants and animals, they could cause disease
disease was thought to be caused by demons and punishment for sins
1865, Pasteur proved silkworm disease caused by microbes
1860’s Joseph Lister begins using phenol (carbolic acid) on surgical wounds to prevent infection.
practice of using phenol drastically reduced number of infections and led other physicians to adopt the
technique
1876, Robert Koch proves that bacteria actually cause disease
- found that bacteria isolated in diseased cattle will cause diseases when injected into healthy animals
- Established a sequence of experimental steps for directly relating a specific microbe to a specific disease.
Steps/Rules are known as Koch’s postulates
o Pathogen must be present in all cases of disease
o Pathogen must be isolated and grown in lab in pure culture
o Pathogen from pure cultures must cause disease when inoculated into healthy,
susceptible lab animal
o Same pathogen must be isolated from the diseased lab animal
- Using his postulates, Koch identified causative agents for tuberculosis, diphtheria, human cholera, typhoid,
tetanus, and other diseases. The postulates are still applied today, although there are some limitations.

Vaccination
Treatment or preventative procedures are often developed before scientists know why they work
smallpox vaccine good example of this
1796, Edward Jenner performed first vaccination
Jenner discovered that if a person was exposed to cowpox they would not contract smallpox
Pasteur later named this technique, vaccination (vacc=cow) in honor of Jenner’s work
1880, Pasteur begins developing vaccines for a variety of diseases, fowl cholera, anthrax, rabies…
Today, most vaccines are produced by weakening the viral particles, killing the viral particles, or
genetically engineering the viral particles

Birth of chemotherapy

chemotherapy – treatment of disease by using chemical substances; also refers to chemical treatment of
noninfectious disease (cancer)
synthetic drugs- prepared from chemicals in a laboratory
antibiotics – produced naturally by bacteria and fungi to act against other microbes
success of chemotherapy is that some chemicals are more poisonous to microbes than to the host
infected by the microbe

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First Synthetic Drug

- First chemotherapy—used substances from nature such as plant products (folk medicine)
Cinchona bark—quinine for malaria
Willow bark—aspirin
Foxglove—digitalis, congestive heart failure and diurectic against dropsy (excess fluid build-up in tissues)
1910, Paul Ehrlich found Salvarsan (arsenic derivative) effective against syphilis
1930’s sulfa drugs (sulfonamides) synethisized
1928, Alexander Fleming discovered penicillin by accident. Fungus infected his bacterial plates and
inhibited the growth of his bacteria
- Penicillin not extensively tested or mass produced until 1940’s

Modern Developments in Microbiology

various branches of microbiology have developed:

o bacteriology – study of bacteria

o mycology – study of fungi
o parasitology – study of protozoa and parasitic worms
o immunology – study of how immunity reacts to various pathogens
o virology – study of viruses
o recombinant DNA technology – tinkering with genes and transference between
species

Recombinant DNA technology

-
 Little progress in molecular genetics and molecular biology took place until we began studying these things in one-
celled organisms in the 1940’s. Then rapid progress was made.
1941---1 gene 1 enzyme
1944---DNA was identified as hereditary material
1953--- Watson and Crick discovered structure of DNA
1960’s--- more about the way DNA controls protein synthesis, discovery of mRNA, regulation of
gene function, "cracked" the genetic code
1970’s----beginning of genetic engineering
- Recombinant DNA- genes can be isolated and inserted into other cells, even from one species to another, and
proteins still be made. This technique is currently used to produce hormones, vaccines, etc. The hope is that
eventually we may be able to insert the needed gene into the cells of a person with a genetic defect---gene
therapy.
 
MORE BENEFICIAL ACTIVITIES OF MICROBES:
1.Recycling vital elements-decomposition of wastes and dead plants and animals by microbes as well
as O2 production by photosynthetic microbes
2.Sewage treatment-bacteria are used in the breakdown of sewage to recycle the water
3.Cleanup of the environment-bacteria can aid in cleanup of pollutants and toxic wastes by
producing enzymes that break down these substances. Some of these bacteria are even genetically
engineered to clean up specific chemicals. This is called bioremediation.
Enzymes produced by bacteria (Bacillus) are used in detergents are used to remove spots

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 4.Insect control - due to concern about toxic insecticides in the environment, there is great interest in
developing microbes that affect only certain insect pests

BIOTECHNOLOGY AND GENETIC ENGINEERING

Biotechnology---commercial use of microorganisms to produce food, chemicals, etc.
The most recent form involves insertion of specific genes into cells to allow productions of new proteins-
for example,Escherichia coli  bacteria are now making human proteins such as insulin.
Diseases caused by genetic defects may one day be cured by insertion of the normal gene into the cells of
the patient (gene therapy)
 
MICROBES AND DISEASE
Although most microbes are harmless and some are even beneficial, a small number do cause disease. These are
pathogens (disease-causing) microbes. Some of these pathogens can be traced as far back as human history. With
the development of antibiotics, vaccines and other modern treatments, there was a time that we believed
infectious disease could be eradicated. Development of resistant bacteria and appearance of new diseases has
ended that belief. Some emerging infectious diseases newly recognized or found in locations where previously
unknown in recent years include:
SARS
West Nile encephalitis
Bovine spongiform encephalopathy (mad cow disease)
Escherichia coli O157:H7
Invasive Group A strep (flesh-eating bacteria)
Ebola fever
Hantavirus pulmonary syndrome
Cryptosporidium
AIDS

Quiz:

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CHAPTER 3 

OBSERVING MICROORGANISMS THROUGH A MICROSCOPE

 Magnification is necessary to observe and study microbes.  Also, we know all microbes are small, but we must still

be able to measure their size.  Units of the metric system are used.  The meter is the standard unit, but since this is

roughly equivalent to one yard, we must use much smaller units for microbes.

