Micro Exam 1

Microscopy:

o microscopes being used to look at the structure and function of microorganisms

How big are microorganisms

o 1000 micrometers = 1 millimeter

o 1000 nano meters = 1 micrometer

bacteria size

1-2 micrometers

virus size

Nanometers

How does a microscope work?

o Lenses

§ Objective & ocular

o Stage light

o Stage for sample

o Focusing knobs (fine and major focusing)

o Van Leuwenhoek

o The first to use and see bacteria on a microscope (only one lens)

Magnification

ability to enlarge an image

Resolution

ability to see two points as distinct and separate

o Numerical aperture is light gathering ability of lens

o Difference wavelengths yield different resolutions

o Higher numerical aperture (more light in scope) better resolution

Resolution equation

o Resolution = 0.5 (wavelength) / numerical aperture

Limit of resolution (human eye)

0.2 mm

Limit of resolution (lab microscope)

0.2 micrometer

Refraction:

§ Light rays bend as go from one medium to another

· Glass to air

§ Different mediums have different refractive indexes

· Oil and glass have the same refractive index

Bright field microscope (AKA light microscope)

§ Stained cells on bright background

Dark field microscope

§ Only light is reflected by specimen

§ Living cells

Small cells

Phase contrast microscope

§ Magnifies differences in density in cells

§ Grey scale

§ Differences in refractive index

§ Living cells (do not need a stain)

Fluorescent microscope

§ Uses one wavelength of light on the specimen (one color)

§ Specimen reflects different color

§ Uses specimen stain with a fluorescent dye

electron microscope

· Do not use visible light, uses electron beam

Transmission Electron Microscope (TEM)

· Detailed internal view

· Thin specimens

Scanning Electron Microscope (SEM)

· Detailed surface view

· Does not need to be a thin specimen

Eukaryotic cells size

8-10 micro meters

Bacterial Cell Anatomy

Domains

o Bacteria

o Archaea

o Eukarya

o Most bacteria have _________.

1 circular chromosome (DNA)

Endospore

o Made by some gram positives

Non-universal internal feature

Creation of spore

sporulation

Endospore

vegetative cell = Germination

Capsule

§ Polysaccharide coat around cell wall

§ FXN: attachment evade phagocytosis by WBC

Flagella

§ FXN: motility

§ IF HAS FLAGELLA: CAN PERFORM CHEMOTAXIS

§ Hollow protein tube in bacteria

§ RUN: movement in 1 direction

TUMBLE: tumble in place

Fimbriae

§ Short many hair-like appendages

FXN: attachment

Pilus/Pili

§ 1-2 per cell

§ FXN: attachment and twitching motility

· Used to exchange DNA

"Sex pilus"

§ Why might it be difficult to develop antifungal drugs?

· Diversity in fungi, tetracycline disrupts ribosomes that fungi do not have.

o Fungi is a eukaryotic cell like our animal/human cells, an antifungal drug who be toxic to us and the fungi.

§ Antifungal drugs target cell membrane

· Fungi have different sterols in cell membrane that human cells

· We have cholesterol and fungi have ergosterol

o Making ergosterol a good target for antifungal drug development because it disrupts their cell membrane

Virus Anatomy

ALL viruses:

§ Have DNA or RNA

§ Have a protein coat (capsid)

§ Multiply inside other cells using cell's machinery

§ Transfer their viral genome into other cells to multiply

§ Envelope: lipid bilayer around capsid

· Derived from host plasma membrane

Viral proteins

Bacteriophage & Lytic Cycle

o Viruses that infect bacteria

o AKA PHAGE

o UV light/other stressors trigger the lytic cycle

Lysogenic Phage:

o Phage incorporates its DNA into the host bacterial chromosome

o Virus does not kill bacterial cell

§ Does not actively multiply

§ AKA: Prophage

o Phage Therapy

o The idea of using bacteriophages to treat bacterial infections

bacteria division

binary fission

o Effect microbial growth:

o Temperature

o pH

o Moisture

o Nutrient availability

o Oxygen

o Sunlight

o Toxic wastes?

o Osmotic pressure

o Spread Plate Pros and Cons

o Pro

§ Can distinguish between live and dead cells

§ Can count individual colonies

o Con

§ Needs to be diluted

o Direct microscopic count

o Same day results

o Volume of fluid over the large square is 1/1,250,000 if a milliliter. If it contains 14 cells, 14*1,250,000= 17,500,000 cells in a milliliter.

o Filtration

o Filter solution, cells get trapped on filter

o Pore size in filter 0.22-0.45 micrometer; smaller than bacteria

o Put filter on petri dish, nutrients attach

o Needs to incubate, no same day results

o Only counts live cells

o Medium has to be more specific to bacteria

o Only works for liquids, dilutes; not lots of bacteria

o Spectrophotometer

o Cloudiness reading

o no cells: 0 absorbance; 100% transmittance

o More cells: higher absorbance, lower transmittance

o Same day results

o Does count dead and alive cells, dead cells absorb light

o Does not tell us cells/mL

o Biofilm

o How most bacteria grow in nature

o Bacteria attached to a surface & encased in a polysaccharide matrix

o Polysaccharide exopolysaccharide (EPS)

o **Lab growth is planktonic

o 70% human infections

positive & negative biofilm

Positive biofilm:

§ Waste-water treatment plan

· Waste water is going over plastic in attempt to have biofilms be created

§ Human intestines

· Biofilm is colonizing on our intestinal tract and processing the nutrients

o Negative:

