Microbio Lab Exam 2

prevents the growth of fungi, targets eukaryotes by inhibiting protein synthesis (ribosome function)

to provide an approximation of the number of viable cells per milliliter or gram of sample

sample must be diluted in water, diluting 1 g of soil in 9 ml of water to a final volume of 10 ml creates a 1:10 dilution
serial dilutions done in 10-fold incriments (reduces the number of cells per volume to a cell density that is easier to count)

CFU/mL (or CFU) = [Number of Colonies/Volume Plated (mL)] × Dilution Factor
Ex. 50 colonies are observed on a plate where 0.1 mL of a 10^-3 dilution was plated:
CFU/ml = (50/0.1) x 1000 = 500,000 CFU/ml

between 30 and 300

spread individual bacteria on a plate to grow into distinct colonies

E. Coli (-) and Staph. Epi (+)
helps find the spectrum of activity of our antibiotic producer

Luria-Bertani (LB): rich medium designed primarily for the growth of fast-growing, non-fastidious organisms
Tryptic Soy Agar (TSA): general-purpose medium capable of supporting the growth of a broad range of organisms; avoids overly rapid growth or competition

Often antibiotic resistant, leading cause of HAIs
Enterococcus faecium
Staphylococcus aureus: Relative of S. Epidermidis
Klebsiella pneumoniae: Relative of E. Coli
Acinetobacter baumannii
Pseudomonas aeruginosa
Enterobacter species

Took ~20 distinct bacterial colonies from one of the dilution plates and put them on a square grid template of whichever agar plate your bacteria grew best on
Isolate bacterial colonies in order to find an antibiotic producer/ acts a library that you can go back to

Took bacteria from square grid master plate and made identical square grid on two different plates; one of each tester organism
Whichever bacteria had a zone of inhibition was an antibiotic producer, method of action determined by which tester organism it inhibited

Narrow: effective against a limited range of bacteria, gram (-) or (+)
Broad: effective against wide range of bacteria, gram (-) and (+)
If antibiotic producer only inhibits one of tester organisims, likely narrow; if antibiotic producer inhibits both of tester organisms, likely broad

to isolate individual bacterial colonies from a mixed population, ensures that each isolated colony originates from a single bacterial cell or a single type of microorganism; no contamination

Gram (+): inhibition of cell wall synthesis, disruption of cell membrane integrity
Gram (-): disruption of outer membrane, inhibition of nucleic acid synthesis
inhibition of protein synthesis and inhibition of metabolic pathways are effective against both

Mixed sample in water with organic solvent (ethyl acetate)
let the phases separate into an organic (nonpolar) layer and and aqueous (polar) layer
extracted organic layer containing antibiotic and evaporated to obtain isolated powdered form of antibiotic
Solvent: a substance that dissolves a solute (e.g. antibiotic) to form a solution, in the context of organic extraction, solvents are used to separate, extract, and purify compounds based on their chemical properties

The ethyl acetate has non-polar and polar characteristics which match the antibiotic we are trying to isolate; the water has polar characteristics which match the agar, proteins, carbs, ions, and nucleic acids
purpose is to islate the purified version of the antibiotic in the ethyl acetate phase and separate it from the other parts of the cell

placed 10 ul of purified antibiotic in methanol in one spot on agar plate 3 times, also did the same with methanol as a control
once dried took 100 ul of tester bacteria and spread it around the plate, covering it entirely
poured top agar once dried
incubated and later assessed for zones of inhibition

To find new bacteria that can produce antibiotics to overcome increasing antibiotic resistance to known antibiotics

To adress the growing problem of antibiotic resistance by discovering new antibiotic producers through student research?