final mbio

Two types of microscopes

light microscope (compound) OR electron microscopes (transmission, scanning, scanning tunneling)

compound light microscope

Any microscope that uses visible light to observe specimens

compound light microscope lenses

objective 100X (by specimen) and ocular 10x (eye piece)

total magnification

objective lense mag times ocular lenses mag \= 1000

what is the charge of the outer surface of the bacteria

net negative

positive stains

adhere to negatively charged bacterium, bacteria turns colour of stain, background is clear, ex: Crystal Violet

negative stains

repelled negatively charged bacterium, bacteria will appear clear, background will be coloured, ex: Nigrosin

simple stains

a single coloured basic dye is used, basic dyes have positively charged colour ion therefore binds to organism staining it that colour, sometimes uses mordant

differential stains

used to differentiate between bacterias, important for diagnostics, exploits differences in cell wall structure and composition, ex: the Gram stain, acid fast stain, endospore, capsule stain

the Gram stain

determines whether a bacterium is Gram positive or Gram negative

acid fast stain

carbolfuchsin used to stain Mycobacterium (waxy/hydrophobic) pink, methylene blue stains all non-acid fast cells (negative)

why will Mycobacterium not Gram stain?

because they're waxy (mycolic acid) - they are gram positive but mycolic acid is outside of this peptidoglycan

capsule stain

2 stains - positive stain will stain cell membrane and negative stain always sticks to background, white halo left is the positively charged halo around cell

endospore stain

positive charged pink stain sticks to all bacterium, malachite green stains endospores. note: only gram positive can produce endospores

what would a negative endospore stain look like

no green, all pink bc no endospore

what would a gram stain on an endospore bacterium look like

it would be purple because only gram positive bacterium can produce endospores

flagella stain

they're too small to see under the light microscope without stain, mordant and stain work together to increase the thickness

Resolution general principle

the shorter the wavelength, the better the resolution

Electron microscope

Use beams of electrons instead of light- better mag than light microscopes - 500000x

Transmission Electron Microscope (TEM)

used to study the internal structure of cells - thin sectioning to cut cell in half and stain is used

scanning electron microscope

Used to view surface of cell, specimen coated in metal- 100000x

chromophore

Positive or negative stain - colour stains

smear

a thin film of a solution of microbes on a slide

Clinical staining procedure

1. Smear on slide
2. Pass through flame
3.apply stain
4.stain removed by rinsing
5.ready to view under microscope

scanning tunneling microscope

Most powerful of electron microscopes, used to visualize atoms

Bacteria at minimum temp

Lowest temp that can support growth of microbe- bacteria at rest ex. Thawing meat

Optimum temp for bacteria

Preferred temp that best supports growth of microbe

Maximum temp for bacteria

Bacteria dies over max

Psychrophiles

cold-loving microbes -5 to 15

Psychrotrophs

Cause food spoil in fridge, temp 15-30

Mesophiles

Love moderate temp, 10-45, causes disease

Thermophiles

45-70, doesnt cause disease in body

Hyperthermophiles

65-110, deep ocean vents

Acidophiles grow in

Low pH

Alkalophiles grow in

High pH

Neutrophiles

5-8 pH

staphylococcus vs streptococcus

staphylococcus grows in clumps - like grapes
streptococcus is like a chain

which cells produce endospores?

Gram positive cells

the Gram + cell wall

-Thick peptidoglycan
-Teichoic acids
- only 1 membrane

Pili

- longer than fimbriae
- only 1-2 per cell
- join bacterial cells together to exchange DNA \= conjugation
- Allows attachment of two bacteria to each other
- Gram +/- can have

bacterial cell wall

- semi-rigid, complex, semi-permiable
- provides cell with shape
- protects cell from environment
- prevents rupture
- can be Gram +/-
- composed of polysaccharide: peptidoglycan

peptidoglycan

- polysaccharide composed of repeating disaccharides
- polysaccharide chains liked together by short polypeptides (lattice, resistant to osmotic changes)
- disaccharides composed of NAG/NAM
- contains amino acids

fimbriae + example

- often found in Gram -
- allows bacterial cell to adhere to surfaces of other cells
- made of pilin protein
- allow bacterium to adhere and colonize \= disease causing
- ex. Neisseria gonorrhoea

flagella in prokaryotes

- filament, hook, basal body
- use to differentiate between stains
- rotate clockwise or counter clockwise
- movement depend on energy production

peritrichous

flagella all over cell surface

monotrichous

single polar flagellum

Lophotrichous

2 or more at one or both ends of cell

amphitrichous

a tuft at each end

glycocalyx

- sugar coat
- composed of polysaccharide and protein (only sugar \= extracellular polysaccharide)

