Download Viruses

Microbiology:
Microorganisms
Continued
Class 2
Domain Bacteria
Staining Procedures
• Three Categories of Staining Procedures
1. Simple stains
2. Structural staining procedures
• Capsule stains
• Spore stains
• Flagella stains
3. Differential staining procedures
• Gram and acid-fast staining procedures.
Domain Bacteria
The Gram Staining Procedure
• Divides bacteria into 2 major groups:
– Gram-positive (blue-to-purple)
– Gram-negative (pink-to-red)
• The final Gram reaction (positive or negative) depends upon
the organism’s cell wall structure.
– The cell walls of Gram-positive organisms have a thick
layer of peptidoglycan, making it difficult to remove the
crystal violet-iodine complex.
– Gram-negative organisms have a thin layer of
peptidoglycan, making it easier to remove the crystal
violet; the cells are subsequently stained with safranin.
Various Gram-Positive Bacteria
Chains of streptococci in
smear from broth culture.
Streptococcus pneumoniae in
blood culture.
Gram-Negative Bacteria
Gram-negative bacilli in a smear
from a bacterial colony.
Loosely coiled Gram-negative
spirochetes, Borrelia burgdorferi,
the cause of Lyme disease.
Domain Bacteria
Staining Procedures (continued)
• Some bacteria are neither consistently purple nor pink
after Gram staining; they are known as Gram-variable
bacteria; example Mycobacterium spp.
• Mycobacterium spp. are often identified using the
acid-fast stain.
• The acid-fast stain
– Carbol fuchsin is the red dye that is driven through
the bacterial cell wall.
– Heat is used to soften the waxes in the cell wall
– Because mycobacteria are not decolorized by the
acid-alcohol mixture, they are said to be acid-fast.
• Ex TB
Domain Bacteria
Acid-Fast Mycobacteria
Many acid-fast mycobacteria in a
liver biopsy.
Acid-fast bacilli in a digested
sputum specimen.
Domain Bacteria
Colony Morphology
• A bacterial colony contains millions of organisms.
• Colony morphology (appearance of the colony) varies
from one species to another.
• Colony morphology includes: size, color, overall shape,
elevation and the appearance of the edge or margin of
the colony.
• Colony morphology also includes the results of
enzymatic activity on various types of media.
• As is true for cell morphology and staining
characteristics, colony morphology is an important “clue”
to the identification of bacteria.
Laboratory Identification of Pathogens
• Obtain and label specimens from patients
• Grow out bacterial cells
• Isolate individual organisms
• Multiply to form colonies
• Stain cells so they can be seen (Gram Stain)
• Perform tests to identify organisms
Methods of Identification
Isolated colonies of bacteria growing on a solid medium
Size of colonies is
determined by the
organism’s generation
time and is another
important characteristic
of a particular bacterial
species.
Formation of a bacterial
colony on solid growth
medium.
Factors Influencing Growth of
Microorganisms

Light
◦ Amount
◦ Type





Temperature
Moisture
Food Availability
Atmosphere-Gas-Oxygen supply
pH
Domain Bacteria
Atmospheric Requirements
• Bacteria can be classified on the basis of their atmospheric
requirements, including their relationship to O2 and CO2
• With respect to O2, bacterial isolates can be classified as:
– Obligate aerobes- require 20-21% O2
– Microaerophilic aerobes -require O2 but less than 21%
– Facultative anaerobes- can live and grow with or without
molecular oxygen. Between 0-21%
– Aerotolerant anaerobes- don’t require O2, grows better
without O2, but can survive in areas with O2.
– Obligate anaerobes- cannot survive in O2 environments
• Capnophilic organisms grow best in the presence of increased
concentrations of CO2 (5 to 10%).
Domain Bacteria
Nutritional Requirements
• All bacteria need some form of the elements carbon,
hydrogen, oxygen, sulfur, phosphorus and nitrogen for
growth.
• Some bacteria require special elements (e.g., calcium,
iron or zinc).
• Organisms with especially demanding nutritional
requirements are said to be fastidious (“fussy”).
• The nutritional needs of a particular organism are
usually characteristic for that species and are sometimes
important clues to its identity.
Domain Bacteria
Biochemical and Metabolic Activities
• As bacteria grow, they produce many waste products
and secretions, some of which are enzymes.
• Pathogenic strains of many bacteria, like staphylococci
and streptococci, can be tentatively identified by the
enzymes they secrete.
• In particular environments, some bacteria produce gases
such as carbon dioxide or hydrogen sulfide.
