Lab Final Study Guide

Bacterial Enumeration
● Binary fission = most common bacteria method of reproduction
○ One divides into two new ones
○ Generation time of bacteria = time from forming of cell to division
■ Some as quick as every 15-20 min
○ Colony doubles every generation (under ideal conditions)
● Standard growth curve
○ Describes the phases of bacterial growth in the lab (important to note that
bacteria don’t behave this way in nature, only lab)
● Lag phase = no cell division nor cell death
○ Bacteria must acclimate first (lag)
● Log phase = bacteria have acclimated and begin dividing exponentially
○ Population doubles every generation
○ Ideal for experiments
● Stationary phase = resources start to run out
○ Death rate is equal to generation
■ Total number remains constant
○ Waste products start to accumulate
● Death phase = waste accumulates and nutrients are almost all gone
○ Death rate surpasses generation rate
○ \When can endospores be studied?
○ In older cultures because sporulation occurs during late stationary phase
● Serial dilution
○ Method of obtaining viable count
● Dilution factor = …
○ Volume added to test tube / new total volume of test tube
● Spread plating
○ Spreading out sample with disposable spreader
● Calculating CFU/mL
○ CFU/mL = # of colonies on plate / [(final dilution factor of tube) * (volume plated
in mL)]
● Required number of colonies
○ each plate must range from 30-300
○ Any more than 300 = TNTC (too numerous to count)
○ E.g.
■ 234 cfu / (10^-3)x(10^-1) mL = 234 x 10^4 = 2.34 x 10^6 cfu/mL
~Study Q’s~
★

SIM, Nutrient Gelatin, Starch agar

● SIM
 ○ Differential medium used to detect sulfide production, indole production, and
motility
○ Differential ingredients: sodium thiosulfate and ferrous ammonium sulfate (S),
casien (I), and 3.5% agar (M)
● Sulfide Production
○ Sodium thiosulfate is reduced by some organism to H2S (hydrogen sulfide),
forms ferrous sulfide which is insoluble in water and appears as a black
precipitate in medium
○ If a black precipitate is present in the medium then the organism produces H2S.
No black = no production
● Indole production
○ SIM contains casein which is rich in tryptophan, can be broken down into indole
by some organisms
■ Will turn red if indole present
○ Kovac’s reagent turns Red = produces indole; not red = does not produce indole
● Motility
○ 3.5% agar added to sim immobilized anything that cannot move, cannot self
propel, is not motile
○ Limited to facultative anaerobes and other organisms that can grow anaerobically
■ Obligate aerobes don't grow well in stabs
○ Haze outside of stab = motile
■ Growth contained to stab = nonmotile
● Nutrient Gelatin
○ Exoenzymes are secreted by bacteria into their environment. They can function
as toxins or provide nutrients to cell
○ Differential medium used to determine if an organism produces gelatinase
■ It contains gelatin, peptone, and beef extract
○ If an organism produces gelatinase, the exoenzymes will break it down and it will
liquify
● Gelatinase
○ Type of protease
■ Protease = group of enzymes that break down proteins
■ Catalyzes the hydrolysis of gelatin into its component amino acids
■ Used widely by bacteria to breakdown proteins for nutrient acquisition
● Or viruses can use this to break down connective tissue as apart
of their invasion process
● Starch agar
○ Amylase breaks down starch (amylose) into maltose and glucose
■ Used in brewing industry, used to make corn syrup
○ Differential medium (soluble starch) used to detect ability to hydrolyze starch
○ After incubation plate is covered with iodine to detect zone clearing
■ Zone clearing = hydrolyzes starch (procuses exoenzyme amylase); no
zone clearing (no amylase) = no hydrolyze
~Study Q’s~
 ★

Phenol Red Carbohydrate

● Fermentation
○ Process that allows to continue to oxidize NADH back into NAD+ and produce
ATPs in the absence of oxygen
○ Organic compounds used as final electron acceptor
● Homolactic vs Heterolactic
○ In homolactic fermentation, the only end product is lactic acid
○ In heterolactic, the end product includes lactic acid, ethanol, and carbon dioxide
● Phenol red broth
○ Used to determine ability to ferment particular sugar, and if so, whether acids or
gasses are produced also
● Ingredients
○ Sugar, such as sucrose, lactose, glucose, or mannitol
○ And Phenol Red
■ Red at neutral PH and yellow at 6.8 or below
● Durham tube
○ Captures gasses that are produced during fermentation
● Yellow colored phenol and durham bubble?
○ Can ferment the sugar and produces acid and gas
○ No bubble still yellow? Ferment with acid and no gas
● Only gives us info on/Doesnt provide any info on
○ Only can conclude whether acid or gas was produced
○ Can’t conclude type of gas
● Faint orange color
○ b/c some obligate aerobes can convert sugars into acid but w/o fermentative
pathway
~Study Q’s~
★

Bacterial Transformation
● Transforming principle and molecule of heredity
○ DNA
● Transformation
○ Uptake of naked DNA from environment
● Competence
○ Describes relative ability of bacterial cell to be transformed
○ Fully competent cells have ability to take in DNA from environment
● Artificial competence
○ Bacteria in active (log) stage of growth
○ In CaCl2 solution (calcium chloride)
○ And subject to heat shock
● Heat shock
 ○ Forms pores in cell wall providing path for plasmid to enter

~Study Q’s~
★

Microbial Control
● Chemotherapeutic Agents
○ Includes topical/oral antimicrobials and antibiotics
■ Antimicrobial = compound that negatively affects growth of microbes
● Depending on target organism, antimicrobials can be antibacterial,
antifungal, antiviral, antihelminthic etc
● Aminoglycoside
○ Includes antibacterials streptomyces, kanamycin, neomycin,
○ Mode of action = bind to bacterial ribosome and prevent protein synth
■ Neomycin is in neosporin
● Bacitracin
○ Antibacterial polypeptide produced by strains of Bacillus subtilis
○ Bacitracin works by preventing cell wall synth
○ also neosporin ingredient
● Beta-Lactams
○ Antibacterial class that includes penicillin and amoxicillin
○ Prevents formation of peptidoglycan ,olecule in the bacterial cell wall
● Chloramphenicol
○ Disrupts protein synth by binding to large ribosomal subunit
● Fluoroquinolones
○ This class of antibacterials works by binding to enzyme DNA Gyrase
■ Prevents replication and killing bacterial cells
○ Ciprofloxacin is commonly used fluoroquinolone
● Macroslides
○ Disrupt protien synth by binding to ribosome
○ Includes Erythromycin
● Tetracyclines
○ Bind to ribosome
● Sulfonamides/Trimethoprim
○ Act as competitive inhibitor of enzymes used in biosythesis of folate by bacteria
■ Often used in combination therapy as each targets different metabolic
enzyme
● Kirby-Bauer Disk Diffusion Assay
○ Used to test susceptibility of a given organism to particular drug
○ Measure diameter of zone clearing around antibiotic
● UV light
○ Forms thymine dimers in DNA molecules
○ If not repaired, they block DNA replication and transcription, aka kill bacteria
○ Does Not penetrate into anything but clear surfaces
~Study Q’s~
★