Microbio Exam 2

2 types of Enumeration of bacterial cells

Direct and Indirect

Petroff-Hausser Chamber

Direct Cell count

Advantages:

1. Direct counting of cells

2. Rapid and low cost

Disadvantages:

1. Limited accuracy

2. Can not differentiate dead vs. live cells

Spread-spread method

Direct cell counting

1. Sample is pipetted onto surface of agar plate (.1 ml or less)

2. Sample is spread evenly over surface of agar using sterile glass spreader

3. Incubation

4. Count surface colonies

Pour plate method

direct cell counting

1. sample is pipetted into sterile plate

2. Sterile medium is added and mixed with inoculum

3. Incubation

4. Count surface colonies

Chemical requirements for growth

Availability of carbon, nitrogen, sulfur, phosphorus, trace elements, organic compounds, oxygen

Physical requirements

pH, Temperature, Moisture, Hydrostatic pressure, osmotic pressure, radiation, light

Hyperthemophile

growth above 80C, 176 F

Thermophile

growth between 50C (122F) and 80 C (176F)

Mesophile

Growth between 15C (59F) and 45 C (113 F)

Psychrophile

Growth below 15 C (59F)

Alkaliphile

Growth above pH 9

Neutralophile

growth between pH 5 and pH 8

Acidophile

Growth below pH 3

Halophiles

Growth in salt, greater than 2 M NaCl

Strict Aerobe

Growth only in oxygen

Facultative microbe

Growth with it without oxygen

Microaerophile

growth only in small amounts of oxygen

Strict anaerobe

Growth only without oxygen

Barophile

Growth at high pressure greater than 380 atm

Barotolerant

growth between 10 and 500 atm

aerobes (Aerobic respiration)

Use oxygen as a terminal electron acceptor in the electron transport chain to extract energy trapped in nutrients and convert it to a biologically useful form

Anaerobe (anaerobic respiration)

Respire without using the electron transport chain without oxygen as the terminal electron acceptor- such as nitrate

Facultative anaerobes

Posses the enzymes to detoxify oxygen radicals and also the machinery for both fermentation and aerobic respiration

aerotolerant anaerobes

Use only fermentation to provide energy but contain superoxide dismutase and catalase or peroxidase to protect them form ROS

Microaerophiles

Will grow only at low oxygen concentrations. They possess a decreased level of superoxide dismutase and/or catalase

Agar

Complex polysaccharide, used as a solidifying agen for culture media in Petri plates, slants, and deeps

Generally, not metabolized by microbes

liquefies at 100C

Solidifies at 40 C

Selective media

Encourage the growth of certain organisms and discourage others

Differential media

enable different species to be distinguished from eachother

Enrichment media

Encourage the growth of a specific organism

Selective vs. differential

Selective medium will have a growth inhibiting additive, such as bile salts, crystal violet or antibiotic, which will limit growth on the medium to only those organisms that are desired

Selective: Mannitol salt agar (MSA) and MacConkey Agar

Differential medium allows for differentiation of particular chemical reaction yield an observable characteristic associated with the growth a particular organism that are able to grow on the medium

Differential: Blood agar

Problem with oxygen

Oxygen is a powerful oxidant and can oxidize numerous biomolecules in the cell, leading to cell injury or death

Toxic forms of oxygen and the enzymes that protect the cell from their activity

Defined media

All components and their ratios are known: broth

Complex media

All components and their ratios are unknown: Solid (agar)

Preserving Bacterial cultures

Deep freezing: -50 to -95 C

Lyophilization (freeze-drying): frozen (-54 to -72C) and dehydrated in a vacuum

Human microbiota

Mutualistic relationship

Two main types: Permanent, Transient

Some parts of the body are considered microbe free

Mutualism

Both benefit

Amensalism

One harmed, One benefiited

Commensalism

One benefitted, one unaffected

Neutralism

Both unaffected

Parasitism

One benefitted, one harmed

Locations of commensal/ mutualistic microorganisms

Skin, oral cavity, upper respiratory tract, intestines, Urogenital tract

Areas and fluids of the body once thought to be sterile have been found to contain microbes