• To identify bacteria in the lab, they are inoculated into
various substrates (i.e., carbohydrates and amino acids)
to determine whether they possess the enzymes
necessary to break down those substrates.
Domain Bacteria
Pathogenicity
• Many pathogens are able to cause disease because they
possess capsules, pili, or endotoxins, or because they
secrete exotoxins and exoenzymes that damage cells
and tissues.
• Frequently, pathogenicity is tested by injecting the
organism into mice or cell cultures.
• Examples of some common pathogenic bacteria:
– Neisseria meningitidis, Salmonella typhi, Shigella
spp., Vibrio cholerae, Yersina pestis, Treponema
pallidum.
Bacterial By-Products
• Endotoxins
– Poison remains within the infected cell until it
disintegrates
– May cause typhoid fever & bacillary dysentery
• Exotoxins
– Poison is excreted by the cell into the
surrounding area
– May cause tetanus, gas gangrene, diphtheria
and scarlet fever
Susceptibility
• Now that the culture has been identified, we need
to know what antibiotic will kill it.
• The growth of the culture will have different
antibiotics applied to determine which will kill the
bacteria.
• If the antibiotic does not kill the bacteria, the
bacteria is called resistant.
• Killed = susceptible to the antibiotic
Unique Bacteria
• Rickettsias, chlamydias and mycoplasmas are bacteria, but
do not possess all the attributes of typical bacterial cells.
• Rickettsias and chlamydias have a Gram-negative type of
cell wall and are obligate intracellular pathogens (i.e.,
they must live within a host cell).
– Rickettsias do not grow on artificial culture media; they
have “leaky membranes.”
– Chlamydias are “energy parasites,” meaning they prefer
to use ATP molecules produced by their host cell.
Rickettsia
prowazekii, the
cause of epidemic
louseborne typhus.
Unique Bacteria (continued)
• Mycoplasmas
– Smallest of the cellular microbes.
– Lack a cell wall and therefore assume many shapes (they
are pleomorphic).
– In humans, pathogenic mycoplasmas cause primary
atypical pneumonia and genitourinary infections.
– Because they have no cell wall, they are resistant to drugs
like penicillin that attack cell walls.
– They produce tiny “fried egg” colonies on artificial media.
SEM of Mycoplasma pneumoniae
Chlamydia
• Energy parasites – use ATP from host
• Gram negative cell wall
• Obligate intracellular pathogen
• Transmission is aerosol or direct contact
• Can cause pneumonia, inclusion conjunctivitis, trachoma
(blindness) as well as sexually transmitted infection.
Photosynthetic Bacteria
• Photosynthetic bacteria include purple bacteria, green
bacteria and cyanobacteria; they all use light as an
energy source, but not in the same way.
– Purple and green bacteria do not produce oxygen,
whereas cyanobacteria do.
– Photosynthesis that produces oxygen is called
oxygenic photosynthesis.
– Photosynthesis that does not produce oxygen is
called anoxygenic photosynthesis.
The Domain Archaea
• Archaea (meaning ancient) were discovered in 1977;
they are procaryotic organisms.
• Genetically, archaeans are more closely related to
eucaryotes than they are to bacteria.
• Archaeans vary widely in shape and live in extreme
environments, such as extremely acidic, extremely hot
and extremely salty environments.
• Archaeans possess cell walls, but they do not have
peptidoglycan (in contrast, all bacterial cell walls contain
peptidoglycan).
Categories of Microorganisms
• Microorganisms can be divided into those that are truly
cellular (bacteria, archaeans, algae, protozoa and
fungi) and those that are acellular (viruses, viroids
and prions).
• Cellular microorganisms can be divided into those that
are procaryotic (bacteria and archaeans) and those
that are eucaryotic (algae, protozoa, and fungi).
• Viruses, viroids and prions are often referred to as
infectious agents or infectious particles.
Acellular Infectious Agents
• Viruses
– Complete virus particles are called virions.
– Most viruses are from 10 to 300 nm in diameter.
– Viruses infect humans, animals, plants, fungi,
protozoa, algae and bacterial cells.
– Some viruses, called oncogenic viruses or
oncoviruses, cause specific types of cancer.
– A typical virion consists of a genome of either DNA
or RNA, surrounded by a capsid (protein coat)
which is composed of protein units called
capsomeres.
– Some viruses (enveloped viruses) have an outer
envelope composed of lipids and polysaccharides.
Acellular Infectious Agents (continued)
Viruses have 5 properties that distinguish them from
living cells:
1. They possess either DNA or RNA – living cells
possess both.