          Centimeter (cm) = 0.01 meter    (2.54 cm = 1 inch)

          Millimeter (mm) = 0.001 meter    (25.4 mm = 1 inch)

          Micrometer (m) = 0.001 mm    (25,400 m = 1 inch)

          Nanometer (nm) = 0.001 m    (25,400,000 nm = 1 inch)

  
MICROSCOPY---THE INSTRUMENTS

Van Leeuwenhoek’s best microscopes had a single magnifying lens and provided approximately 300X

magnification.  About 1830, an effective compound (2 lens) microscope was developed.  Improvements in this led

to the modern compound light microscope.  Using light rays to illuminate the specimen, magnifying lenses in the

ocular and the objectives can provide a total magnification of up to 1800 - 2000X, although most of these go up

only to 1000X.

 
LIGHT MICROSCOPY

Modern microscopes are compound light microscopes. This means that there are 2 lenses that magnify, the ocular

lens and the objective lens. Light rays provide the illumination. 

Brightfield microscopy---this is the most common use of the light microscope. Specimens are usually stained to

make them more easily visible.  Light rays pass though the condenser lens and are directed straight up into the

objective lens, passing through the specimen on the way.  The field of view, seen through the ocular, is brightly

illuminated, with the specimen appearing slightly darker if unstained, or brightly colored due to dyes.

The total magnification provided can be determined by multiplying the ocular magnification times the objective

magnification. The very best compound light microscope might have a top magnification near 2000X.

 Magnification alone is not the only concern--- a clear picture in which fine detail can be seen is also required.  This

is known as resolution (resolving power)--- ability of the lenses to distinguish between 2 points a specific distance

apart.  For light microscopes, this is 0.2 m.  This is the limiting factor in useful magnification by light microscope.

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Resolving power is a function of the wavelength of light from the light source and the numerical aperture of the

condenser and objective lens. The shorter the wavelength, the greater the resolution. Blue light has the shortest

wavelength, so that is the reason for the blue filters.

•          Refractive index is the light-bending ability of a medium.

•          The light may bend in air so much that it misses the small high-magnification lens.

•          Immersion oil is used to keep light from bending.

Unstained specimens may be difficult to locate and observe with bright-field microscopy, but in some situations

stains are undesirable.

         All stains kill the microbes, so living microbes must be observed unstained.

         Some microbes do not stain well.

         Staining procedures may distort the microbe or slightly alter the size. 

If stains are not appropriate, one of the following methods would work better than bright-field:

 1.  Darkfield Microscopy---this type of microscope uses a modified condenser.  Instead of directing all light rays

straight through the specimen into the objective, only those rays which are reflected off the specimen reach the

objective.  The result of this is that the objects on the slide glow against a dark background.

 2.  Phase-Contrast Microscopy---this technique permits better viewing of internal structures of cells without use

of stains, so living cells can be viewed. Areas and structures of different densities appear as various shades of gray. 

This also requires a special condenser.

 3.  Differential Interference Contrast (DIC) Microscopy---similar to phase-contrast but gives higher resolutions and

brighter color.

 SPECIAL TYPES OF LIGHT MICROSCOPES

         Fluorescence Microscopy-Special dyes (fluorochromes) can absorb short-wave length light and give off light

at a longer wave length.  Observed through a fluorescence microscope, which uses an UV light source, objects

stained with these dyes glow with neon colors against a dark background.  Some bacteria can be directly

stained with these special dyes to show their presence in a sample, but the more common use is the

fluorescent-antibody technique (immunofluorescence).  Antibodies against certain organism are stained with a

fluorescent dye.  These antibodies are added to a slide containing the specimen.  The slide is gently washed.  If

the antibodies did not match the bacteria or viruses in the specimen, the antibodies are washed away.  If the

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 antibody matches, it remains on the slide and can be viewed as it fluoresces.  This technique is a rapid, reliable

diagnostic test.

          Confocal Microscopy-Used in conjunction with a computer to construct 3D images of the specimen, sort of

a CT scan through a microscope.

 
ELECTRON MICROSCOPY

Objects smaller than 0.2 m cannot be clearly viewed with a light microscope.  They require the use of an electron

microscope.  Viruses and tiny internal cell structures would be examples.  These microscopes use a beam of

electrons, with its much shorter wavelength, for illumination instead of light rays, so the resolution is much greater

(2.5 nm compared to 0.2m for light microscopes). Images produced appear on a screen and are photographed.

Types of electron microscopes:

         Transmission Electron Microscopy (TEM)--- the beam of electrons is passed through a very thin-sliced

specimen.  Electromagnets are used to focus.  Objects are most often magnified 10,000- 100,000X or even

more.  Since the beam of electrons has much less penetrating power than light rays, the specimen must be

sliced very thin.  Even most bacteria would have to be sliced. Specimens are “stained” with thin coats of metal

salts to make them more clearly visible.  The image is focused on a fluorescent screen or photographic plate. 

Disadvantages:          

      1) Specimens are dead 

      2)Preparation may shrink and distort the specimen

         Scanning Electron Microscopy (SEM)---this technique “scans” the outside of the specimen and gives a 3-D

view.  Objects are usually magnified 1000-10,000X.  Entire cells, bacteria and viruses can be viewed.

 SCANNED-PROBE MICROSCOPY

         Scanning Tunneling and Atomic Force Microscopy---both produce 3D images of the surface of a molecule.

  

PREPARATION OF SPECIMENS FOR LIGHT MICROSCOPY

It is not impossible to view unstained microbes with bright-field microscopy.  This is usually done by one of the

following methods:

         TEMPORARY WET MOUNT---a drop of liquid containing the microbes is placed on a slide.  A cover slip is placed

over the drop and the slide is viewed.  Lowering the condenser and dimming the light a little may help. 

