COA270: Exam II

random sampling

surface soil sampling

chooses random points within a plot of interest

sampled at a defined depth

transect sampling

surface soil samping

collection of samples in a single direction

Two Stage sampling

subdivision of a plot of interest into primary units

random or systematic sampling AFTER division

Grid Sampling

systematic sampling across regular intervals and fixed spacing

Air Rotary Drilling

subsurface soil sampling technique in unsaturated soil

→samples up to several hundred meters

→ large compressor forces air down drill pipe, out drill bit, and outside through the bore hole

→ overheating prevented by air through borehole and water/ surfactant injfection

Hollow stem Auger drilling

technique used to sample saturated subsurface soil

→ bore hole msut be kept open during sample collection

→ reaches depth up to 30 meters

→ Hollow tubes with rotating drill bit

→ reverse threaded casing hat pushes cutting up and out

Mud Rotary Drilling

technqiue to sample saturated subsurface soil sampling

depths >30 m

utilities drilling fluids to prevent borehole collapse

borehole mst be kept open during collecyion

• lower colony counts

• not normally exposed to atmosphere

• expensive to repeat

contaimation prevention is necessary for subfurcation samples because they

Plating

culture-based anaylsis of bacteria

_> utilizes different mediums (PYTG, TSA, BEA) to select for speific populations

→ allows for enumeration

Direct counts

culture based anaylysis of bacteria

determines total bacterial number present

→ does not determine the number of populations

Air dry Sample

Filter soil through 2mm mesh to remove large drbis

store remaining sample at 4C for up to 21 days

soil sample anaylsis processess

/mass of soil

units for purified dna

Coumminity DNA Anaylsis of Bacteria

anaylsis techeniue that determines populations present an genetic potential

→ DNA extracted in situ, lysis occurs releasing DNA for extraction, cell debris, and soil particles precipitated out via centrifugation

→ DNA purification process: supernatant is transported to another container, DNA precipitated out

→ quantification UV spectroscopy and fluorometry

→ relate total extracted DNA to number of microbes in sample

lysis methodology

combintion of physical and chemical means to cause lysis

phscial: bead beating, sonification, freeze thaw cycle

chemical: enzymes(lysozyme)

Soil washing metholody

method for isolating fungal hyphae and spores

→ saturate small soil sample

→ Aggregates are teased up with fine jet of water

→ Heavier particles sediment, fine particles decant our

→ Heavy particles spread on thin film of water and observed under dissecting microscope

→ Observed hyphae extracted

storage of soil sampels with fungi

samples are stored in sterile boxes containing sieves of varying grade sizes

sample is then washed vigorously for 2 minutes

Each wash is plated onto media and results are recorded

hyphae are retained by sieves so growth on media is dormant spores from samples

sewage sludge

soil or marine sediments contaminated with wastewater or sludge

used to determine pathogen risk in environment

Viruses Sampling

collect sewage sludge and reduce pH to 3.5 by adding Hcl or AlCl3

Sludge is centrigued and soils extracted

resuspended in netural beef extraction to detach viruses

beef extract removes by flocculation of proteins and the remaning viruses are neturalized

lower

concentrations of microbes in water is ___ compared to soil

eluation

step for processing water samples

→ removal of virus from collection filter

→ virus absorption elutation filters can be electronegative or electroposotiive to attrach viruses

hose lines and pumps

tools used in collection of water samples

Membrane filtration

processing water samples to find bacteria

collection and concentration of bacteria via filter

cultured after completion

most probably number

processing water samples for bacteria

serial dilution at different aliquot levels

Cartridge filters

used to detect protozoan parasites in water samples

Eluation buffer is added after filtration, and the solution is agitated for 5 minutes ona shaler to remove cysts and oocysts

protozoan are pelleted by centrifugation and resuspended in buffer

size exclusion

protozoans are filtred by

foam filters

for proecessing water samples to find prpotozoan parasites

eluavation buffer added after filtration, protozoan are squeezed out with plunger

immunomagentic seperation of protozoan from partiuclate matter

stained with flursecent mono clonal antibodies

Impingement

collecting air samples; trapping airborne particles in a liquid matrix

seperate by flow rate

Impaction

air samples; forced deposition of airbones particlesonto a solid surface

size of particles and flow rate

increasing impacation potenital of airbone particles determines

centrifugation

Mechanically forced deposotion of air bone particles through intertial and gravitonal forces