Normal microbiota

Collection of bacteria, archaea and eukaryotic microbes usually colonizing our body

Traits for bacterial classification

Comparison of 16S rRNA structure and sequences

Whole genome sequencing data

Metabolic traits

Ecological traits

Mycelia

Dense, branching, filamentous networks formed by the genus of Nocardia, Streptomyces, frankia and actinomyces

vectors

biological and mechanical

Hosts

Definite hosts (reproduction), Intermediate hosts (parasite developes), Reservoir hosts ( harbor parasite)

Host specificity

range of hosts in which a parasite can mature

Division of parasites

1. Ectoparasites and endo parasites

2. Obligate and facultative parasites

3. Permanent, temporary and accidental parasites

Hyperparasitism

parasites have their own parasites

Can a mechanical vector also be a definite host?

no

Mechanisms to evade host defenses

1. Encystment- formation of an outer coasting covering that protects from environmental conditions

2. Changing surface antigens quicker than the host can produce antibodies

3. by invading host cells

Fungi

Kingdom: Fungi

Nutrition type: Chemoheterotroph

Multicellularity: All, except yeast

Cellular arrangement: Unicellular, filamentous, fleshy

Food acquisition method: Absorptive

Characteristic features: sexual and asexual spores

Why are fungi important?

1. Beneficial decomposers

2. Some fungi secrete to metabolic wastes which are toxic to other microorganisms - antibiotics

3. Can be destructive when invading other organisms

4. To invade they require

   1. Proximity to host

   2. Ability to penetrate host

   3. Ability to digest and absorb nutrients from cells

Single celled yeast

Advantage of being single celled: Rapid growth and dispersal in aqueous environments

Some yeasts are asexual, whereas others alternate between diploid and haploid forms

Both diploid and haploid forms are single cells

Haploid cells undergo several generations of mitosis and budding (vegetative growth)

Under various stress conditions such as nutrient limitation, haploid cells may develop into gametes and undergo sexual reproduction (mating)

Fungal life cycle

1. Asexual- Hyphae fragmentation, spores, condiospores, sporangiospore

2. Sexual- fusion of nuclei from 2 opposite matting strains of the same fungal species

Some fungi show dimorphic growth, filamentous in one condition while unicellular in other

Reproduction of Eukaryotic cells

• All have linear chromosomes that must divide during mitosis

• Mitosis ensures that each daughter cell recives a full set of daughter chromosomes

• Stages of eukaryotic mitosis:

• 1. G1

• 2. S phase

• 3. G2

• 4. Mitosis

  • Prophase

    • Chromatin to chromosome

  • Metaphase

    • Chromosome align to center of the cell

  • Anaphase

    • Chromosome align in opposite side

  • Telophase

    • Chromosome to chromatin

• Meiosis generates haploid cells that contain half the number of chromosomes of the original diploid cell

Reproduction of Eukaryotic cells

Many eukaryotic microbes can proliferate indefinitely by mitosis (asexual reproduction)

Most eukaryotes, single or multicellular, also have the option of sexual reproduction

Sexual reproduction requires the reassortment of genetic material from different chromosomes

Sexual life cycles alternate between cells that are diploid and sex cells that are haploid

Two of the haploid sex cells (gametes) join each other by fertilization to regenerate a diploid cell

Germination

Mycelia form, if the two mating types (+ and -) are in close proximity, extensions called gametangia form between them

Plasmogamy

Fusion between + and 0 mating types results in a zygosporangium with multiple haploid nuclei. The zygosporangium forms a thick, protective coat

Karyogamy

The nuclei fuse to form a zygote with multiple diploid nuclei

Mitosis and germination

A sporangium grows on a short stalk. Haploid spores are formed inside

Eukaryotic cells (overview)