Dabbing vaseline around the edges of the cover slip will keep the specimen wet longer.

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         HANGING DROP---a special slide with a hollowed out “well” in the center is used. A drop of liquid containing the

microbes is placed on a cover slip which has dabs of Vaseline on the corners. The special slide is lowered over

it, concave side down, until it touches the Vaseline. The slide is then flipped over and viewed. .

Most of the time, microorganisms are stained by applying colored dyes to make them more easily viewed.  This

usually begins by spreading a thin film of the specimen over a slide.  This is called a smear.  It is allowed to air dry,

and then is passed through the flame of a Bunsen burner to heat fix the smear.  This is mainly to stick the

organisms to the slide, but the heat and drying also kill most of them.  The slide is now ready for the desired

staining procedure.

Some dyes are attracted to the outside of bacterial cells, which are usually slightly negative in charge. These dyes

are called basic dyes, and the colored portion of their molecules is positively charged. These include:

         Crystal violet         Methylene blue          Safranin          Carbolfuchsin          Malachite green

Other dyes are not attracted to the bacteria and color the background, leaving the bacteria unstained. These are

called acidic dyes. The colored portion is negatively charged, and is repelled by the outside of bacterial cells. These

include

         Nigrosin          India ink          Eosin

  
STAINING TECHNIQUES

    1.     Simple stains---one stain is applied to a heat-fixed smear and rinsed off.  All microbes would take on the

color of the dye.

      2.   Differential stains---stains will react differently with different types of bacteria

 
A. GRAM STAIN

This is the most important staining technique in microbiology.  It was developed in 1884 by Dr. Christian Gram.  It is

a differential stain used to sort bacteria into 2 groups, gram-positive and gram-negative, which is the first big step

in identification.  The gram reaction may also aid in determining the proper treatment of the disease.

Steps in the gram stain:

         Crystal violet is applied to a heat-fixed smear for 30 seconds and gently rinsed off.  This is the primary stain.

         The smear is thoroughly covered with Gram’s iodine, which is left for 1 minute and then rinsed.  The iodine

acts as a mordant, fixing the crystal violet into the cell wall of gram-positive bacteria.

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         95% ethanol (alcohol) is then applied with the slide at an angle, washing over the stained area.  This is the

decolorizing step.  The purple dye will wash off of the Gram-negative bacteria, but remain in the cell wall of

Gram-positive bacteria.

         Safranin is applied for 30 seconds.  This is the counter-stain.  Then rinse, blot dry and examine.

Gram-positive organisms will be purple (blue), Gram-negative will be red (pink).  This difference is due to

differences in the structure of the cell wall between the 2 groups.

 
B. ACID-FAST STAIN

This stain identifies bacteria that incorporate a waxy substance called mycolic acid into their cell wall. The main

acid-fast bacteria are members of the genus Mycobacterium, which includes the bacteria that cause tuberculosis

and leprosy, and the genus Nocardia, which are mostly soil organisms but occasionally show up as pathogens in

lung and skin infections..

Steps:

         Carbolfuchsin is steamed into a heat-fixed smear for 5 minutes. (Primary stain)

         Slide is cooled and gently but thoroughly rinsed.

         Acid-alcohol is applied (decolorization) to remove fuchsia color from all bacteria except acid-fast.

         Methylene blue is applied as the counterstain. Rinse, blot dry and examine

         Acid-fast organisms will be fuchsia (hot pink); non-acid-fast will be blue.

3.SPECIAL STAINS

1. CAPSULAR STAIN----some types of bacteria are surrounded by a slimy outer covering called a capsule. The capsule

does not accept colored dyes, so the procedure involves a negative staining technique. 

Steps:             

         A drop of nigrosin or India ink is applied to one end of a clean slide.

         Bacteria are mixed into the drop

         The mixture is spread to form a thin film over the slide, which is allowed to air dry.

         The slide is gently heat-fixed.

         Apply crystal violet for I minute and very gently rinse. Blot dry and examine.  The capsules

should appear as clear areas against a dark background.  Inside each capsule, a purple

bacterium should be seen.

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2. ENDOSPORE STAIN-----Endospores are special dormant forms of bacteria that are highly resistant to adverse

conditions such as heat, drying, toxic chemicals, lack of nutrients, etc.  Since only  a few genera of bacteria are able

to form endospores, this can be a good identification feature.

                        Steps in Schaeffer-Fulton Endospore Stain:

         Malachite green is steamed in to a heat-fixed smear for 5 minutes.

         The slide is gently but thoroughly rinsed.

         Safranin is applied for 30 seconds. The slide is washed, blotted dry and examined. 

Endospores retain the green dye and appear as tiny round to oval bodies.  Regular

vegetative cells will stain pink from the safranin.

3. FLAGELLA  STAINING---this is a tedious, difficult procedure which we won’t attempt.  It involves a special procedure

to deposit a thickening material on the flagella, which are too fine to be seen by light microscope

otherwise. (Electron microscopy could show them easily).

