1. Microbiology: Bacterial Structures and their Functions (3) Flashcards
What is resolving power?
Refers to the ability of the lenses to discriminate between two adjacent objects.
What is light microscopy?
Refers to the use of any kind of microscope that uses visible light to observe microorganism.
Most used today are Compound Microscopes, containing two sets of lenses: the objective lens and the ocular lens.
What is the absolute limit of the resolving power?
Roughly 1/2 the wavelength of light used.
What is the range of the wavelength of visible light?
Ranges from 400nm to 800nm, and thus the smallest object that can be observed in a light microscope must be at least 200nm (0.2 um)
Why can light microscopes not be used to provide information on the internal structures of bacterial cells?
Because of the size and length of the bacteria.
most bacteria range in size from 0.2 micrometers to 2 micrometers and 2 micrometers to 8 micrometers in length.
What are the different types of light microscopy?
Brightfield microscopy phase-contrast microscopy darkfield microscopy ultraviolet microscopy fluorescence microscopy
What is electron microscopy?
an electron microscope uses a beam of electrons rather than visible or ultraviolet light to illuminate the object.
The shorter wavelengths of electrons at the voltages commonly used does what to the resolving power?
increases the resolving power to about 0.001 micrometers (1nm).
What are the types of electron microscopes?
the transmission electron microscope (TEM)
the scanning electron microscope (SEM)
Most microorganisms usually appear what when viewed through a light microscope?
Colorless
Most stains are composed of what?
are salts composed of a positive and a negative ion, one of which is colored (referred to as the chromophore)
What are basic dyes?
colored cation (+ charge) colorless anion (- charge) includes crystal violet, safranin, malachite green, and methylene blue
What is acidic dyes?
colorless cation (+ charge) colored anion (- charge) includes Cango red, eosin, and nigrosin
At pH 7, bacteria are charged how?
negatively charged
therefore, the colored positive ion in a basic dye is attracted to the negatively charged bacterial cell.
What is known as negative staining?
the colored negative ions in an acidic dye are repelled by the negatively charged bacterial cell, so the stain colors the background while the bacterial cell remains colorless.
How do you prepare bacteria for staining?
a small amount of culture is dispersed in a drop of water on a glass slide.
The smear is allowed to dry at room temperature and the bacteria are fixed to the glass by gentle heating in a flame, smear side up.
Fixing also kills the bacteria.
Facts about gram stains
was developed in 1884 by the Danish bacteriologist Hans Christian Gram.
the basis of the gram reaction is the differences in the structure of the cell walls of gram-positive and gram-negative bacteria.
What is the primary stain?
Crystal violet (violet to purple)
What is the counterstain?
Safranin (red to pink)
What is the Decolorizing agent?
Alcohol or acetone-alcohol
What is the Mordant?
Iodine.
a mordant increases the affinity of a stain for a biological specimen; it can also be used to coat a structure (eg. flagellum) to make it thicker and easier to see after it is stained with a dye.
List the steps of a gram stain
1) application of crystal violet (purple dye)
2) application of iodine (mordant)
3) alcohol wash (decolorization)
4) application of safranin (counterstain)
Why can the crystal violet-iodine complex inside the cells of gram-positive bacteria not be washed out by alcohol?
because of the thick peptidoglycan layer and thus the gram-positive cells retain the dye and stain purple.
How do gram-negative bacteria remain colorless when crystal violet-iodine complex is used?
the decolorizing alcohol disrupts the lipopolysaccharide layer and the crystal violet-iodine complex is washed out through the peptidoglycan layer. This leaves the gram-negative bacteria colorless until counterstained with safranin, after which they appear pink.
When are the best results obtained during staining?
when young, growing cultures are used.
old cultures are easily decolorized because autolytic enzymes attack the bacteria cell wall.
Information obtained in the gram reaction in used in what?
bacterial identification
selection of antibacterial agent(s)
and in clinical evaluation of a disease
What are the three basic shapes of bacteria?
1) spherical= coccus (plural: cocci=berries)
2) rod-shaped= bacillus (plural: bacilli= little staffs)
3) spiral
Describe Cocci shaped bacteria
mostly round
however some cocci are oval, elongated, or flattened on one side.
incompletely separated cocci may appear in a number of different patterns, depending upon the planes in which they divide.
Describe Bacilli shaped bacteria
They divide only across their short axis.
What is the Glycocalyx?
sugar coat
is the polysaccharide-containing gelatinous material external to the cell wall.
it is synthesized inside the cell and excreted to the outside.
it varies among bacteria, but usually contains glycoproteins and various polysaccharides.
What is the Extracellular polysaccharides (EPS) and what role do they play?
the outer polysaccharide layers.
play significant role in the attachment of certain pathogenic organisms to their hosts and other bacterial cells.
Glycocalyx can bind to water to do what?
protect it from dehydration.
also depending on its viscosity, it may inhibit the movement of nutrients out of the cell.
What is the Capsule?
is a dense, well-defined, mostly glycocalyx or polypeptide layer firmly adherent to the bacterial cell.
can be demonstrated using negative staining methods.