Cells have a true nucleus with a nuclear membrane

Membrane bound organelles are present

DNA is organized into chromosomes inside the nucleus

Some eukaryotes can be unicellular (protists), while others are multicellular (plants and animals)

Protozoa

Kingdom: Various

Nutrition type: Chemo heterotroph

Cellular arrangement: Unicellular

Food acquisition method: Absorptive and ingestive

Characteristic features: motility, some form cysts

Protozoa- Asexual reproduction

• Binary fission

• Budding

• Schizogony

  • schizogony the nucleus of a cell divides multiple times before the cell divides into many smaller cells. The products are called merozoites and they are stored in structures known as schizonts

  • Trophozite: Active, feeding, motile stage of a protozoan parasite

  • Sporozoite: The infective stage of a parasite that is transmitted from one host to another

Protozoa- sexual reproduction

Conjugation- fusion of 2 cells, the micronucleus from each cell migrates and eventually fuse

Protozoa- Metamonada

• Group of flagellated protozoan parasites

• Mostly lack conventional mitochondria

• Usually anaerobic or microaerophilic, like gut or other oxygen-poor environments

Protozoa- Euglenozoa

• Group of flagellated protozoa

• Members can be free-living or parasitic

• Having one or more flagella for motility

• Some are photosynthetic (like Euglena), while others are parasitic (like Trypanosoma)

Move by flagella:

• Euglenoids

• Hemoflagellates: Hemoflagellates are flagellated protozoan parasites that live in the blood or tissues of their hosts.

Trypanosomatida

• Consist of an elongated cell with a single flagellum

• They have a unique organelle called the kinetoplast, which consists of a specialized mitochondrion that provides energy for the flagellum

• Sleeping sickness, Chagas disease, and Leishmaniasis

Amebozoa

• Amebozoa is a majorly characterized by movement via pseudopodia

• Most are free-living, but some are parasitic

• Voracious predators, consumes other protists and bacteria, and can reach several mm in size

• Infective stage Trophozoites

IN trypanosoma what is the intermediate host?

fly

What is the definite host of amebozoa?

no definite host

Apicomplexa

• Major group of parasites of humans and other animals

• Apical complex is a specialized structure that facilitates entry of the parasite into the host cell

  • Micronemes: Release adhesion proteins and help parasite attach to host cells

  • Rhoptries: Inject proteins into host cell and modify host cell membrane to allow parasite entry

• Underwent extensive reductive evolution, losing their flagella and cilia

Babesia microti- babesiosis

Definite host: Tick, Resoivoir host: Mouse, Accidental host: Humans

Helminths

Kingdom: animalia

Nutrition type: Chemoheterotroph

Multicellularity: yes, all

Cellular arrangement: tissues and organs

Food acquisition method: Ingestive; absorptive

Characteristic features: Life cycle

Helminths

Aka worms are bilaterally symmetrical. Have a head and tail end and its tissues are differentiated into 3 distinct tissue layers (Triploblastic): ectoderm, mesoderm and endoderm

Nematodes (roundworms)

are dioecious, cylindrical and a digestive tube that ends in an anus

Reproduction:

1. Human ingests pinworm eggs

2. In the small intestine the larvae hatch and grow

3. Female worms migrate to the anus, where they release eggs

4. Scratching of irritated anus promotes egg transmission to new host

Trematodes (flukes)

Are hermaphrodite, oval- shaped flatworms with a digestive tube that ends in a cecum (no outlet)

• Infect many kinds of animals, but most require a mollusk as their primary host

• Flatworms with internalized mouth, pharynx and digestive tube, but the tube ends in one or more pouches called caeca

• Life cycle is complex and often involves several hosts

• Types of fluke infections in humans:

  • Tissue flukes: attach to the bile ducts, lungs, or other tissues. Lung- Paragonimus westermani and liver Fasciola hepatica

  • Blood flukes- Found in blood in some stages of their life cycle. various species of the genus Schistosoma

Cestodes (tapeworms)

