microbio exam 1 study guide

what is refraction

the bending of light as it passes from one substance to another

especially substances of different densities

this is what allows eyes and microscopes to focus light into a visual image

Why does refraction happen?

happens because light changes speed and direction when passing at an angle between materials of different densities

what is the normal line?

an imaginary line of 90 degrees that is perpendicular to the surface, separating two different media at the specific point where light strikes it

serves as a reference for measuring the angle of incidence and the angle of refraction

how does light bending depends on whether it is going from an area of low density to high density?

light beams will bend towards the normal line

how does light bending depends on whether it is going from an area of high density to low density?

light beams will bend away from the line

what is refractive index?

the ratio of speed of light in a vacuum to its speed in a specific medium

how do you calculate refractive index?

n= speed of light in a vacuum ( c ) divided by the speed of light in a medium ( v )

what is the lowest possible refractive index?

1 in a pure vacuum

what does increasing the refractive index means for how that material can bend /refract light

the higher the number, the stronger the material bends light

material slows down more, causing it to bend or refract more towards the normal line when entering

what is resolution?

the smallest distance at which 2 different objects can be seen as separate from each other in an image

for this, smaller numbers are better

the smaller the resolution the sharper the image

how do you calculate resolution?

what are the factors that can affect resolution?

1. the wavelength of light used

2. the refractive index of the lens

3. the lens half angle

what is the resolution of the human eye?

0.1 millimeters or 100 micrometers

why do the systems that we use must provide both magnification and a greater resolution than the human eye

human eye has limitations so we must use those systems to provide greater magnification because it enlarges the image to make it easier to see and resolution to help distinguish between adjacent small features

what are the functions of the condenser?

focus and align light into a single beam

what is the objective lens?

creates a magnified image from focusing the refracted light onto the retina

what is the ocular lens?

further magnifies the image made by the objective lens

how does the microscope creates an image?

1. condenser lens focus and align light into a single beam

2. the light will pass thru the microbial sample or specimen

3. light will refract as it passes through the specimen

4. refracted light from the sample is focused to create a magnified image by objective lens

5. the image made by objective lens is further magnified by ocular lens

what is contrast?

the darkness of the image background relative to the specimen

what are the ways that we can provide contrast to a microscopic image?

thru manipulation of light or chemical treatment

what is staining?

using chemical dye to give an image color and contrast

has two groups which are simple and differential stains

what are simple stains?

using a single dye that chemically binds to cellular structures

what are differential stains?

multiple dyes that bind differently to cellular structures

allows us to classify microbes based upon their staining properties

what are the drawbacks of staining?

the staining process kills microbes and can’t visualize live cells

how does a dark-field microscope work?

works by using an Abbe condenser with a dark-field stop to block direct light

the only light that enters the objective is the light that passes through the specimen itself

what kind of images does dark-field microscope produce

images that will always have a bright image of the specimen on a dark background

what does dark-field stop do?

creates a hollow cone of light

what does the Abbe condenser do?

focuses the cone of light at an angle rather than directly onto the specimen

How does interference microscopy provide contrast?

by using a modified optical system to create contrast using constructive and destructive interference

what are the two types of interference microscopy?

constructive and destructive interference

what is constructive interference?

areas that are especially bright

what is destructive interference?

areas that are especially dark

how could you tell a phase contrast from a DIC image?

DIC images do not show phase halo

what does phase contrast provide?

produces a strong 2D contrast with very bright edges

what is phase halo?

bright, fuzzy edge to the images

what is differential interference contrast?

produces a slightly weaker contrast that appears 3D

How does fluorescence microscopy work?

by using dyes that give off light when struck by visible light of a specific color

filters in the microscope allow only the emitted light from the specimen to reach the eye

what are the advantages of staining live cells?

fluorescent dyes typically do not kill live cells

dyes can be attached to antibodies to identify and stain specific proteins

why do light microscopes have a maximum limit of resolution?

because light microscopes can only reliably interact with object bigger that its wavelength

½ of wavelength of light being used

what is the max resolution?