 CHAPTER 4

FUNCTIONAL ANATOMY OF PROKARYOTIC AND EUKARYOTIC CELLS

 
All cells share numerous characteristics, but cells of living things can be divided into 2 basic types. Here is a quick
comparison.
CHARACTERISTIC PROKARYOTIC EUKARYOTIC
Size Smaller---typical size is 0.2 - Larger---typical size is 10 -
2  m in diameter 100  m in diameter
Nucleus No nuclear membrane or nucleoli True nucleus with nuclear
(nucleoid) membrane and nucleoli
Membrane-enclosed organelles Absent Many, including lysosomes, Golgi
complex, ER, mitochondria &
chloroplasts
Flagella Consist of 2 protein building Complex; consist of multiple
blocks microtubules
Phagocytosis None Some can carry on phagocytosis
Glycocalyx Present as a capsule or slime Present in some cells that lack a
layer cell wall
Cell wall Usually present, chemically When present, usually simple,
complex (peptidoglycan) NO peptidoglycan
Plasma membrane No carbohydrates, almost all lack Sterols and carbohydrates
sterols incorporated
Cytoplasm No cytoskeleton Cytoskeleton
Ribosomes Smaller size (70S) Larger size (80S) except those
within organelles
Chromosome (DNA) Single circular chromosome, no Multiple linear chromosomes
arrangement histones with histones

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Cell division Binary fission Mitosis
Sexual reproduction No meiosis; transfer fragments of Meiosis
DNA only
 
PROKARYOTIC CELLS
Size----most range from 0.2 to 2  m in diameter, 2 - 8  m in length
Shape----Coccus (sphere), Bacillus (rod), Spiral

Arrangement:

a. Cocci---many will be single, but sometimes when cells divide they may remain attached to form:
1) Diplococci (pairs)
2) Streptococci (chains)
3) Tetrads (groups of 4)
4) Sarcinae (groups of 8)
5) Staphylococci (grapelike clusters)

b. Bacilli----mostly single, but some form:

1) Diplobacilli (pairs)
2) Streptobacilli (chains)
3) Palisades (rods side by side)
Rods may vary in shape-long rods to just slightly oval (coccobacilli). Some are irregularly shaped----club-shaped, for
example
Some species may exhibit the ability to have different shapes under different conditions. Some that are normally
quite rod-shaped may change to very short rods, so short they look almost round, for example. This is frequently
seen in cultures grown in artificial media and is called pleomorphism.

c. Spirals are always single. They vary from:

1) Vibrios-----just somewhat curved

2) Spirilla----spirals like a cork-screw and stiff
3) Spirochetes----spirals but flexible

A few bacteria do not fit into the 3 basic shapes: star-shaped, square, triangular. Some are filamentous
(threadlike). These are mostly soil bacteria.
 
PROKARYOTIC CELLS IN DETAIL

A. STRUCTURES EXTERNAL TO THE CELL WALL

1. GLYCOCALYX--this is a sticky mass that surrounds many prokaryocytes. It may be made of polysaccharides or
polypeptides.
a. Capsule-glycocalyx that is organized and firmly attached to the cell wall.
b. Slime layer-unorganized and loosely attached.
c. Functions of glycocalyx
1) May protect bacteria from phagocytosis, making them more virulent (able to cause more serious disease)—
Bacillus anthracis, Streptococcus pneumoniae
2) May allow bacteria to attach to a surface---Streptococcus mutans  on teeth
3) May serve as a source of nutrients and protect against dehydration

2. FLAGELLA-long filamentous (thread-like) appendages used by bacteria for movement

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a. Arrangement of flagella
1) Monotrichous--- 1 flagellum at one en
2) Amphitrichous- cluster of flagella at each en
3) Lophotrichous—2 or more flagella at one end
4) Peritrichous- over entire cell

b. Structure of flagella (3 parts):

1) Filament-- long, threadlike, outermost part. Made of a protein called flagellin (which may not be exactly
the same in all bacteria). Chains of this protein twist around a hollow core.
2) Hook--- connects filament and basal body
3) Basal body---anchors flagellum to cell wall and plasma membrane. Consists of a central rod inserted into
rings.

c. Movement is produced by rotation of the basal body. Bacteria possessing flagella can use them to move toward
a favorable environment or away from harm.
d. Since the proteins of the flagella may vary slightly species or even among different strains of the same species,
this can be used for identification.
1) The flagellar protein called H antigen can be used for distinguishing serovars, or variations within a species
of gram- negative bacteria.
2) Escherichia coli  has at least 50 different H antigens. One strain identified this way, E. coli 0157:H7 is the
one in the news for causing severe food poisoning

3. AXIAL FILAMENTS (ENDOFLAGELLA)--- these are found only on spirochetes. These are bundles of fibrils that
arise at the ends of the cell and wrap around the length of the cell under a layer called the outer sheath. These
axial filaments rotate like flagella, but since they are under the outer sheath this causes the outer sheath to move
and gives the bacteria a corkscrew motion.

4. FIMBRIAE AND PILI--these are made of the protein pilin. They are shorter and thinner than flagella, and are used
for attachment (not motility). They are found on some gram-negative bacteria.
a. Fimbriae-vary from a few to several hundred---used for attachment to surfaces, such as mucous
membranes. Neisseria gonorrhoeae (gonorrhea) and Eshcerichia coli use these in "hanging on" to establish urinary
tract infections.
b. Pili—longer--only one or two per cell. Used to join bacterial cells in preparation for the process of conjugation
(transfer of DNA). They are also sometimes called sex pili.
 
B. CELL WALL
Complex, semi-rigid structure that gives shape and protection to the cell.
1. Functions
► a. Prevents cell from rupturing due to entrance of too much water.
b. Helps give shape
c. Point of anchorage for flagella
d. May contribute to ability to cause disease
e. Site of action for some antibiotics
f. Chemical composition may be used in classification

2. Composition and Characteristics

a. Eubacterial cell walls are mainly made of peptidoglycan, a substance found nowhere else in nature. It is in the
form of one macromolecule that surrounds the entire cell. It consists of disaccharide units connected by
polypeptides to form a lattice.

1) Gram-positive cell walls

a) These consist of many layers of peptidoglycan, forming a thick, rigid structure.
b) Also included are teichoic acids, which consist of an alcohol and phosphate.

