Prokayrotic Lab Quiz 5

resident microorganisms

microorganisms that we carry on us all the time

several layers of skin, cilia, mucous membranes, sneeze/cough

list the 4 mechanical defenses

salt, acidic compounds, enzymes

list the 3 chemical defenses

microorganisms in our skin and intestines create environments that prevent harmful organisms from growing, quorum sensing

microbial defenses

cutaneous infections

infections that are usually restricted to the site of entry and are usually more irritating than harmful

parenteral inoculation

if epidermis and dermal layers of the skin are actively broken, the mo’s have access to deeper tissues and could be distributed to other body parts in the bloodstream

epidermis-outer epithelial, dermis-inner sensitive vascular layer

two parts of the skin

hair follicles, sebaceous glands, sebum, sweat glands

4 things embedded in dermis

stratum corneum

out layer of epidermis, outer layer rich in water insoluble protein-keratin

moist areas such as the armpit

where do you most likely find skin organisms?

staphylocci and proprionobaceria

What species are normally found on skin?

staphylococci-gram(+), non motile, non spore forming, facultative aneraobe

propionobacteria-gram(+) rods of irregular shape

What are characteristics of live species on skin?

mechanical mechanism: impenetrable stratum corneum and rapid shedding of dead epithelial cells

chemical factor: less role, make pH of 3-5, lack of moisture and chemicals that inhibit growth

two most important factors for preventing excessive microbial skin growth

microcolonies

name for resident microorganisms that inhabit the skin of 1000 to 10,000 cells

Transient microorganisms

where we pick up organisms from out environment like e. coli and Bacillus fragillus from fecal contamination

-resident microorganism compete more effectively for nutrients

-poor adapted to our skin surface

why do transient organisms often never survive long in our system

corticosteroids

drug given to decrease skins rate of epithelial shedding, gives pathogens longer time to become established(but if administered on already present infection, infection becomes worse)

• low ph-inhibits only bacteria

• Yeast-moist, shiny, elevated surfaces

• mold-fuzzy powdery extensive growth

Sabouraud Agar is good for growing yeast and mold

pathogenic mo’s find their way to sterile parts of UT system and proliferate

how do UTI occur

bacteria, fungi, protozoa, and viruses

UTI’s can be caused by mo’s that include:

urethritis

inflammation of urethra

cystisis

inflammation of the bladder

urethrocytosis

inflammation of both the urethra and the bladder

prostatitis

inflammation of the prostate gland

pyelonephritis-kidneys inflammation

glomerulonephritis-inflammation of the glomeruli of the kidneys

two problems that start in the lower urethra and ascend upwards

-midstream urine specimen must be collected

-must be cultured immediately due normally occurring lower urethra bacteria outgrowing possible pathogens→false negative results

midstream urine process

1000→normal

100,000→significant urinary infection

10³- 10^5→marginal(may or may not)

urine mo’s: 1,000→

urine mo’s: 100,000→

urine mo’s:10³- 10^5→

First-Void or First-Pass Sample Catch

Designed to primarily test for STIs since it focuses on microbes on external genitalia or lower urethra

Mid-stream Catch

More commonly used for UTI diagnosis and designed to give an appropriate snapshot into the microbiome of an individual

dark metallic growth on EMB→strong lactose fermentation-E.coli present(gram(-) lactose fermenting bacterium consistent with a UTI

Halo of clearing around a colony on blood agar→bacteria completely lyses RBC’s→Beta hemolysis

What does dark metallic growth on an EMB plate mean about a urine sample? What about a halo of clearing around a colony on a blood agar plate?

if positive→staphylococcus sp.

if negative→streptococcus sp.

purpose of catalase test in urine screening

the metabolism and growth of several types of microorganisms rise and fall in succession before the final product(sauerkrat) is produced

What is microbial succession and how does it work?

lactic acid bacteria

Cabbage fermentation results from the metabolism of __________ that exist naturally on healthy cabbage plants

controlled natural fermentation

unlike yogurt, sauerkraut results from a ___________ means that human create an environment that favors growth and metabolism of lactic acid bacteria only

1.)adding sodium chloride to leaves

2.)creating a anaerobic environment

what 2 ways is sauerkraut fermentation accomplished

pulls nutrient fluid(brine) from leaves by osmosis that suppresses growth of undesirable microorganism’s and favors lactic acid growth

in sauerfraut fermentation salt(NaCl) does what?

-Enterobacter cloacae and Erwinia herbicola, both faculatative anaerobic, gram neg, rods

-Salt tolerant microbes respire and use up dissolved oxygen→makes system anaerobic

-they ferment the plant sugar and produce acid-lowering pH

early stage sauerkraut fermentation, bacteria and function

-Leuconostic mesenteroides, gram postive, coccus shaped, strictly ferment, salt and acid tolerant, lactic acid bacteria, all heterolactic fermentors

-produce equal lactic acid, CO2, ethanol using PPK pathway, unline regular Emben-Meyerhoff Parnas pathway

intermediate stage sauerkraut fermentation, bacteria and function

-Lactobacillus species, gram postive, rods, strictly fermentative, salt tolerant, lactic acid bacteria

-homolactic sugar fermentation

-oxidizes remaining sugar through Embden-Meyerhoff-Parnas

final stage sauerkraut fermentation, bacteria and function

pH decreases and makes a more sour acidic taste

What are the general trends in terms of pH and flavor as the cabbage ferments?(sauerkraut fermentation)

process of exogenous genetic matieral introduced and taken up by bacterial cell

What is bacterial transformation and how does it work?