How does the capsule protect a bacteria?
it contributes to bacterial virulence by protecting pathogenic bacteria from phagocytosis by neutrophils and macrophages.
Capsules inhibit what?
inhibit the uptake of some antibiotics and promote adherence to other bacteria or to host tissue surfaces.
What are Flagella?
singular: flagellum
are long, thin, filamentous appendages composed of helically coiled protein subunits called flagellin.
are anchored in the bacterial cell membrane through hook and basal body structures.
What are the four attachments of flagella?
Amphitrichous= each end Monotrichous= one Lophotrichous= multi on one end Peritrichous= multi all over (trichous= hair like)
How can flagella be demonstrated?
sing flagella stains.
they are readily observed with the electron microscope.
Flagellum is an organ of what?
organ of motility.
motility allows the bacteria cell to move toward a favorable region or move away from an adverse one in its microenvironment.
What are Fimbriae?
singular: fimbria; latin for “fringe”
are hairlike appendages (up to several hundred per cell) that may be evenly distributed over the bacterial surface or they can accur at the poles of the bacterial cell.
are attached to the cytoplasmic membrane.
Fimbriae are composed of what?
composed of protein subunits called PILIN
The tips of fimbriae may contain what?
proteins called LECTINS that bind to specific sugar residues (eg. mannose) present on host cell membranes. once colonization occurs, the bacteria can cause disease.
What happens when fimbriae are absent?
(this is due to genetic mutation)
colonization is impaired and the bacteria are unable to cause disease.
What is the Cell Wall of a bacteria?
is a chemically complex, semirigid structure that is found in most bacteria except Mycoplasma spp. and Ureaplasma spp.
What is the main function of the cell wall?
is to prevent bacterial cells from rupturing when the water pressure (~2 atmosphere; roughly the same as in an automobile tire; due to dissolved solutes inside the bacterial cell) inside the cell is greater than that outside the cell.
The cell was also functions in what?
to give shape and rigidity to the bacterial cell.
The cell wall plays an essential role in what?
in cell division: cross-wall formation
bacteria are divided into what based on differences in cell wall structure?
gram-positive and gram-negative cells
The strength of the cell wall resides primarily in the what?
peptidoglycan layer
What is the peptidoglycan layer?
a macromolecular network found only in bacteria.
is a thin sheet composed altering two sugar derivatives, N-acetylglucosamine and N-acetylmuramic acid, and either lysine or diaminopimelic acid (DAP).
the amino acids are attached to N-acetylmuramic acid.
What also provides additional strength to the peptidoglycan?
the glycan chains formed by the sugars are connected by peptide cross links formed by amino acids.
How does cross-linkage occur in gram-negative bacteria?
cross-linkage occurs by direct peptide linkage of the amino roup of DAP to the carboxyl group of the terminal D-alanine.
In gram-positive bacteria, cross-linkage is usually by?
by a peptide crossbridge, the kinds and numbers of cross-linking amino acids vary among gram-positive bacteria.
Describe the peptidoglycan in gram-positive cell wall
multiple layers extensively cross-linked, resulting in the formmation of a thick, rigid structure.
makes ups 50-90% of the cell wall.
Describe Teichoic acid in gram-positive cell wall
is an acidic polysaccharide consisting primarily of an alcohol such as ribitol or glycerol and phosphate.
it is covalently linked to the peptidoglycan and i essential to cell viability.
Because of their negative charges, teichoic acids can what?
can bind and regulate the movement of cations into and out of the cell.
they also promote adherence to other bacteria and to specific receptors on mammalian cell membranes.
Describe the peptidoglycan layer of gram-negative cell wall
consists of one or a few layers and is cross-linked in only two dimensions. It is cross-linked to the outer membrane by lipoproteins.
What is the periplasmic space of gram-negative cell wall?
is a fluid-filled space between the outer membrane and the plasma membrane.
it contains a high concentration of hydrolytic enzymes, eg, Beta-lactamases (inactive penicillins), specific carrier molecules, etc.
Describe the outer membrane of gram-negative cell wall?
it is a phospholipid bilayer and embedded proteins.
the phospholipids of the outer leaflet are replaced by lipopolysaccharide.
its strong negative charge impairs phagocytosis by neutrophils and macrophages (repulsion between phagocytic cell and bacteria).
The outer membrane is a barrier to what?
antibiotics (eg, penicillin G) digestive enzymes such as lysozyme detergents heavy metals bile salts and certain dyes
What do Porin Proteins do?
form ion channels, permitting the passage of molecules such as nucleotides, peptides, disaccharides, amino acids, iron, and vitamin B12.
LPS: the polysaccharide portion consists of what?
consists of repeating sugars that function as antigens (somatic, “O” antigen).
they are used in the serotyping of gram-negative bacteria, eg, E. coli O157:H7.
The Lipid A portion of the LPS is embedded in what?
embedded in the outer leaf of the outer membrane.
it is toxic (endotoxin) when in the host’s bloodstream or gastrointestinal tract. It is associated with fever, septicemia, shock, etc.
What is Lysozyme?
is a mucopeptidase enzyme present in serum, tears, salivia, nasal secretions, mucus, and other body fluids as well in lysosomal granules of neutrophils and macrophages.