Are hermaphrodite, parasitic flatworms, absorbing nutrients through their skin

• Tapeworms are transmitted through larvae embedded in uncooked meat

• Different species are found in pork, beef and fish

• Consist of a scolex- head with suckers that attach to the intestine wall, and a long chain of hermaphroditic proglottids which contain reproductive organs of both sexes

• Each body segment contains a complete set of male and female reproductive organs

Arthropods

A large and diverse phylum of invertebrate animals characterized by a segmented body, jointed appendages and an exoskeleton made of chitin. Include insects, crustaceans, arachnids, and myriapods

Arachnid parasites

• Mites cause mange (in animals) and scabies (In animals or humans)

• Scabies in humans is caused most often by Sarcoptes scabiei

• Mites attach to skin by suckers and burrow under the skin using special mouthpart and cutting surfaces on their forelegs

• As they burrow, they lay eggs that hatch into larvae. Larvae come out of the skin to attach to a hair follicle where they feed and molt until they reach adult stage

Ticks

• Resemble mites in their eight-legged form, but are larger and usually do not burrow completely into the skin

• Ectoparasites that suck blood for nutrients to produce eggs, then fall off to disperse their progeny

• THe deer tick is famous for carrying the spirochete of lyme disease

Insect parasites

• Sucking lice are wingless ectoparasites that suck blood and then produce eggs

• Tend to be highly specific to one host species, and different species have a preference for different body sites: head lice, body lice, pubic lice

• Difficult to eliminate because the eggs are attached firmly and are not killed by normal shampoos

Bed bugs

• they detect the host mainly from the exhaled carbon dioxide

• They suck the hosts blood and depart to lay eggs unnoticed

• bedbugs were nearly eradicated in the US in the mid-20th century by the use of DDT and other pesticides

• Today, bedbugs resist the older pesticides and are killed by newer chemicals with toxic side effects

What is a virus?

• Infectious agents too small to be seen under a light microscope are not considered cells

• Viruses need a living host to replicate and multiply - obligate intracellular parasites

Host and viral transmission

• Each species of virus infects a particular group of host species, known as the host range

• Transmission- direct contact, indirect contact, or via a vector

• Zoonose- when a virus is transmitted from an animal host to a human host

• Reverse zoonose- when a virus is transmitted from a human host to an animal host

• Tissue tropism- refers to the range of tissue types that a virus can infect

Viral components

Virion: complete virus particle

Nucleic acid: DNA and RNA, single or double stranded; linear or circular

Capsids: Compounds of capsomers and protects and encloses the nucleic acid

Envelope: Lipid bilayer acquired when they bud from the host cell. Composed of lipids, proteins and carbohydrates

Spikes: Glycoproteins mediate attachment to the host

Bacterial viral components

Head: Iscosahedral protein shell

Genome: Nucleic acid, contains viral genetic information

Collar/neck: Connects head and tail

Tail sheath: Contractile protein tube that injects DNA into host

Tail fibers: recognize and attach to bacterial receptors

Base plate: Anchors phage to host and triggers DNA injection

Pins/spikes: Short projections on base plate, helps penetration of bacterial cell wall

Viral structure

Viral shape and size are determined by wither their capsomeres or their envelope

Non-enveloped polyhedral virus

Capsid made up of capsomeres, nucleic acid inside

enveloped helical virus

envelope with spikes, capsomere inside, nucleic acid inside there

Viral classification: Baltimore method

Group I: Double stranded DNA is transcribed to RNA

Group II: Single stranded DNA generates double stranded form within the host cell, which is transcribed to mRNA

Group III: Double stranded RNA makes mRNA by using RNA-dependent polymerase

Group IV: Single stranded RNA (+) makes a complementary strand , which is transcribed to mRNA

Group V: Single stranded RNA is transcribed to mRNA

Group VI: Single stranded RNA is reverse transcribed to DNA, which is transcribed to mRNA

Group VII: Double stranded DNA is transcribed to mRNA, which is reverse transcribed to make viral genomes for packaging into virons