FIltration

trapping of airbones particles through size exusions

→ uses vacuum and filters that can differ in pore size and flow rate

Deposition

collection of airborne particles through natural deposition forces (gravitational setting)

-< low sampling efficiency

Resolution

smallest distance between two points that are viewed

resolving power

ability to distinguish two points

Magnification

ability to enlarge the aparent size of an image

contrast

ability to distinguish an object from its surrodnings

bright field microscopy

type of microscopy where light is transmitted THROUGH specimen, producing an image

→ increases in mangfication and morphology requires staining

→ used to exam cell morphology

Positive(Basic) Stain

type of simple stained used in bright field microscopy

uses a single dye to stain cells

background is left unstained

used for determinig cell size, morphology, number present

ex: Methylene Blue

Negative(Acidic) Stain

simple stain used in bright field micrscopy

→ single dye to stain BACKGROUND

→ cells repel dye (H+) and are left unstained

→ silluthote apperance

differential stains

type of stain used in bright field microscopy

two dyes (primary and counterstain)

used to determine Gram -/ Gram + bacteria

pink

stain color of gram - bacteria

purple

stain color of gram + bacteria

Dark field microscopy

type of mircoscopy

bright speciment is seen within a dark background, increases contrast of transprent organisms

determines bacterial and protozoan mobility as well as microcolony grwoth

used to visuzlie alive, unfixed cells

Phase control microscopy

type of microscopy

internal cell components are distinguished from surrodning background

differing densities of cell compenets interacted dirrectly with light → changing how they are going to be viewed on the scope

increases contrast

wet mount

suspended cells are placed between a glass slide and cover slip

cells are living and locomotion can be observed

Smears

preperation for microscopy

tecnqhie better for determing cell morphology and cellualr compoents using staining

cells are air dried, heat fixed and stained→cells are killed

Buried slide tehcnqiue

soil samples for micrscopy

galss slide is inserted into sample, slide is incubated in the sample for a set time

slide is removed with least disturbance possible and observed under micrscope

Pedoscope technque

soil sample for microscopy

varation of th buired slide techniqe that contains opitcally flate capillary tubes

fluoresecent microscopy

type of light micrscopy that uses UV and a combo of fluroscent dyhes

=> Direct Count: provides number of total cells present

→ estimating bio mass

fluorescent immunolabeling

type of labeling where microbes are detected through fluorescently labled antibodies

observed by fluorescent microscopy

Flouresent in stiu hybridization

type of labeling where fluroresecently labeled nucleic acids target species DNA/ RNA sequences

universal probe

observed by fluoresent microscopy

Flow Cytometry

microscopic detection of cells perfomed as they pass a laser detector

measures flursen light emisison of cells

separate target cells/ particles from others

Scanning electron microscopy

type of electron microscopy where electrons itneract with speciment surfaces

used to produce 3d images of surface characteristcs

Transmission electron microscopy

type of electron microscope where electrons pass through speciment

used for viewing fine internal cell stucture

electron microscopy

type of microcopy that utllzies electrons isntead of ligh to form images

electron beam is focused on the specimen with coiled electromagnets in a vaccum

50%

amount of atmospheric oxygen provided by aquatic microbes

1 meter

length light can penerate in areas with high particulates

photic zone

where light is able to peneratre

aphotic zone

where light does not reach

Neustone zone

zone of thin, gel like amtrix of biomolecules at the air water interface of sea surface

