Prokaryotic Structure and Form

What does monomorphic mean?

Maintaining the same shape throughout their life

What does pleomorphic mean?

capable of changing shape in response to environmental cues

Are most bacteria monomorphic?

yes

What word equates to rod-shaped?

bacillus

What word equates to spherical?

coccus

What word equates to spiral shaped?

spirillum

What word equates to having a boomerang-like shape?

vibrio

Provide an example of a bacteria with a boomerang-like shape?

Vibrio cholerae

What word equates to having multiple curves in the shape?

spirochete

What genus of bacteria are star-shaped?

Stella

What makes some bacteria look fungal like?

they have budding, stalks or hypha

What prefix is given to bacteria that have cells arranged in pairs?

diplo

What prefix is given to bacteria that have cells arranged in clusters?

staphylo

What prefix is given to bacteria that have cells arranged in chains?

strepto

What prefix is given to bacteria that have cells arranged in branches?

streptomycetes

Where does E.coli get its name from?

The scientist who discovered it and its habit (colon/large intestine)

Where does Saccharomyces cerevisiae get its name from?

Saccharo = sugar

Myces = fungal

Cerevisiae = beer

It’s a microorganism that does fermentation

Where does Staphylococcus aureus get its name from?

Staphylo = clustered

Coccus = spherical

Aureus = gold coloured colonies

If a microscope has only one lens…

It’s a simple microscope

How many lens does a compound light microscope have?

2

The total magnification of a compound light microscope is…

objective lens x ocular lens

What is magnification?

increasing the apparent size of an object

What is resolution?

the shortest distance between two points on a specimen that can still be distinguished

Is it better to have a microscope with a small or big resolving power?

Small because it lets you see more detailed things

What is light microscopy?

the use of a microscope that uses visible light to observe a specimen

What’s the highest magnification of a light microscope?

1000

What are the different types of compound light microscopy?

brightfield, darkfield, phase-contrast, confocal, fluorescence and differential interference contrast

Compare brightfield and darkfield microscopy

In BF, the background is lighter than the specimen whereas in darkfield the background is dark

What’s the best microscope to use if you want to view bacteria in a natural state?

phase-contrast microscope

What does a phase-contrast microscope do?

Accentuate the diffraction of the light that passes through a specimen for greater detail

Are the images created by a phase-contrast microscope or DIC colourful?

No

What is the purpose of fluorescent microscopy?

Make it easier to see cells and distinguish between them and the background OR track specific proteins within a cell

How does fluorescence microscopy work?

• Stain cells with fluorophores

• illuminate specimen with specific wavelength of light

• fluorophores absorb that and emit a different wavelength of light

What’s a commonly used fluorophore?

Green fluorescent protein (GFP)

Where was GFP first observed?

In jellyfish

What wavelength of light excites GFP and what does it emit in response?

its excited by violet and emits wavelengths with a green colour

How have different dyes been made from GFP?

by modifying the amino acids in the GFP protein

What’s one way that fluorophores are used?

add the sequence that encodes for GFP to the sequence for your protein of interest

What’s a second way that fluorophores can be used?

attach the fluorescent dye to the antibody and use antibody to tag a protein of interest

Why are Indirect Fluorescent Antibodies used more?

allows the antibody to be used for multiple processes and provides greater amplification of the dye

What do confocal microscopes do?

take images of a specimen at different depths and compile them into a 3D image (Z-stack)

What is electron microscopy used for?

visualising organelles and viruses

Are the specimen for electron microscopes alive or dead?

Dead because they must be fixed to preserve shape

How does an electron microscope work?

electron beam is shot at the surface of an object and it picks up the electrons scattered back and turns that into an image

What’s the highest magnification of an electron microscope?

100,000 to 300,000x

What is a smear?

a thin film of solution of bacteria on a slide

What is fixation?

killing cells in a manner that preserves their physical structure to prevent decay and lysis, and adhering them to the slide

How can fixation be done?

with heat or chemicals

What is staining?

colouring the microbe with a dye to emphasise structures and provide contrast to the surrounding medium

What is the purpose of a wet mount?

to see bacteria in its live state and observe whether it’s motile

How is a wet mount done?

place liquid onto a slide and put a coverslip on top

What kind of compound is a stain?

a salt compound with a positive and negative ion

In a basic dye, the chromophore is a…

cation

Why does a basic dye stain the cells?

most microbes have a negative charge which attracts the cation chromophore

In an acidic dye, the chromophore is a…

anion

What is a simple stain?