§ Infected knee replacement

Growth Control, physical control

o Heat

§ Moist heat: autoclave and pasteurization

§ Dry heat: flaming/bacticinerator

o Filtration

o Radiation

§ Ionizing: high energy waves (X rays, gamma rays)

Non-ionizing: UV light

o Sterile

o Free from all life forms, including viruses

o Mainly using autoclave

o Pasteurization

o 72 C for 15 seconds

o Kills disease causing bacteria

o Autoclave

o Steam under pressure

o 121 C, 15 lbs pressure, 15 minutes

Disk diffusion method

· Spread bacteria all over a petri dish but with four small paper disk with a chemical on each of them. They will repel/create zones in the bacteria, the bigger the zone the more effective

Minimal inhibitory concentration (MIC)

· Incubation with same # of cells

· how much you can put in without

inhibiting growth

o Especially hard to kill

o Endospore formers

o Mycobacterium (waxy cell wall)

o C diff (TB/tuberculosis)

o Antibiotic

o A chemical that is made by a microbe and inhibits bacteria

Sulfa drugs are _________, but not ___________

antimicrobial, antibiotics

Mainly (Sulfa Drugs)

sulfa drugs inhibit folic acid synthesis

PABA is an....

o PABA is an intermediate in folic acid synthesis pathway

Where do we get folic acid?

our diets

Bacteria can....

synthesize folic acid from scratch

PABA process

A --> B --> PABA --> D --> Folic Acid

What is blocked by sulfa drugs?

PABA --> D

Are sulfa drugs harmful? Why or Why not?

No, they are synthetic chemicals that are microbe specific

Distinction between antibiotic and antimicrobial drug

drugs that target microbes (sulfa drug/antimicrobial drugs)

lysozyme

body secretions (tears, saliva, etc.)

breaks down peptidoglycan

Two types of immunity

innate and adaptive

Innate immunity examples

break down peptidoglycan

Lysozyme --> body secretions

Normal microbiota --> 1st line

Cilia (Ciliary escalator)

Traps and moves mucous along so pathogens get moved

ciliary escalator and cystic fibrosis

o Most common genetic inherited disease in Caucasians

o Median survival with CF is 37 years (death from respiratory failure). A.

Cystic fibrosis demonstrates the effect of lack of ciliary escalator

Second line of defense

Phagocytocytosis, inflammation, interferons

phagocytosis

§ Two main phagocytic cells (neutrophils & macrophages)

Toll-like receptor (TLR) on WBC recognize pathogens via PAMP (pathogen associated molecular patters)

inflammation

§ Blood vessel dialate & become leaky B.

§ Bacteria goes to bacteria

Phagocytic cells go to that location and eat the bacteria

interferons

§ proteins made by virus-infected cell to signal neighboring healthy cells

Fever

o speeds up metabolism & enhances interferon effect

Adaptive vs Innate

Adaptive: tailored to the pathogen, takes time to develop, slower to respond, has memory

Innate: nonspecific, present at birth, quicker to respond no memory

types of humoral immunity

humoral and cellular

humoral

b cell response

cellular

t cell response

B AND T cells...

have cell surface protein receptors to recognize specific pathogens

B cells have...

antibodies on the cell surface (aka immunoglobulins)

T cells have...

T receptors (TCR) on cell surface

Both hace variable at constant regions -->

referring to amino acids in proteins

AB & TCR recognize....

epitopes (small part of the antigen bound by the AB or TCR) on antigens (foreign proteins & sugars)

Antibody structure

§ 4 polypeptide chains

§ 2 identical light chains

§ 2 identical heavy chains

§ Variable & constant regions

§ 2 identical antigen binding sites

we always have ____ of different B cells in our body

MILLIONS

These B cells are _______ b/c each B cell has a uniquely shaped antibodies on its ____________.

different, cell surface

All the antibodies on the _________ B cell have the __________ shape

same, same

§ Antibody diversity allow us to recognize ______ pathogen

ANY

Examples of pathogens

viruses, bacterium, fungus

· Antibodies on B cells recognize __________________ pathogens (outside our human cells)

EXTRACELLULAR

§ Antibody proteins are uniquely shaped to recognize one specific ___________

antigen

§ B cell needs two things to divide:

· Antigen match

· Cytokine help from T helper cells

agglutination

§ Reduces # of infectious units for immune system to deal with

opsonization

§ Coating antigen with antibody enhances phagocytosis

Activation of Complement

§ Cause inflammation & cell lysis

Antibody-Dependent Cell-Mediated Cytoxicity

Antibodies attached to target cell causes destruction by macrophages, eosinophils, & NK cells

Neutralization

§ Blocks adhesion of bacteria & viruses to mucosa; Blocks attachment of toxin

o Memory cells do not fight off _______________________, but does make the response ____________ when/if they return

current infection, stronger

o Two types of T cells

§ T helper cells

§ T cytotoxic cells

o Both Types need to recognize antigen on _________ to become activated

MHC

When Activated (T helper)

§ secrete cytokines to activate other immune cells

When Activated (T cytotoxic)

§ differentiate into two cell types

· Cytotoxic T lymphocytes (CTL) which lyse altered self (human) cells; INTERNAL ANTIGEN

o Kills human cells that are messed up

o Kills cancerous cells before formed

· Memory T cells

MHC

major histocompatibility complex

MHC 1

· on all nucleated cells and present internal antigen to T cytotoxic cells