Microbio Lab Exam 2

what is a plate count used for

estimating the number of microbes in a sample and the risk of infection from that sample

what is the procedure of a plate count

1. a sample is diluted through a series of bottles or tubes

2. a set volume/aliquot of the dilutions is then put onto plates

3. the number of colonies on the plates is counted after incubation for 24 hours or more

in a plate count, what number of colonies is considered to be valid

30 to 300, fewer is labeled TFTC (too few to count), any more are labeled TNTC (too numerous to count)

how can you calculate the number of bacteria in an original sample in a plate count

by multiplying the number of colonies on the plate by the dilution factor

what were the dilution factors in our plate count

1st tube = 10^-1

2nd tube = 10^-2

3rd tube = 10^-3

what is a colony forming unit (CFU)

what counts on a surface plate count method are considered, as the assumption that each colony in a plate count arises from a single bacterial cell may not always be true

what is an advantage of the surface plate count method

under a microscope, living and dead cells look the same, but only living cells will grow on a plate (therefore, plate counts are a better indicator of disease risk)

what is the formula for calculation a dilution

sample/(volume of sample + volume of diluent)

what are serial dilutions and how do you calculate them

one dilution followed by another; you multiply all the dilutions used to get the final dilution of that sample

what is the purpose of doing a standard plate count

to find out the concentration of bacteria in the sample being tested

what is the formula for calculating colony forming units in the original sample

the number of colonies on the plate x 1/(final dilution)

at lower dilutions in a plate count…

you may see plates with more than 300 colonies (TNTC)

at higher dilutions in a plate count…

you may see plates with less than 30 colonies (TFTC)

what is osmosis

water movement across a semipermeable membrane regulated by the concentration of solutes in a cell

what is a solute

a compound that dissolves in a solution (ex: proteins, carbohydrates, salts/ions)

what is a hypertonic solution

the concentration of solutes outside the cell is higher than inside the cell (water moves out of the cell, causing the cytoplasm to lose volume and the plasma membrane to shrink)

what is plasmolysis

excessive water loss occurring in a hypertonic solution that causes a cell’s plasma membrane to pull away from the cell wall (makes it difficult for a bacterial cell to carry out essential functions required for growth)

what is a hypotonic solution

the concentration of solutes outside the cell is lower than inside the cell (water moves across the cytoplasmic membrane to the inside of the cell until equilibrium is achieved, will have increased internal pressure)

what is turgor pressure

pressure created by the influx of water into a bacterial cell, required to sustain life (bacteria store/import ions in order to maintain turgor pressor)

what are halophiles

bacteria that can grow at higher salt concentrations due to their ability to maintain high enough cytoplasmic concentrations of solutes, prevent excessive loss of water

what are facultative halophiles

bacteria that grow in both low-salt and high-salt environments

what is an obligate halophile

a bacterium that can grow only in the presence of high salts

what is an isotonic solution

water concentration is equal inside and outside the cell (water moves in and out of the cell at the same rate)

what are the two forms of radiation used in control of microbial growth

gamma radiation and UV radiation

what is gamma radiation

ionizing radiation that collides with molecules in a cell, creating toxic free radicals

what is UV radiation

nonionizing radiation that primarily kills microbes by damaging DNA and RNA by affecting bonding relationships within the molecules (ex: formation of thymine or cytosine dimers)

what are thymine/cytosine dimers

bonds between two adjacent pyrimidines in a DNA strand that can lead to mutations

what type of UV light is the most lethal

UV light with a wavelength between 200 and 290 nanometers (nm), these are the wavelengths absorbed the most by DNA

describe exposure to UV-C light

UV-C light is radiation with a wavelength between 100-280 nm and it causes more damage than cell systems can repair, leading to death of the cell

what are limitations of UV light as a disinfecting/sterilizing method

poor penetrating ability: UV light will not go through plastic, glass, and many other materials, so it is mainly used for killing microbes on surfaces, in air purifiers, or in drinking water

what does antimicrobial mean

general term for something that kills/inhibits microorganisms

what is the purpose of antimicrobial susceptibility testing

to find out whether the bacteria being tested are sensitive (killed by an antibiotic) or resistant to it (the drug will be ineffective for treating the patient) - helps medical providers avoid treating patients with an antibiotic that has only intermediate effectiveness or to which the bacteria are resistant

what is the Kirby-Bauer method of testing bacteria sensitivity to antimicrobials

1. cover the surface of a plate with the bacteria being tested

2. paper discs containing different antimicrobials are placed on the surface of the plate