• Resist ampicillin because of bla gene

• Produce GFP(green flurescent protein) when GFP gene is turned on by arabinose

the pGLO plasmid transformed bacteria to do what?

small circular DNA pieces that exists separately from bacterial chromosomes

What are plasmids?

1.)add transformation solution containing CaCl2

2.)Ice cells

3.)Add the pGLO plasmid

4.)incubate on ice

5.)Heat shock

6.)Add LB broth and allow recovery

7.)Plate bacteria

order of transformation steps

calcium ions reduce the negative repulsion between the bacterial cell surface and negatively charged plasmid

-helped plasmid associate with bacterial cell and make cells competent

(pGLO transformation)

purpose of adding CaCl2 reagent

cold treatment help stabilize cell membrane and helped plasmid associate before heat shock

(pGLO transformation) purpose of keeping cells on ice

rapid temp change helps promote uptake of plasmid DNA by competent bacteria

(pGLO transformation) purpose of heat shock

helped bacteria recovered from just being heat shocked so they can recover and can express the ampicillin resistance gene

(pGLO transformation)purpose of LB broth being added

ability of the bacteria to take up DNA from the environment

-CaCl2 treatment+cold incubation followed by heat shock

What is competency? How did we make bacteria competent in this exercise?

select transformed bacteria by using antibiotic resistance gene carried by plasmids

-if they have bla gene→ampicillin resistance

How can you select for transformed bacteria?

encodes B-lactamase-gives bacteria ampicillin resistance

(pGLO transformation) bla function

encodes green fluoroscent protein→produces green fluorescence under UV light

(pGLO transformation) GFP function

encodes AraC regulator protein→regulates expression from P-BAD promoter in response to arabinose

(pGLO transformation)araC function

arabinose-regulated promoter→controls arabinose operon(transcription of GFP gene)

(pGLO transformation)P_BAD function

microbial quality

The number of microorganisms indicates the foods general __________

Food Infection

microorganism is ingested and colonizes our digestive trac

Food Intoxication

microorganisms is ingested and produces toxins that cause illness usually within 6-12 hours after

ingestion

Campylobacter jejuni

found in Raw poultry, unpasteurized milk, sometimes water

Causes spontaneous abortion. Most common organism cause of food infection

Salmonella

in raw poultry, eggs

Second most common cause of food infection

Listeria monocytogenes

found in unpasteurized milk, fresh soft cheese

Causes swelling of the brain, meningitis of newborn, encephalitis, spontaneous abortion in

second/third trimester, still birth

Vibrio cholerae

Found in water, raw oysters

Causes enteritis called vibriosis

Escherichia coli

in raw meats and on raw vegetables

Strain 0157 produces verocytotoxin, causes damage to intestinal lining, kidney

hemorrhage

Staphylococcus aureus

on the skin in infected cuts, transferred to food via hand and nose droplets

Causes food intoxication via enterotoxin

Bacillus cereus

in soil and dust, often found in rice dishes – spores.

Causes food intoxication via enterotoxin

Clostridium perfingens

produces spores and causes food intoxication via enterotoxin

Clostridium botulinum

produces neurotoxin, can cause respiratory paralysis, double vision

1.)Clean food, Clean your hands, Store properly

2.)Washing-removes most of soil borne microbes, Blaching-denatures enzymes that cause post harvest tissue breakdown,quick freezing-prevents microbial growth after blanching

What can you do to minimize food bacterial contamination

enrichment-culture technique/enrichment selection

makes use of conditions designed to enable a particular type of mo to grow faster than other in a sample(opposite of selective media)

Chemical strategies

(Enrichment)techniques such as the use of specific carbon sources(like cellulose)to enrich for organisms with specific degradative abilities, or the use of an acidic pH to enrich for fungi

Physical strategies

(Enrichment)methods like incubation at high temperature to enrich for thermophilic organisms or incubation in the absence of oxygen to enrich for anaerobes

Biological strategy

(Enrichment)the addition of live host cells to enrich for specific type of bacterial virus

-play a role in recycling sulfur in our environment

-anaerobic, chemoheterotrophs

-oxidize compounds and pass high energy electrons to ETC, sulfur is final electron acceptor

-usually gram negative

Characteristics of sulfate-reducing bacteria

Van Delden’s medium

effective enrichment medium for sulfate reducing bacteria

1.)Anaerobic conditions

2.)presence of carbon and nitrogen

3.)toxic effects of H2S that is produced by sulfate reducers

3 selective factors in Van Delden’s medium

-photosynthetic bacteria

-chemoautotrophs

-use CO2 as a carbon source

-can grow in absence of organic carbon compounds

characteristics of algae and cyanobacteria

-fix carbon and nitrogen from atmosphere

-heterocysts

-vegetative cells

cyanobacteria characteristics only

Bristol’s medium

-enrich algae and cyanobacteria that live in soils and other natural environments

Bristols

Chemoheterotrophs don’t grow on this medium because it lack reduced source of organic carbon

photosynthetic chemoautotrophs

incubating under bright light gives __________________ an advantage due to unlimited energy source

atmospheric Nitogen(N2)

cyanobacteria use only _______________ as a sole nitrogen source for synthesis and growth

Chu’s medium

medium that lacks fixed nitrogen so the cells must fix atmospheric nitrogen to grow