A stain consisting of one dye

What is a direct simple stain?

applying dye to the cells

What is a simple indirect stain?

applying dye to the background

What is a differential stain?

Using two or more dyes to distinguish between bacteria

Provide examples of differential stains

Gram stain, endospore stain and acid-fast stain

What is the average size of a prokaryotic cell?

0.2 –1.0 µm x 2 – 8 µm

What happens if you double the radius of a cell?

the volume becomes 8x larger

What are the advantages of small cell size?

higher surface area to volume ratio, faster growth and greater nutrient exchange

What determines a cell’s upper size limit?

rates of diffusion for import and intracellular transport

What determines a cell’s lower size limit?

molecular dimensions (having enough space for genetic material and ribosomes)

What is the bacterial cell wall made of?

peptidoglycan

What is peptidoglycan?

a polymer of disaccharide and amino acids

What sugars are found in peptidoglycan?

–N-acetylglucosamine (NAG)

–N-acetylmuramic acid (NAM)

What kind of bond is found in the disaccharide?

A 1,4. glycosidic bond

Describe the structure of peptidoglycan

a polymer of disaccharide linked together by amino acid cross bridges

Why are the amino acid cross bridges important?

they provide strength and make the cell wall a solid surface

What is the purpose of the cell wall?

prevent osmotic cell lysis and provide structure and shape

What makes peptidoglycan a good target for antibiotics?

it’s a feature of cells that eukaryotes don’t have and there are many enzymatic steps to synthesizing Peptidoglycan means there are many antibiotic targets 

How does penicillin work?

it prevents bacterial transpeptidase enzymes from forming the peptide crosslink

What are beta-lactams?

a class of drugs that inhibit penicillin-binding proteins

What are lysozymes?

innate immune system component that break the 1,4 glycosidic bond in the disaccharide of peptidoglycan

How else do lysozymes attack bacteria?

they are highly cationic and can puncture membranes

Describe a gram-positive bacteria

a cytoplasmic membrane with a thick layer of peptidoglycan

What’s an additional feature of a gram positive bacterial cell wall?

teichoic acid which can be immunogenic

Give an example of a gram positive bacteria

Staphylococcus aureus

Describe a gram negative bacteria

Cytoplasmic membrane, then a thin layer of peptidoglycan and an outer membrane on top

What are additional features of a gram-negative bacterial cell wall?

lipopolysaccharide and porins

What are the 3 components of LPS?

Lipid A, core polysaccharide and O-specific polysaccharide

What is the lipid A component of LPS?

a heat-stable toxin associated with the outer membranes

Due to being an endotoxin, what effects does lipid A have on the body?

when recognised by TLR-4, it stimulates an immune response, inflammation, and can lead to septic shock

What does the O-polysaccharide component do?

interacts with the extracellular space so it can be immunogenic. On its own it can trigger a full-on immune response with the same effect as the whole bacteria

What are porins?

transport proteins of the outer membrane that passively import and export molecules

What are the advantages and disadvantages of having a large porin?

can import more nutrients but have greater susceptibility to antibiotics

What are the advantages and disadvantages of having a small porin?

lower susceptibility to antibiotics but harder to import nutrients

Give an example of a gram negative bacteria

Escherichia coli

If a bacteria is gram negative, what colour will it express after a gram stain?

pink

If a bacteria is gram positive, what colour will it express after a gram stain?

purple

Why do we use an acid fast stain?

Some bacteria are gram-indeterminate because they have mycolic acid in their cell wall

What are the steps of an acid-fast stain?

fix the bacteria, drive carbol fuschin stain into cells with heat or phenol, decolorise with acid-alcohol, counterstain with methylene blue

Why are gram-indeterminate bacteria pink after an acid-fast stain?

the mycolic acid resists the blue counterstain because it isn’t driven in with heat and phenol

Why do bacteria without mycolic acid appear blue after an acid-fast stain?

the acid-alcohol punctures through the peptidoglycan which allows the counterstain to get in

Give examples of gram-indeterminant bacteria

Mycobacterium (e.g tuberculosis) and Nocardia

Describe the composition of the cytoplasmic membrane

40% phospholipid and 60% proteins for communication and transport

What feature do almost all archaea have on their cell surface?

an s-layer

What is the s-layer?

A part of cell wall consisting of a lattice formed by one or two proteins or glycoproteins. It’s highly variable