2. They are unable to replicate on their own.
3. Unlike cells, they do not divide by binary fission,
mitosis or meiosis.
4. They lack the genes and enzymes necessary for
energy production.
5. They depend on the ribosomes, enzymes and
metabolites of the host cell for protein and nucleic
acid production.
Model of an icosahedral virus: adenovirus
Acellular Infectious Agents (continued)
• Viruses are classified by:
• Type of genetic material (either DNA or RNA)
• Shape and size of capsid
• Number of capsomeres
• Presence or absence of an envelope
• Type of host it infects
• Disease it produces
• Target cell
• Immunologic/antigenic properties
Herpesviruses acquiring their envelopes as they
leave a host cell’s nucleus by budding.
Comparative sizes
of virions, their
nucleic acids and
bacteria.
Acellular Infectious Agents (continued)
Viruses
• Origin of Viruses
– One theory states that viruses represent ancient
derivatives of degenerate cells or cell fragments.
– Most scientists agree that viruses are nonliving entities.
• Bacteriophages
– Viruses that infect bacteria are known as bacteriophages
or simply, phages.
– Virulent bacteriophages always cause what is known as
the lytic cycle, which ends with the destruction of the
bacterial cell.
– Temperate (lysogenic) bacteriophages do not cause
the lytic cycle, but the DNA integrates into the bacterial
chromosomes
A partially lysed cell of Vibrio cholerae with
attached virions of phage CP-T1.
The bacteriophage T4 is an
assembly of protein components.
20
facets,
filled with
DNA
Viral DNA enters the
cell through the core.
Acellular Infectious Agents (continued)
– The steps in multiplication of viruses are:
• Attachment
• Penetration
• Biosynthesis -when the viral nucleic acids take
over the host cell and dictates what is produced
• Assembly -fitting the viral pieces together to
produce complete virons
• Release
– Inclusion bodies are remnants or collections of
viruses; often seen in infected cells and used as a
diagnostic tool to identify particular viral diseases.
Infectious Nature of Viruses
• Contact can be airborne or contact
• Virus settles into/ onto the body and enters an open
portal.
• *Virus attaches to cell membrane, injects nucleic acids,
“cons” cell’s DNA into synthesizing the new material. New
viruses are made until the cell bursts (lyses), sending
more viral material into the interstitial space. The
process repeats
Acellular Infectious Agents (continued)
• Latent Virus Infections
– Viral infections in which the virus is able to hide from
a host’s immune system by entering cells and
remaining dormant.
– Herpes viral infections are examples.
– Once acquired, herpes virus infections (e.g., those
that cause cold sores, genital herpes, and
chickenpox/shingles) never completely go away; for
example, chickenpox may be followed, years later, by
shingles - both the result of the same virus.
Acellular Infectious Agents (continued)
• Antiviral Agents
– Antibiotics are not effective against viral infections.
– Antiviral agents are drugs that are used to treat viral
infections.
– These agents interfere with virus-specific enzymes
and virus production by disrupting critical phases in
viral multiplication or inhibiting synthesis of viral
DNA, RNA, or proteins.
Acellular Infectious Agents (continued)
• Oncogenic Viruses or Oncoviruses
– Viruses that cause cancer.
– Examples include Epstein-Barr virus, human
papillomaviruses and HTLV-1.
• Human Immunodeficiency Virus (HIV)
– The cause of acquired immunodeficiency syndrome
(AIDS).
– It is an enveloped, double-stranded RNA virus.
– The primary targets for HIV are CD4+ cells.
Human Immunodeficiency Virus (HIV)
Retroviruses
• HTLV virus (RNA) – Leukemia
• RNA tumor virus – Tumors
• HIV (main one of this generation) – AIDS
Acellular Infectious Agents (continued)
• Viroids and Prions (smaller and less complex infectious
agents than viruses)
– Viroids
• Viroids are short, naked fragments of singlestranded RNA, which can interfere with the
metabolism of plant cells.
• Viroids are transmitted between plants in the same
manner as viruses.
• Examples of plant diseases caused by viroids:
potato spindle tuber, and citrus exocortis.
Acellular Infectious Agents (continued)
• Prions
– Prions are small infectious proteins that cause fatal
neurologic diseases in animals; examples: Scrapie,
Bovine Spongiform Encephalopathy (“Mad Cow
Disease”) and Creutzfeldt-Jacob disease.
– Of all pathogens, prions are the most resistant to
disinfectants.
– The mechanism by which prions cause disease
remains a mystery.
Algae
Characteristics and Classification
• Algae are photosynthetic, eucaryotic organisms.