15
c) Exact structure of the teichoic acids varies among species. This is the part that often acts as an antigen and is
used for identification.
d) Cell walls of  Streptococcus are covered with various polysaccharides, which are used to sort them into groups.
e) Cell walls of acid-fast bacteria contain large amounts of mycolic acid and this is what makes them acid-fast.

2. Gram-negative cell walls--these have a much thinner layer of peptidoglycan. Outside the peptidoglycan layer is
the outer membrane, which consists of lipoproteins, lipopolysaccharides and phospholipids. The fluid-filled space
between the outer membrane and the plasma membrane is called the periplasm. The peptidoglycan is in this
space.
a. Functions of outer membrane
1) Help the bacterium avoid phagocytosis and effects of complement
2) Provides a barrier to certain antibiotics and harmful chemicals
3) Allows nutrients to enter through channels called porins
4) Lipopolysaccharides of the membrane are important in 2 ways:
a) O polysaccharides act as antigens—this is the rest of the E. coli strain O157:H7
b) Lipid A is an endotoxin----causes fever and shock

3. Atypical cell walls

a. A few prokaryocytes do not have a cell wall.
1) Members of the genus Mycoplasma have no cell walls. These organisms are also extremely tiny.
2) Since they lack a cell wall, their plasma membranes need extra strength. To provide this,  Mycoplasma organisms
incorporate sterols into the membrane.
b. Cell walls of archaea do not contain peptidoglycan.
1) Most have a cell wall which contains pseudomurein instead.
2) A few archaea lack a cell wall.

4. Damage to the Cell Wall

a. Cell walls can be damaged by exposure to the enzyme lysozyme (found in tears, mucus, saliva). Cell walls of
Gram-positive bacteria are more susceptible.
b. If the Gram-positive cell loses its wall and is in hypotonic surroundings, it will undergo lysis (burst) due to
entrance of water
c. If the surroundings are isotonic, the gram-positive cell can exist with no cell wall as a protoplast.
d. Lysozyme does not affect Gram-negative cell walls to the same extent. A Gram-negative cell treated with
lysozyme becomes a spheroplast. It retains its outer membrane and remnants of the cell wall.
e. Since animal cells do not have cell wall, antimicrobials which target cell walls often do not harm the cells of the
patient (host). This makes the cell wall an ideal target for antibiotics.
f. Penicillin is an example of an antibiotic that works this way. Gram -negative organisms are not usually highly
susceptible to penicillin because the outer membrane gives them some protection.
 
C. STRUCTURES INTERNAL TO THE CELL WALL

1. PLASMA (CYTOPLASMIC) MEMBRANE-----lies inside the cell wall and encloses the cytoplasm.

a. Consists of phospolipids and proteins. Phospholipids form a bilayer that consists of 2 layers of phospholipid
molecules. A phospholipid molecule looks like this:
 Head-polar and water soluble, hydrophilic (glycerol + phosphate) 2 tails, non-polar, hydrophobic, not water
soluble (fatty acids)
 

 b. Membrane proteins are placed in 2 groups

1) Peripheral proteins---loosely attached to the inner or outer surface of the membrane. Some are enzymes and
some give support to the membrane. These are easily removed.

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 2) Integral proteins are built into the membrane and cannot be removed without damage. Some of these extend
from one side of the membrane to the other (transmembrane proteins). Some proteins contain channels
through which substances entering/leaving the cell can pass.

c. The membrane is highly flexible and allows membrane proteins to change position if necessary. This idea of the
structure of the membrane is known as the fluid mosaic model.

d. Functions:

1) The plasma membrane serves as a selective barrier to substances entering or leaving the cell. Some things are
allowed to cross while others are not. This is selective permeability.

Permeable to a substance-----it can cross

Impermeable to a substance-----it cannot cross
Selectively permeable---some can cross and some cannot

Some general statements regarding permeability:

a) Large molecules mostly cannot cross (proteins would be an example). Smaller molecules cross more easily.
b) Lipid-soluble substances cross easily since they just dissolve in the phospholipid of the membrane. (O 2 and
CO2 are examples)
c) Some non-lipid-soluble smaller molecules cross with the aid of integral protein channels. (Some simple sugars,
water)
d) Ions usually require the aid of a transporter protein and cross slowly.
2) In bacteria, the plasma membrane is associated with the breakdown of nutrients and the synthesis of ATP. In
photosynthetic bacteria, required pigments and enzymes for the process are associated with the plasma
membrane.
 

DAMAGE TO THE PLASMA MEMBRANE BY ANTIMICROBIALS

The plasma membrane is vital to the survival of the bacterial cell. Some chemical disinfectants act by damaging the
plasma membrane. This includes aldohols and quaternary ammoniums. In addition, polymixin antibiotics also act
on the plasma membrane.
Before discussing movement across the plasma membrane, we need to briefly review solutions. A solution consists
of a dissolving medium, called the solvent, and one or more dissolved substances, called the solutes. In biological
solutions, the solvent is water. In crossing cell membranes, substances are moving from one solution to another.

MOVEMENT OF MATERIALS ACROSS MEMBRANES

Processes are divided according to whether or not they require energy.