3. after incubation, the plates are examined for sones of inhibition and the zones are measured

4. the measurements are compared to standards to determine if the bacteria are sensitive, intermediate, or resistant to an antibiotic

what is a zone of inhibition

if an antimicrobial inhibits the growth of the bacteria, a clear, circular area appears are around the disc 

what type of agar is used in the Kirby-Baur test for antimicrobial susceptibility

mueller-hinton agar

what does no zone of inhibition mean

bacteria in sample is resistant to that specific antibiotic

what does a large zone of inhibition mean

bacteria in sample are sensitive to that specific antibiotic

what is binary fission

the generation of offspring in bacteria that produce organisms genetically identical to the parent cell (unless there is a mutation)

how do bacteria generate genetic diversity

horizontal gene transfer: conjugation, transformation, and transduction

what is the significance of horizontal gene transfer

individuals in a population can easily spread and acquire new traits, as well as bacteria populations can become antibiotic resistant

what is conjugation

transfer of bacteria DNA (a plasmid or a chromosome) from a donor cell to a recipient cell via direct contact through a sex pilus

what is transduction

a process by which bacterial viruses (bacteriophages) transfer DNA from one bacterium to another

what is transformation

bacterial cells take up DNA from the surrounding environment that is then transported through the bacterial cytoplasmic membrane into the cytoplasm

what does cell competency mean

a cell is able to take in genetic material across their membrane (required for transformation)

what are plasmids

small, circular, self-replicating DNA molecules that continue to replicate once transferred into a new bacterium (can also carry antibiotic-resistance genes

what is nitrate

an inorganic form of nitrogen that can be used as a nutrient of as a terminal electron acceptor in the respiratory chain

what are nitrogen-fixing bacteria

bacteria that convert nitrogen gas into a combined or organic form of nitrogen

what are nitrifying bacteria

bacteria that convert ammonium into nitrite and then to nitrate

what are denitrifying bacteria

bacteria that reduce nitrate to nitrogen gas or to a nitrous oxide under anaerobic conditions (this decreases the amount of nitrogen available for plants and decreases plant productivity)

what is nitrate reductase

enzyme that convert nitrite to nitrous oxide or nitrogen gas

what is nitrate reduction

the conversion of nitrate to nitrite, allowing the nitrite to be further metabolized into various other nitrogenous forms

what are the results of nitrate reduction

broth turns red with the addition of reagents A and B: nitrite is present and nitrate reduction occurred (positive reaction)

broth does not turn red/remains colorless with the addition of reagents A and B: nitrate was not reduced OR all nitrate has been converted to nitrogen gas/nitrous oxide (add zinc and HCl to differentiate between these two possibilities)

broth turns red after the addition of zinc and HCl: nitrate was not reduced by bacteria and is sill in tube (negative reaction)

broth turns clear after the addition of zinc and HCl: nitrate reduction occurred and the final product was nitrogen gas or a nitrous oxide (positive reaction)

what does zinc do in nitrate reduction

zinc catalyzes the conversion of nitrate to nitrite

what is an extracellular enzyme

enzyme produced within the cell and secreted outside of the cell wall, once secreted they diffuse into the environment and act upon their specific substrate

what is DNase

an extracellular enzyme that breaks down/digests macromolecular DNA to its nucleotide subunits

why do bacteria produce DNase

nucleotides are the building blocks of nucleic acids and can serve as nutrients for a bacteria; production of DNase has also been linked to an organism’s ability to degrade biofilms, affecting an organism’s ability to invade a host or spread from a site of origin