• All algal cells consist of cytoplasm, a cell wall (usually),
cell membrane, a nucleus, plastids, ribosomes,
mitochondria and Golgi bodies.
• Some have a pellicle, a stigma and/or flagella
• Algae range in size from unicellular microscopic
organisms (e.g., diatoms) to large, multi-cellular
organisms (e.g., seaweeds or kelp).
• Algae produce their energy by photosynthesis.
• Some may use organic nutrients.
Algae: Medical Significance
• One genus of algae, Prototheca, is a very rare cause of
human infections.
• Causes protothecosis.
• Algae in several other genera secrete substances called
phycotoxins.
• Poisonous to humans, fish, and other animals.
• If ingested by humans, the phycotoxins produced by the
dinoflagellates can lead to paralytic shellfish poisoning.
Protozoa
Characteristics
• Protozoa are nonphotosynthetic, eucaryotic organisms.
• Most protozoa are unicellular and free-living, found in soil
and water.
– Most protozoa are more animal-like than plantlike.
– All protozoal cells possess a variety of eucaryotic
structures/organelles.
– Protozoa cannot make their own food; they ingest
whole algae, yeasts, bacteria and smaller protozoa
for nutrients.
Protozoa
Characteristics (continued)
• Protozoa do not have cell walls, but some possess a
thickened cell membrane called a “pellicle,” which serves
the same purpose – protection!
• A typical protozoan life cycle has 2 stages – a trophozoite
and a cyst.
– The trophozoite is the motile, feeding, dividing
stage.
– The cyst is the dormant, survival stage.
• Some protozoa are parasites.
– Parasitic protozoa cause many human diseases, such
as malaria, giardiasis and trypanosomiasis.
Typical Pond Water Algae and Protozoa
Voticella sp.
Volvox sp.
Stentor sp.
Euglena sp.
Paramecium sp.
Amoeba sp.
Protozoa
Characteristics (continued)
• Protozoa are divided into groups, based on their
method of locomotion:
– Amebae move by means of pseudopodia (“false
feet”) – example: Entamoeba histolytica, the cause
of amebic dystentery.
– Flagellates move by means of whiplike flagella –
example: Giardia lamblia, the cause of giardiasis.
– Ciliates move by means of hairlike cilia – example:
Balantidium coli, the cause of balantidiasis.
– Sporozoa have no visible means of locomotion –
example: Plasmodium spp., which cause malaria.
Giardia lamblia – a Flagellate
Drawing of Giardia lamblia trophozoite
TEM of Giardia lamblia trophozoite
Fungi
Characteristics
• The study of fungi is called mycology.
• Fungi are found virtually everywhere.
• Some fungi are harmful, some are beneficial.
• Fungi represent a diverse group of eucaryotic organisms
that include yeasts, molds and mushrooms.
• Fungi are the “garbage disposers” of nature.
• Fungi are not plants – they are not photosynthetic!
Fungi
Characteristics (continued)
• Fungal cell walls contain a polysaccharide called chitin.
• Some fungi are unicellular, while others grow as filaments
called hyphae.
– Hyphae intertwine to form a mass called a mycelium.
• Some fungi have septate hyphae (the hyphae are divided
into cells by cross walls or septa).
• Some fungi have aseptate hyphae (the hyphae do not
have septa).
• Whether or not a fungus is septate or not is an important
clue to its identification.
Fungal Colonies and Terms
Relating to Hyphae
Fungi
Reproduction
• Depending on the species, fungal cells can reproduce by
budding, hyphal extension, or the formation of spores.
– There are 2 general categories of spores:
• Sexual spores
• Asexual spores (also called conidia)
– Some fungi produce both asexual and sexual spores.
• Fungal spores are very resistant structures.
Microscopic Appearance of Various Fungi
Aspergillus fumigatus
Penicillium sp.
Scopulariopsis sp.
Aspergilus flavus
Curvularia sp.
Histoplasma capsulatum
Fungi - Yeasts
• Yeasts are eucaryotic, unicellular
organisms that lack mycelia.
• Individual yeast cells, also referred to
as blastospores or blastoconidia, can
only be observed under a microscope.
• Yeasts usually reproduce by budding,
but occasionally by a type of spore
formation.
• A string of elongated buds is known as
a pseudohypha (not really a hypha).
• Some yeasts produce thick-walled,
spore-like structures called
chlamydospores (or chlamydoconidia).