1) Passive processes---- all share 2 major characteristics:
Do not require energy. Can move only from greater concentration to lesser concentration (with the
concentration gradient)

a) Simple Diffusion----- net movement of molecules or ions from an area of higher concentration to an area of
lower concentration. This movement continues until the 2 concentrations become equal.

b) Osmosis- this is really still simple diffusion but this time water (solvent) molecules do the moving instead of the
solutes (dissolved material). Osmosis occurs across a membrane that is permeable to water but relatively
impermeable to the solutes. Water molecules move from an area of higher water concentration to an area of
lower water concentration. Here are the 3 ways osmosis can affect a bacterial cell:

17
1] Isotonic surroundings-water and solute concentration are equal inside the cell and in the solution that
surrounds the cell. Since concentrations are already equal, no net movement occurs.
 Water = Water
Solutes = Solutes
2] Hypotonic surroundings---- water concentration is higher and solute concentration is lower outside the cell than
inside. Bacteria often find themselves in these surroundings. In these conditions, water would enter the cell, even
enough to cause the cell to burst, if not for the cell wall.
Lower Water Higher Water . Higher Solutes Lower solutes
3] Hypertonic surroundings- water concentration is lower and solute concentration higher outside the cell than
inside. Water would tend to leave the cell. If enough leaves, this could kill the cell. This is the principal behind
salted meats and jellies.
 Higher Water Lower Water Lower Solutes Higher Solutes
c) Facilitated diffusion---substance to be transported combines with a plasma membrane carrier protein called a
transporter. Transporters are specific to one substance. As the substance molecule moves into the open end of the
transporter molecule, its presence causes the carrier molecule to change shape. The open end of the carrier in now
on the inside of the cell, so the substance moves on in. The carrier releases the substance and returns to the
original shape, ready to repeat the process. Movement can continue as long as the substance is moving from
higher concentration to lower concentration.
d) Passive processes are less important in bacterial cells than in eukaryotic cells. One reason is that bacterial cells
frequently find themselves in surroundings where nutrients are in low concentration, so passive processes are
unable to meet their needs. Another point is that only relatively small molecules can enter cells by passive
processes. In some cases, bacteria produce enzymes and release them out into their surroundings (extracellular
enzymes). These enzymes break down larger molecules into a size that can enter passively.
 
2) Active Processes---all share 2 major characteristics
Require energy from ATP . Can move substances from lower concentration to higher concentration (against
a) Active transport---this is quite similar to facilitated diffusion in that a specific transporter protein molecule in
the plasma membrane is required. The difference is that ATP energy is required, and that substances can be moved
from lower concentrations to higher concentrations. In this process the substance is not changed as it
enters the cell.
b) Group translocation- this occurs only in prokaryocytes. It is a special form of active transport in which the
substance being transported is chemically changed as it is transported. One example is glucose, which is changed
to glucose phosphate as it is brought in. The plasma membrane is completely impermeable to glucose phosphate,
so it cannot leave the cell, even if the concentration inside the cell becomes quite high.
c) Phagocytosis and pinocytosis do not occur in prokaryotic cells.
 

2. CYTOPLASM----(prokaryotic definition)---cellular material inside the plasma membrane. (There is no nuclear

membrane for it to be outside of!) It is about 80% water and is thick, semitransparent and gel-like. It contains
dissolved and suspended materials:
Proteins (many of these are enzymes) Carbohydrates Lipids Inorganic ion.
It is the site of many chemical reactions.

3. NUCLEAR AREA (NUCLEIOD)---prokaryotic cells do not have a separate, membrane-enclosed area for the DNA.
Their DNA, in the form of one circular chromosome without associated histones, is not separated from the
cytoplasm. The chromosome’s DNA twists into a tight helix and is connected to the plasma membrane. As in
eukaryocytes, the chromosome must replicate (make a copy of itself) before the cell divides, so that each daughter
cell can receive one. Proteins of the plasma membrane are believed to be responsible for this replication of the
DNA.

18
4. PLASMIDS--not all bacterial cells have these, but many do. Plasmids are small, circular DNA molecules separate
from the chromosome. They usually contain 5-100 genes, which code for proteins that may give the cell resistance
to certain antibiotics, resistance to toxic metals, ability to produce certain toxins, or ability to synthesize enzymes
not included in the chromosomal genes. Proteins coded on plasmid DNA are usually not required for cells under
ideal conditions, but they can give the cell that possesses them advantages for survival and also makes those cells
more dangerous as pathogens. Copies of plasmids can be transferred from one cell to another and once a cell has a
plasmid, it will replicate the plasmid and pass a copy on to each daughter cell during cell division.

5. RIBOSOMES---ribosomes are the site of protein synthesis. In a living cell, nothing but a ribosome can make a
protein. In prokaryotic cells, the ribosomes are mostly scattered in the cytoplasm.
a. Ribosomes are composed of two subunits, which fit together. Each subunit consists of protein and ribosomal
RNA.
b. Bacterial ribosomes are described as 70 S ribosomes. The two subunits are one 30 S subunit and one 50 S
subunit. Ribosomes of eukaryocytes are slightly different.
c. The S stands for Svedberg unit. This measures the rate at which particles sink when centrifuged.
d. Since bacterial ribosomes are not quite the same as eukaryotic ribosomes, this makes another possible target for
antibiotics.
Streptomycin and gentamicin---30 S subunit
Erythromycin and chloramphenicol---50 S subunit

6. INCLUSIONS----these are reserve deposits of chemicals within the cytoplasm. They tend to accumulate when
nutrients are plentiful.
a. Metachromatic granules (volutin granules)---phosphate reserve that can
be used in synthesizing ATP. These large granules stain red with methylene blue. They are characteristic
of Corynebacterium diphtheriae.  These granules are also found in algae, fungi, and protozoa.
b. Polysaccharide granules---glycogen granules (which iodine stains reddish brown) and starch granules (which
iodine stains blue).
c. Lipid inclusions- most common is poly- -hydroxybutyric acid (PHB). Found in Mycobacterium, Bacillus  and
others.
d. Sulfur granules- bacteria whose metabolism involves oxidizing sulfur may deposit these as an energy
reserve. Thiobacillus  is an example.
e. Carboxysomes-bacteria that use CO2 as their only carbon source form these granules containing an enzyme
required in the process.
f. Gas vacuoles- these are adjusted so that the cell that contains them can float at the proper depth in water.
g. Magnetosomes- inclusions of iron oxide that act like magnets. Function uncertain, but they apparently can
decompose hydrogen peroxide.
7. ENDOSPORES- these are dormant cells, formed when certain genera (mainly Clostridium and Bacillus) find
themselves facing adverse conditions such as lack of nutrients, drying, presence of toxic materials, etc. Once
formed, endospores can withstand extreme conditions and survive for apparently unlimited periods of time.

a. Regular forms of bacteria are known as vegetative cells. These are the ones that take in nutrients, carry out
metabolic activities, reproduce, cause disease, etc. They are susceptible to heat, drying, lack of nutrients, etc., and
most of them are relatively easy to kill.

b. Sporulation or sporogenesis is the process of endospore formation.