How does the lysozyme work?
it cleaves the glycan chain of peptidoglycan by hydrolyzing the glycosidic bond between MurNAc and GlcNAc, making the bacterial cell susceptible to osmotic lysis.
Gram-positive bacteria are much more susceptible than gram-negative bacteria to osmotic lysis why?
due to selective permeability of the outer membrane.
How does Penicillin G work?
Blocks synthesis of peptidoglycan by inhibiting the enzyme responsible for the formation of the peptide cross-bridge in gram-positive bacteria.
What is a protoplast?
gram-positive bacterium treated to remove the cell wall.
is usually spherical and if the environment is balanced, it does not undergo osmotic lysis and can carry out metabolic activities.
What is a Spheroplast?
a spherical, osmotically sensitive gram-negative cell that has lost some, but not all, of its cell wall; some of the outer membrane remains.
What is the plasma membrane (cell membrane)?
also called inner membrane in gram-negative bacteria.
is a typical “unit membrane” composed of a phospholipid bilayer and proteins.
The cell membrane is responsible for?
many of the functions carried out by various eukaryotic organelles.
What is selective permeability?
certain molecules and ions can pass through the membrane while others are prevented from passing through it.
Active transport of metabolites is facilitated by what?
membrane-bound permeases
binding proteins
and various transport systems
The cell membrane is the site for what?
site of electron transport and energy production
The cell membrane contains the enzymes and carrier lipids that function in what?
in DNA replication, phospholipid biosynthesis, and cell wall biosynthesis
What does the nucleoid contain?
contains the bacterial chromosome.
the chromosome is attached to the plasma membrane.
Proteins in the plasma membrane may be responsible for what?
for replication of the DNA and segregation of the new chromosomes to daughter cells during cell division.
Some bacteria may contain what?
plasmids (extrachromosomal genetic elements)
What are ribosomes?
are composed of two subunits, each of which consists of protein and ribosomal RNA (rRNA).
the cytoplasm contains several thousand ribosomes.
Ribosomes are the site of what?
protein synthesis
a number of antibacterial agents work by inhibiting protein synthesis on ribosomes.
What are Inclusions?
are reverse deposits present in he cytoplasm of prokaryotes.
they include nutrient reserves (eg, lipid inclusions, polysaccharide granules), energy reserves (eg, sulfur granules), etc.
What are bacterial Endodpores?
are small, highly resistant, thick-walled, “resting” cells produced in response to exhaustion of nutrients (eg, absence of carbon or nitrogen source) or adverse environmental conditions.
How are endospores formed?
are formed when the bacterial cells are unable to grow, hence actively growing cells do not form endospores.
Endospores are unique to what?
are unique to bacteria and are formed within the cell.
Most spore-forming bacteria are inhabitants of what?
soil
Bacterial spores are found where?
almost everywhere
Bacterial spores are produced by what?
certain gram-positive bacteria such as those in the nera Clostridium and Bacillus.
What kind of structure is an endospore?
a dehydrated, multilayered structure containing a complete copy of the bacterial chromosome, minimal concentrations of essential proteins, ribosomes, and calcium dipicolinate.
Depending on the bacterial species, endospores vary in what?
size, shape, and location of the spore in the vegetative cell.
Endospores are resistant to what?
heat (can be killed by moist heat at 121 degrees Celsius for 15 minutes) drying radiation acids and chemical disinfectants
The resistance of endospores is attributed to?
to their complex structure, their dehydration state (they contain much less water than vegetative cells), their negligible metabolic activity, and to the presence in the core of high concentrations of calcium bound to dipicolinic acid (not present in vegetative cells)
Endospores can remain what for centuries?
they can remain dormant as viable spores.
however, in soils low in calcium or in acidic soils, calcium may leach from spores, shortening their survival times.
Why was the discovery of bacterial endospores significant?
because the knowledge of such remarkably heat-resistant forms was essential for the development of adequate methods of sterilization, not only of bacterial culture media but also foods and other perishable products.
What is Sporulation?
the process of endospore formation within a vegetative (parent) cell.
One vegetative cell gives rise to a single endospore.
What are the evnets that lead to endospore formation?
transcription of spore mRNA while other mRNAs are turned off
synthesis of dipicolinic acid
duplication of the chromosome, etc.
the entire process may take 6 to 8 hours for completion.
What is spore germination?
is the growth of an endospre to become a vegetative cell.
a single spore germinates to form a single vegetative cell.
What are the three stages of spore germination?
Activation
Germination
Outgrowth
Describe activation stage of spore germination
this may occur in response to disruption of the spore coat by mechanical stress, sublethal heat, pH, etc, and requires water and triggering nutrient (eg, alanine)
Describe the Germination stage of spore germination
this occurs in the presence of specific nutrients.
it involves the uptake of water, enzymatic degradation of the spore cortex and coat, loss of calcium dipicolinate from the spore, and production of a new vegetative cell identical to the original vegetative cell.
Describe the Outgrowth stage of pore gemination
this is the period of active biosynthesis of new RNA, proteins, and DNA, terminating with division of the new vegetative cell.