0.2 micrometers

how do electron microscopes allow us to overcome the resolution and magnification limits?

they replace visible light with electrons

they use electromagnets rather than glass lenses to focus

How are electrons focused in an electron microscope

electromagnets focus electrons to provide both magnification and resolution

How do transmition electron microscopes work?

produce 2D images that are a slice through a microbe

allow us to visualize the internal structures of microbial cells and viruses

magnifies structures more than 100000x

How do scanning electron microscopes work?

uses an electron bean to scan the surface of a microbe

produces a 3D image of the outer surfaces of a microbe

what are the pros and cons of TEM and SEM?

TEM can produce images with higher magnification and better resolution

limitations are that it can only visualize small sections of a microbe

SEM can visualize lower magnification and worse overall resolution

How does Cryo-EM work?

freezing samples in liquid nitrogen

How does Cryo-EM produce images?

an electron beam takes images of the specimen at multiple angles

what is the smallest resolution achieved so far with cryo-em?

2.8 angstrom

what is are bacteria?

single celled organisms that lack a nucleus and most other internal organelles

what are archaea?

superficially similar to bacteria but are an evolutionary distinct domain of life

typically live in an extreme or unusual environments

genetically closely related to eukaryotes than bacteria

what are eukarya?

all eukaryotic cells

in microbio those are fungi and protists

what is plasma membrane?

flexible outer covering of a cell

what is the cytoplasm?

highly organized, water-based inside part of a cell

what are ribosomes?

machines used by the cells to make proteins

what are chromosomes?

genetic information that is stored on one or more chromosomes

what are the basic two shapes of bacterial cells?

coccus (spherical) and bacillus ( rod-shaped)

what are the arrangements of the bacterial cells?

duplo: pairs

strepto: chains

staphylo: clusters, like grapes

polisades: side-by-side arrangements of bacilli

what are the major functions of cell walls in bacteria?

1. providing cell shape and resistance to osmotic stress

2. cell wall prevents distortion of the cell membrane

3. provides resistance against physical damage

4. allows bacteria to stick to other cells

5. protects bacteria from being identified by the immune system

what are the two types of cell wall?

gram positive and gram negative

what is special about gram positive cell walls?

made almost entirely of a thick layer of peptidoglycan

has teichoic acid

what is so special about gram negative cell walls?

consists of thick layer of peptidoglycan and a second plasma membrane, the outer membrane

do not have teichoic acid but have lipopolysaccharides or LPS, that can also stick to cells

what is peptidoglycan?

is a mesh made of polymers of sugars

peptido -is partially made of protein

glycan - partially made of sugars

what is teichoic acid?

able to attach pr stick to molecules on the surface of another cell

what is the backbone?

the rigid, helical, sugar chain that forms the main scaffold of the bacterial cell wall

made of alternating sugars NAM and NAG that are liked together into a long, straight chain

what are the functions of the backbone?

main structural framework

gives the cell wall physical shape and integrity

resists osmotic pressure

provides the base structure that everything else attaches to

what are stem peptides?

attach to each NAM sugar or a short peptide chain ( a little tail)

connection points between sugar chains

allows the wall to be cross-linked

what is a cross linkage?

a bond between stem peptides on different peptidoglycan chains

they are relatively loose connections so this allows the cell wall to have some flexibility and elasticity

what is a lipopolysaccharide?

lipo : fat

polysaccharide: part sugar

different molecule but same function as teichoic acid, sticking to cells

Why can LPS/endotoxin make Gram negative infections very serious?