1) Bacterial chromosome replicates. One copy plus a little cytoplasm are isolated in a small area of the cell as the
plasma membrane forms a partition called the spore septum.

2) Spore septum becomes a double-layered membrane that surrounds the chromosome and bit of cytoplasm and
this is now called the forespore. This is inside the original cell.

19
3) Thick layers of peptidoglycan are produced between the two membrane layers of the spore septum.

4) A thick protein spore coat forms around the outside membrane.

5) Water in the cytoplasm of the spore is removed throughout the process, leaving very little in the cytoplasm.
Metabolic reactions cease. This cytoplasm will contain high levels of dipicolinic acid and calcium ions.

6) When the endospore is complete, the vegetative cell that formed it will rupture and release the endospore.
While it is still contained in the vegetative cell, the endospore will be located in a characteristic part of the cell:
 terminal
 subterminal
 centrally
  
c. Germination is the return of the endospore to the vegetative state. One endospore produces one vegetative cell.
Exactly what triggers germination is uncertain, but enzymes within the endospore break down the wall and spore
coat and metabolism resumes.

d. Endospores are not easily killed by heat, chemicals, drying, radiation, etc. They can survive hours of boiling, for
example. This is important in the food industry as well as in medical sterilization.
They can survive for thousands of years.

EUKARYOTIC CELLS

This is the type of cell found in all living organisms except bacteria.

Here is the comparison chart again:

CHARACTERISTIC PROKARYOTIC EUKARYOTIC

Size Smaller---typical size is 0.2 - Larger---typical size is 10 -
2  m in diameter 100  m in diameter
Nucleus No nuclear membrane or nucleoli True nucleus with nuclear
(nucleoid) membrane and nucleoli
Membrane-enclosed organelles Absent Many, including lysosomes, Golgi
complex, ER, mitochondria &
chloroplasts
Flagella Consist of 2 protein building Complex; consist of multiple
blocks microtubules
Phagocytosis None Some can carry on phagocytosis
Glycocalyx Present as a capsule or slime Present in some cells that lack a
layer cell wall
Cell wall Usually present, chemically When present, usually simple,
complex (peptidoglycan) NO peptidoglycan
Plasma membrane No carbohydrates, almost all lack Sterols and carbohydrates
sterols incorporated
Cytoplasm No cytoskeleton Cytoskeleton
Ribosomes Smaller size (70S) Larger size (80S) except in
organelles
Chromosome (DNA) Single circular chromosome, no Multiple linear chromosomes
arrangement histones with histones
Cell division Binary fission Mitosis

20
Sexual reproduction No meiosis; transfer fragments of Meiosis
DNA only

In brief, eukaryotic cells are larger and more complex.

DETAILS OF A EUKARYOTIC CELL

A. FLAGELLA AND CILIA- -when present, these are used to provide motility for the entire cell or to move
substances along the surface of the cell.

1. Flagella occur as one or a few and are relatively long

2. Cilia are short and occur in large numbers over the entire surface the cell.
3. Structure of eukaryotic cilia and flagella are similar to each other, but quite different from bacterial flagella.
a. Covered with plasma membrane
b. Filled with cytoplasm
c. Contain microtubules--long, hollow tubes made of the protein tubulin.
d. Move in a wavelike motion (bacteria flagella rotate)
e. Use energy from ATP
f. Functions
1) Flagella--Euglena  uses flagella to swim, so do sperm cells
2) Cilia- Paramecium use to swim; cells that line the respiratory tract use them to sweep mucus
fast and out---cigarette smoke kills the cilia
B. CELL WALL---some have one, many do not. Most plant cells have them, animal cells do not.
1. Plants have cell walls made of the polysaccharide cellulose. Some have other chemicals as well.
2. Most fungal cell walls are made mainly of another polysaccharide, chitin, but may contain some cellulose as
well.
3. Protozoa do not have a true cell wall, but may have a flexible covering called a pellicle.
4. Animal cells lack a cell wall.
5. No peptidoglycan in eukaryocytes.
C. GLYCOCALYX-layer of sticky carbohydrate may cover the plasma membrane and strengthen it.
D. PLASMA (CYTOPLASMIC) MEMBRANE---structure is quite similar to prokaryotic plasma membranes.
1. Phospholipid bilayer still forms "backbone" of membrane.
2. Peripheral and integral proteins are still present, but they will be unique to the various species of eukaryocytes.
3. Eukaryotic membranes also contain carbohydrates, which may be in the form of glycoproteins and glycolipids.
These are used for cell to cell recognition and provide attachment sites.
4. Sterols are present to strengthen the membrane. (Only Mycoplasma, which lack a cell wall, contain sterols
among the bacteria).
E. MOVEMENT OF MATERIALS
1. Simple diffusion, osmosis, facilitated diffusion and active transport occur as in prokarytic membranes. Facilitated
diffusion is of greater importance in eukaryocytes than in prokaryocytes.
2. Group translocation does not occur.
3. An additional ACTIVE process, endocytosis, occurs in eukaryocytes, although it does not in prokaryocytes. In
endocytosis, a segment of the plasma membrane surrounds a particle, large molecule, or droplet and draws it into
the cell. Unfortunately, viruses are sometimes brought into cells this way.
a. Phagocytosis ("cell-eating")--projections called pseudopods surround a solid. This is an important body defense
against invaders, as white blood cells ingest and destroy bacteria, etc.
b. Pinocytosis ("cell-drinking")--no pseudopods-a liquid droplet is brought in. Many kinds of cells do this.
F. CYTOPLASM- the eukaryotic definition is cell substance inside the plasma membrane but outside the nucleus. In
many ways eukaryotic cytoplasm is similar to prokaryotic cytoplasm, but one difference is that eukaryotic cells
have a complex internal structure called a cytoskeleton, made of three kinds of protein filaments:
1. Microfilaments
2. Intermediate filaments
3. Microtubules

