Cells to organisms practical exam

potassium hydroxide test

• differentiates bacteria based on gram positive vs negative

• uses differential lysis

• weaker envelope of gram negative can be destroyed by K-OH, gram positive stays intact

• how to preform

  • flame loop

  • place a colony of bacterial isolate on microscope slide

  • place a drop of 3% K-OH onto bacteria

  • mix bacteria with loop for 30-60s

  • observe consistency of bacteria as pull loop away

    • if mix gets viscous and stringy, organism is likely gram negative if not likely gram positive

oxidase production test

• tests for the presence of cytochrome C oxidases

• tests if the bacterium can use oxygen for energy via the etc

• uses N,N, N’, N’- tetramethyl-p-phenylenediamine (TMPD) as a redox indicator

• blue → oxidase positive (obli. o2 or fac ano2)

• colorless → oxidase negative (obli. ano2)

oxidase-fermentative test

• tests if bacteria can grow in the presence of oxygen

• uses a pH indicator to show when bacteria have acidified in the media

• how to preform

  • two test tubes innocculated with bacteria

  • one subsequently sealed w/ paraffin

• yellow→ positive

• purple→ negative

• if both tubes are yellow → fac anO2

• if only non sealed tube is yellow → obli O2

• if only sealed tube is yellow → obli anO2

• genera of gram negative, rod shaped

catalase production

• tests if bacteria possess catalase

• H2O2 is made as an end product of aerobic breakdown of sugars

• H2O2 is toxic to bacteria if it accumulates

• bacteria produce catalase to convert H2O2 to water and O2

• how to test:

  • colony on microscope slide

  • place a drop of 3% H2O2 onto slide

  • Mix and see if bubbles form

• Bubbles appear → catalase positive (obli O2 or fac anO2)

• No bubbles → catalase negative (obli anO2)

• differentiation of gram positive

salmonella

• survives highly acidic environments due to asr gene which is upregulated at low pH

• has ~4500 genes

pPasr::gfp

p - indicates that the genetic information is present on a plasmid

P - indicates that only the promoter sequence of the asr gene is present in the plasmid

• the plasmid pPrpsM::gfp contains the rpsM promoter sequence

:: - indicates that pPasr is fused to gfp

gfp - indicates the presence of the complete gfp gene

how to see if asr gene is expressed

• transform the bacterium by adding gfp gene under the asr gene promotor

• when asr gene is activated (under acidic conditions) bacteria will express gfp and fluoresce under blue light

salmonella experimental controls

positive → ecoli carrying the pPrpsM::gfp plasmid under a constitutive promoter

• ie. always expressed

negative → untransformed e coli

non pathogenic ecoli used is strain DH5 alpha

tetraethylammonium

blocks potassium voltage channels

longer depolarizations due to no repolarization

tetrodotoxin

blocks sodium voltage channels

• can be bypassed by electrical depolarization

neurotoxins

dendrotoxin → blocks K+ channels

latrotoxin → massive Ach release (black widow spider venom)

alpha bungarotoxin → blocks Ach nicotinic receptors (cuare)

embryonic age

measured in days post coitum (dpc) for mice and rats

embryonic stage

assessed through a set of morphological criteria

most commonly used criteria known as

• Theiler Stages for mice

• Carnegie Stages form humans

electromyography

measure of electrical pulses of muscles

• for accurate read of muscles

  • green (negative) lead on lower forearm

  • red (positive) lead on upper forearm

  • black (ground) lead above elbow

pacemaker (Sinoatrial node)

• right atrium

• initiates an electrical sequence

• impulse passes down conduction pathways between atria to the atrioventricular node and from there to both ventricles

• conduction pathways facilitate orderly spread of the impulse and coordinated contraction of first the atria and then the ventricles

blood pressure

• varies from peak pressure produces by contraction of the left ventricle, to a low pressure maintained by closure of the aortic valve and elastic recoil of the arterial system

• diastole - pressure maintained even while left ventricle is relaxing

Mean Arterial Pressure (MAP)

a measure of the adequecy of blood supplied to vital organs when blood pressure is dangerously low

Tidal Volume (TV)

volume of air breathed in and out without conscious effort

Inspiratory Reserve Volume (IRV)

additional volume of air that can be inhaled with maximum effort after a normal inspiration

Expiratory Reserve Volume (ERV)

additional volume of air that can be forcibly exhaled after a normal expiration

Vital Capacity (VC)

The total volume of air that can be exhaled after a maximum inhalation

VC = TV + IRV + ERV

Residual volume (RV)

volume of air remaining in the lungs after maximum exhalation

Total Lung Capacity (TLC)

TLC = VC + RV

Respiratory muscles for active breathing

• sternocleidomastoid

• scalenes

• internal intercostals

• abdominal muscle groups