• consists of lipid A, core polysaccharide, and o antigen

• when gram negative bacteria die, or break, or multiply, lipid A gets released

What are the potential effects of endotoxin in the body?

can cause a severe inflammatory response

extremely high fever with chills and shivering

organ stress can cause sepsis

What are the first step of the Gram stain?

add crystal violet

it stains all bacteria purple

gram + is purple

gram - is purple

EVERYONE IS PURPLE

What is the second step of Gram stain?

add iodine

mordant makes dye less soluble so it adheres to cell walls, clumps crystal violet

gram +: still purple

gram -: still purple

EVERYONE IS PURPLE BUT THE DYE IS LOCKED IN

What is the third step of Gram stain?

add alcohol

alcohol dissolves crystal violet

gram +: remains purple

gram -: loses its dye, becomes colorless

GRAM + AND GRAM - SEPARATE

what is the fourth step of Gram stain?

add safranin ( pink or red dye that stains any colorless cells)

gram +: stays purple ( purple masks pink)

gram - : takes up safranin and becomes pink/red

What is the structure of acid-fast cell wall

• contains thick layer of peptidoglycan

• has a special layer of fat-based molecules, mycolic acid

• also has a plasma membrane

how is acid-fast cell wall similar to gram + and gram - cell walls?

gram +: thick layer of peptidoglycan

gram -: outer membrane behaves like thick wax

how to identify acid fast and non-acid fast bacteria on an acid fast stain?

instead of gram staining, you perform acid-fast staining

acid fast appears red or pink

non-acid fast appears dark blue or purple

How does the acid-fast cell wall affects the ability of bacteria to be resistant to disinfection, the immune system, and to antibiotics?

unique chemistry of acid-fast cell wall makes mycobacteria resistant to disinfection

can resist dehydration by heat, most water-based cleaners, most typical antibiotics

acid fast cell walls resist destruction by the immune system

what is mycobacteria?

the most common acid-fast bacteria belong to a group of rod-shaped bacilli bacteria

the most dangerous is mycobacterium tuberculosis

How does the immune system handle tuberculosis bacteria that it can’t destroy?

immune system walls off TB by forming granuloma

it prevents spread thru the body and keeps TB in a sleeping state to limit the tissue damage elsewhere

can’t kill it → cages it → granuloma

What can happen if TB left untreated?

lung failure

How do you treat TB?

special antibiotics can penetrate the waxy mycotic acid layer

What are the clinical problems related to the difficulty of treating TB?

treatment is expensive and poor patient compliance

what is the cell envelope?

a protective, multilayered structure surrounding the cytoplasm

primarily consisting of the inner cell membrane, a peptidoglycan cell wall, and, in Gram-negative bacteria, an outer membrane with lipopolysaccharide (LPS)

what structures can be a part of the cell envelope?

slime capsule, slime layer, peptidoglycan, lipoteichoid acid, cytoplasmic membrane

what is the slime layer?

adhesion structure made of loosely packed and unorganized sugars

how does the slime layer look like on a typical microscope?

lack of organization makes it appear fuzzy on microscope images

what does the slime layer provide?

adhesion because the concentrated sugars have a chemical stickiness

what is a capsule?

an adhesion structure made of densely packed and organized sugars

what does capsule do?

just like slime layers, it has chemical stickiness

how do capsules look like on a microscope?

appears sharp because of how organized it is

white border around cells because its density prevents it from being stained

what is an s-layer?

a protein-based outer surface covering

what is the basic structure of s-layer?

is built from many copies of a single s-layer protein that bind together in a layer 1 protein thick

are able to self assemble into a crystal like surface

what are the types of proteins s-layer is build from?

built form glycoproteins that are sometimes modified in certain species to add carbohydrate chains

how is the s-layer anchored to the cell?

in gram + is anchored to teichoid acid

in gram - is anchored to lps

what are adhesion structures used for?

sticking to cells and surfaces

why does bacteria need adhesion? ( especially ones that cause disease)

they can be easily removed by physical forces or fluid flow

some bacteria can work together as a community to adhere by forming a biofilm

what are the three major adhesion structures?

fimbriae

pilus

biofilm

what are fimbirae and what are the made from?

adhesion structure made from proteins

is think, hair like, usually dozens or 100s per cell

what are pilli and what are the made from?

adhesion structure that is also made of protein

usually larger and fewer in number that fimbriae