Longitudinal Section of a
Budding Yeast Cell
Microscopic appearance of
Candida albicans
A = Chlamydospores
B = Pseudohyphae
C = Budding yeast cells
(blastospores)
Fungi - Yeasts (continued)
• Yeasts are found in soil and water and on the skins of
many fruits and vegetables.
– Yeasts have been used for centuries to make wine
and beer.
– Saccharomyces cerevisiae is used in baking.
– Candida albicans is the yeast most frequently
isolated from human clinical specimens, and is also
the fungus most frequently isolated from human
clinical specimens.
Fungi - Yeasts (continued)
• Yeast colonies may be hard to distinguish from bacterial
colonies.
– A simple wet mount can be used to differentiate
yeast colonies from bacterial colonies.
• Yeasts are larger than bacteria and are usually
oval-shaped.
• Yeasts are often observed in the process of
budding.
• Bacteria do not bud!
Fungi - Molds
• Also spelled “moulds.”
• Molds are fungi that are often seen in water and soil and
on food.
• Molds grow in the form of cytoplasmic filaments called
hyphae.
– Aerial hyphae extend above the surface of whatever
the mold is growing on.
– Vegetative hyphae grow beneath the surface.
• Reproduction is by spore formation, either sexually or
asexually, on the aerial hyphae.
Fungi – Molds (continued)
• Molds have great commercial importance.
1. Some produce antibiotics.
• Examples: Penicillium and Cephalosporum
2. Some molds are used to produce large quantities of
enzymes that are used commercially.
3. Ferments wine and beer
4. Use in water filtration to reduce bacterial or chemical
wastes in water supplies
5. The flavor of cheeses like bleu cheese, Roquefort,
camembert and limburger are due to molds that grow
in them.
Fungi – Molds (continued)
• Fleshy fungi
– Include mushrooms, toadstools, puffballs and
bracket fungi.
– Consist of a network of filaments or strands (the
mycelium) that grows in soil or on a rotting log.
– The fruiting body that grows above the ground forms
and releases spores.
– Some mushrooms are edible; some are extremely
toxic!
Fungi – Medical Significance
• A variety of fungi including yeasts, molds and some
fleshy fungi, are of medical, veterinary and agricultural
importance because of the diseases they cause in
humans, animals and plants.
• Mycoses are infectious diseases of humans and animals
that are caused by molds.
• Fungal infections of humans are categorized as
superficial, cutaneous, subcutaneous and systemic
mycoses.
Superficial and Cutaneous Mycoses
• Superficial mycoses are fungal infections of the
outermost areas of the human body – hair, nails and
epidermis.
• Cutaneous mycoses are fungal infections of the living
layer of the skin, the dermis.
– A group of molds collectively referred to as
dermatophytes cause tinea (“ringworm”) infections.
– The yeast, Candida albicans, can also cause
cutaneous, oral, and vaginal infections.
Subcutaneous and Systemic Mycoses
• Subcutaneous and systemic mycoses are more severe
types of fungal infections.
• Subcutaneous mycoses are fungal infections of the
dermis and underlying tissues. Example: Madura foot.
• Systemic mycoses are fungal infections of the internal
organs of the body.
– Spores of some pathogenic fungi may be inhaled with
dust from contaminated soil or dried bird or bat
feces. They may also enter through wounds of the
hands and feet.
Subcutaneous and Systemic Mycoses
(continued)
• Examples of deep-seated pulmonary infections include
blastomycosis, coccidioidomycosis, cryptococcosis, and
histoplasmosis.
• Inhalation of common bread molds like Rhizopus and
Mucor spp. can cause disease and even death in
immunosuppressed patients.
• Diagnosis of mycoses is accomplished by culture
techniques and immunodiagnostic procedures.
– Yeasts are identified by a series of biochemical tests.
– Molds are identified by a combination of macroscopic and
microscopic observations.
Dimorphic Fungi
• A few fungi, including some pathogens, can live as either
yeasts or molds, depending on growth conditions. The
phenomenon is known as dimorphism and the fungi are
called dimorphic fungi.
– When grown in vitro at body temperature (37oC),
dimorphic fungi grow as yeasts and produce yeast
colonies.
– When grown in vitro at room temperature (25oC),
dimorphic fungi exist as molds, producing mold
colonies.
– In vivo, dimorphic fungi exist as yeasts.
Dimorphic Fungi
(continued)
• Dimorphic fungi that cause
human diseases include:
– Histoplasma capsulatum
(histoplasmosis)
– Sporothrix schenckii
(sporotrichosis)
– Coccidioides immitis
(coccicioidomycosis)
– Blastomyces
dermatitidis
(blastomycosis)