capsules

- virulence
- protect from phagocytosis
- allows organisms to adhere and colonize
- prevents dehydrations and keeps nutrients in

wall teichoic acids

extends out from peptidoglycan

lipoteichoic acids

connect plasma membrane to peptidoglycan

the Gram stain procedure

- apply crystal violet (positively charged purple): Gram+/-\=purple, eukaryote \= purple
- apply iodine (mordant): all\= purple
- add alcohol: gram + \= purple, gram -\=colourless, eukaryotic\=colourless
- add safranin (pos charge pink): gram +\=purple, gram-\=pink, eukaryotes\=pink

genome

all of DNA preset within a given cell or plasmids

the Gram negative cell wall

- thin peptidoglycan layer
- contain both plasma membrane AND OUTER membrane
- outer membrane \= lipids, proteins, lipopolysaccharide (endotoxin)

what is the only cell that has peptidoglycan?

bacteria

peptidoglycan is a target to whom?

- host defences
- chemotherapies
- penicillin

plasmids

- circular double stranded DNA - replicates autonomously
- contain gene for antibiotic resistance \- - \---they are an advantage not essential

prokaryotes ribosome large and small sub unit and total

large \= 50s
small \= 30s
total \= 70s

eukaryotes large small and total sub units

large\= 60s
small\= 40s
total \= 80s

eukaryotic cell wall

- not in animal cells
- composed of single polysaccharide
- cellulose\=plants and algae
- chitin\=fungi
- not made of the polypeptide peptidoglycan

what do eukaryotic cells have in their plasma membrane that prokaryotes don't?

sterol (cholesterol)

eukaryote flagella/cilia

move in whip like motion
contain protein and cytoplasm (prokaryotes are hollow)
for motility

endospores

- only in gram positive
allow bacteria to resist heat and other danger

inclusion bodies

- deposits of nutrient granules stored for later use

sporulation

- bacteria replicates DNA
- septum divides cell
- larger compartment engulfs smaller forming a forespore in mother cell
- spore coat forms
- spore freed from mother cell

if a microscope has 6nm resolving power...

it can distinguish between 2 points if they are 6nm apart

electron vs light (compound) microscope

comp/light: 0.20um.
electron: 0.20nm
- electron more clarity

osmosis

movement of solvent molecules across a membrane
- water moves from high to low concentration

hypertonic

- solution highly concentrated with solute
- when cell is placed in hypertonic solution, water rush out of cell
- cell shrivelled and dry

hypotonic

- solution has low concentration of solute
- when cell is placed in hypotonic solution, water rushes into cell
- cell burst

isotonic

- 2 solutions are of equal concentration
- no net movement of water

honey or salted fish is an example of

hypertonic - bacteria lose water and can't replicate

extreme halophiles

live in salty environments

heterotrophs vs autotrophs

obtain carbon from organic material(ex sugars); obtain carbon from inorganic matter (CO2)

Chemical requirements for growth

- carbon
- nitrogen
- sulfur
- phosphorus
- iron

trace elements required

- zinc
- copper

cell respiration / aerobic respiration

use oxygen to produce ATP

fermentation / anaerobic respiration

enable cells to produce ATP without the use of oxygen

facultative anaerobes

compete for oxygen but can use fermentation

obligate anaerobe

organism that cannot live in the presence of oxygen

obligate aerobe

organism that requires a constant supply of oxygen in order to live

aerotolerant

do not utilize oxygen but can survive and grow in its presence, use fermentation

Micro-aerophiles

requires low levels of oxygen to grow, but high concentrations will kill them

culture

microbes continuously growing and multiplying

inoculum

microbes introduced to a culture

batch culture

never feed them again
never clean waste
see journey from growth to death

continuous culture

fed
wastes removed
support sindefinite growth

solid media

uses agar to grow colonies

culture medium

nutrients prepared for microbial growth - chemically defined and undefined
must be sterile

selective media

suppress unwanted microbes and encourage desired microbes
ex: intestinal bacteria, add bile salts to inhibit all non intestinal bacteria

differential media

distinguish between different bacterias

bacteria reproduction

binary fission
- elongates and duplicates chromosomes
- wall forms between 2 chromosomes
- two cells separate

it takes 10min for bacteria to divide how many cells will you have after 20min? 30min?

4, 8

E. coli generation time

20 minutes

average generation time for bacteria

1-3 hours

Mycobacterium tuberculosis generation time

24 hours

lag phase

growth \= death
nutrients ^
deaths low

log phase

growth \> death
nutrients ^
death low

stationary phase

growth \= death
nutrients low
waste^