• high UV radiation, nutirents and chmiecals

Pelagic zone

water column./ planktonic habitat subdidied by depth

epipelagic, meso pelagic, bathypelagic, abyssopelagic

Oceans Pealgic zones

profundal

lake pelagic zone with < 1% light peneration

limmetic

lake pelagic zone that recieives >= 1 light peneration

benthos zone

sediment habitat underlying the water column

epibiotic

microroganism attached to the surface of other organisms

→ Episilon proteobacteria and euglenozoans

Endobiotic

micrograonsim living with another organism’s tissues

ex: Virbri fischeri and Hawiian Bobtail squid

Bacterioplankton

arcahe/bacteria in pelagic zone

phototrophs, chemotrophs, and heterotophs

phytoplankton

photoautrophic microbes and eukayotes

(cyanobacteria and eurkaryotes)

Zooplankton

larger, hetertrophic microbes

ex: protozoan, copepods

Particulcate Organic Matter

large macromolecules (ex: polymers) that form cell structural components

prodicued by plankton

dissolved organic matter

smaller, soluble, matter that a rapidly taken up and metabolized by microbes(amino acids, carbohydrates)

→ passes thorugh filter w/ pore size of .7micro m

benthos

microbes readily grow in this oceanic zone due to nutrients and carbon sources becoming more plentiful from organic matter settlement or deposiiton from terrestial sources

Anaerobic methane oxidation

fermentation of carbon → organic acid and CO2→ methane (Ch4)→ Co2

ammonia oxidation

ammonia produced by decomposer → oxidized as energy source

Nitrifciation

ammonia → nitrite → nitrate

Biofilms

interfacial habit of a strong association of microbes with the surface through the prodction a polymer matrix

usally aqutic

role in water purification (removes DOM)

Reversible attachment

stage in biofilm formation

transitory physiochemical attraction from hydrophobic and electrostatic interactions

irreversible attachment

stage of bio film attached of biologically mediated stabilizaion reaction

→ attached bacteria sevrete extracellular polymers that forms strong chemical bridge with surface and allows addition of more cells

void spaces

part ot the biofilm structure that transports nutrients and icnrease biofilm surface area

Extracellular polymer matrix

part of the biofilm structure that surrounds the cell

filters and collects essential nutrients

protection aganist enviromental stressors

barrier for antibiotic/disinfectant treaments

Microbial Mats

specialised type of biofilm that is arranged in colored stratified layers

found in extreme enviroments

self-sufficient due to use of many major biogeochemical cycles”

cycling of aerobic and anaerobic conditions partly by oxygenic photosynthesis and anaerobic sulfate reduction

inorganic nutrient availability

water temperature

turbidity of water

level of verticla mixing

enviromental factors that affect primary producers

high

coastal region production levels

POM/DOM from river outflows and upwelling

low

ocean production levels

due to lack of inorganic nutrients

DIssolved Organic matter

plantkon excretion, organic matter released during feeding and viral lysis of plankton

used in bacterioplankton biomass production (secodnary production) and the microbial loop

microbial loop

bacterioplankton remineralize DOM→ CO2 and nutrients → more primary production

Karenia brevis

dinoflagellate that produces toxic red tides in the gulf of mexico

viruses

most abundant biological entities in the ocean

limnology

study of freshwaer habitats

springs

running water that forms when subterranean water reaches surface

dominated by photosynthesis bacteria and algae

initially low heterotrophic activity, can increase with DOM additions

Gammaproteobacteria and Deltaproteobacteria

most abudant microbial phyla in soft bottom sediment communities

decreases

in soft bottom communities, thenumber of microbial cells ____ with depth

chemolithoautotrophs

in hard bottom ocean crusts, these microbes are in abudance

Rivers

running water

primary producer communities: biofilms, benthic sediment communities. aerobic/facultatively aerobic microbes

increase DOM = increase heterotrophic activity

Lakes

bodies of stagnant water

dominant microbial group: filamentous and epiphytic algae, phytoplankton dominate central area of lake

Oligotrophic lake

type of lake environment where high rates of primary production occur due to deeper light penetration

secondary production linked to amount of primary production

Eutrophic lake

type of lake where primary production is low; higher rates of secondary production

Zoophagus insidians

type of lake fungi that form fishing lines to catch rotifers

brackish waters

area in which salinity is between fresh and sal water

low primary production (high turbidity); higher heterotrophic and secdonary production