21
The cytoskeleton provides support and shape, as well as assisting in transport within the cells.
G. ORGANELLES--specialized structures within cells that have distinct functions and specific shapes.
1. NUCLEUS- characteristic of eukaryotic cells, it is the largest organelle and contains the DNA. The nucleus is
surrounded by a double-layered membrane, the nuclear envelope. This membrane has tiny openings called nuclear
pores, which allow large molecules to enter and leave the nucleus. While the cell is not dividing, the DNA
molecules and associated proteins form a tangled threadlike mass called chromatin. (During divisions, the
chromatin threads will shorten to form visible chromosomes.)
2. NUCLEOLUS (NUCLEOLI)- one or more will be found within the nucleus. These are condensed regions of
chromosomes where ribosomal RNA is synthesized.
3. ENDOPLASMIC RETICULUM (ER)- this is a network of flattened membranous sacs called cisterns. The ER is
continuous with the nuclear envelope. ER synthesizes lipids. It stores and modifies lipids and proteins. The ends of
the cisterns can pinch off and form vesicles, which can transport substances enclosed in them.
a. ROUGH ER- has ribosomes attached to it. These ribosomes synthesize proteins which will leave the cell, enter
lysosomes, or become part of the plasma membrane. Inside the cisterns of rough ER, carbohydrates or
phospholipids may be attached to the protein molecules.
b. SMOOTH ER- no ribosomes-synthesis of various lipids and steroids, detoxification, conversion of glycogen to
glucose, and storage of calcium in relaxed muscles.
4. RIBOSOMES- some are attached to rough ER, others are free in cytoplasm. Their function is protein synthesis.
Eukaryotic ribosomes are slightly different from those of prokaryocytes. Eukaryocytes have 80 S ribosomes, with
one 60 S and one 40 S subunit. The subunits are made in the nucleolus and leave the nucleus separately. They join
in the cytoplasm.
a. Free ribosomes--free in cytoplasm, synthesize proteins used inside the cell.
b. Membrane-bound ribosomes--attached to rough ER , synthesize proteins which will:
1) Leave the cell
2) Be inserted into the plasma membrane
3) Become the enzymes in lysosomes
Sometimes 10 - 20 ribosomes may join in a string--this is called a polyribosome.
5. GOLGI COMPLEX- consists of several flattened sacs called cisterns, stacked on top of each other. The ends of the
cisterns can detach to form vesicles to transport substances within the cell or to the plasma membrane for export
from the cell. The Golgi complex receives proteins and lipids from the ER and then sorts, packages, and delivers
them.
a. These proteins and lipids may become part of the plasma membrane
b. Enzymes may be placed in vesicles to form lysosomes.
c. Proteins may be sent out of the cell by exocytosis.
6. LYSOSOMES- vesicles formed in the Golgi complex containing digestive enzymes. These can break down most
organic molecules, including bacteria taken into cells by phagocytosis.
7. VACUOLES-spaces or cavities inside a cell, enclosed by a membrane called a tonoplast.
a. Some temporarily store proteins, sugars, organic acids, and ions.
b. Form during endocytosis to bring food into the cell.
c. Some contain waste and toxins.
d. May take up water, making plant leaves and stems rigid.
8. MITOCHONDRIA- these are the powerhouses, where most of the cell’s ATP is produced. A mitochondrion has 2
membranes, a smooth outer membrane and an inner membrane that lies in deep folds called cristae. The inner
cavity is the matrix. The process of cellular respiration is carried out by enzymes associated with the cristae.
Mitochondria have a small circular piece of DNA that codes for these enzymes and their own 70 S ribosomes to
synthesize them. When more mitochondria are needed, existing ones grow and then divide.
9. CHLOROPLASTS- this is an organelle found in algae and green plants that carry on photosynthesis.
a. Chlorophyll is found in membrane sacs called thylakoids.
b. Contain 70 S ribosomes
c. Enzymes required for photosynthesis are also present.
10. PEROXISOMES--contain enzymes that oxidize various organic substances such as amino acids and fatty acids in
normal metabolism. The enzymes also detoxify harmful substances such as alcohol. This results in production of

22
peroxide, which is also toxic, but the enzyme catalase is present and breaks down the peroxide. New peroxisomes
form by the division of existing ones.
11. CENTROSOME--located near the nucleus, this organelle has 2 components:
a. Pericentriolar area--cytosol region with a dense network of protein fibers. It is the center of organization for the
mitotic spindle and all cellular microtubules.
b. Centrioles- cylindrical structures made of microtubules. Each non-dividing cell has one pair, lying at right angles
to each other within the pericentriolar area.
1) Play a role in cell division
2) Help form cilia and flagella.
 
ENDOSYMBIOTIC THEORY
Proposes that mitochondria and chloroplasts originally were very small prokaryocytes which invaded early
eukaryocytes and remained, gaining protection and a food source and providing much more efficient energy
production for the larger cell.

23