what is DNase agar made of

powdered DNA mixed into nutrient agar

what is the procedure of a DNase test

1. a DNase plate is inoculated and incubated

2. HCl is poured onto the surface of plate

what is HCl and what is its purpose in a DNase test

hydrochloric acid is a potent denaturant; this reagent denatures the structure of DNA on a DNase test plate and allows the visibility of DNA remaining in the agar (turns a white/opaque color)

what are the results of a DNase test

positive test: a clear zone around the colony after the addition of HCl (DNase has been secreted and the DNA in the agar surrounding the colony has been degraded)

negative test: whitening of the agar surrounding a colony (no DNase has been produced so DNA is still present in the medium)

what is gelatin

a protein derived from collagen that when chilled forms cross-links to create a semi-solid gel; provides a rich source of amino acids and peptides to bacteria but is too large to be transported into a cell whole

what is gelatinase

extracellular protease that hydrolyzes bonds in gelatin to produce smaller peptides and amino acids that diffuse easier into a cell

what is the purpose of a nutritional gelatin medium

used to detect the production of an extracellular gelatinase

what is nutritional gelatin medium made of

nutrient broth and gelatin, no agar (at room temp, can liquefy to slightly viscous; when refrigerated, the gelatin molecules cross-link and form a semisolid gel)

what are the results of gelatin hydrolysis

tube forms a semisolid gel after refrigeration: organism is negative for gelatinase (tube gels as expected)

tube is liquid after refrigeration: organism is positive for gelatinase (enzyme was secreted into medium and hydrolyzed the gelatin)

how does gelatinase play a role in bacterial pathogenicity

the ability to break down connective tissue increases the ability of the pathogen to spread through the body and cause tissue damage

what is starch

an abundant, naturally occurring plant polysaccharide composed of amylose and amylopectin that is a rich source of carbon and simple sugars

what is amylase

an extracellular enzyme that catalyzes the hydrolysis of amylose and some amylopectin (aka starch) through the breaking of glycosidic bonds to produce maltose, glucose, and short-chain polysaccharides

what is starch agar made of

soluble starch added to a general growth medium such as nutrient agar

what reagent is used to identify the presence/absence of starch in starch agar

iodine

what are the results of starch hydrolysis

positive test: absence of a blue/blue-black color around the colony after the addition of iodine (no starch present, bacteria produces amylase)

negative test: agar around colony turns blue/blue-black after the addition of iodine (starch is present, bacteria does not produce amylase)

what is casein

large, globular protein that gives milk its white color (a good source of amino acids)

what are casein/milk agar plates made of

general nutrient agar plates to which evaporated skim milk is added

what is casease

an exoenzyme that hydrolyzes casein into amino acids and short peptides to be better imported into a cell

what are the results of casein hydrolysis

positive reaction: clearing around the colony (casease is produced and breaks down casein in agar)

negative reaction: agar around colony remains milky white color (casease is not produces and casein remains whole)

what is the function of carbohydrates

function in cells as structural and energy molecules: monosaccharides and disaccharides are used in cellular respiration to produce ATP

what is glycolysis

catabolic process of converting glucose to two (net) molecules of pyruvate and ATP

what is the function of phenol red broth

determines an organism’s ability to utilize a specific sugar

what does phenol red broth contain

pH indicator phenol red, the sugar of interest, salts, and a peptone, as well as a Durham tube that collects gas

what are the results of phenol red sugar broth

pH indicator changes from red to yellow: acid by-products are present (bacteria breaks down sugar)

pH remains red: alkaline reaction occurs (bacteria does not break down sugar, but instead the amino acids in the peptone - protein breakdown increases pH)

gas in Durham tube: fermentation of sugar occurs

what is reversion

phenol red reverts from yellow back to original red as the bacteria have exhausted the available carbohydrates and begin to break down the peptone in the medium, increasing pH

what is TSIA

triple-sugar-iron-agar is a differential medium used for the differentiation and identification of gram-negative enteric bacteria (tests an organism’s ability to ferment selected carbohydrates and to produce hydrogen sulfide)

what is in TSIA

3 sugars (glucose, sucrose, and lactose), a pH indicator (phenol red), 2 sources of sulfur (cysteine and thiosulfate), an iron salt, and a general nutrient base such as peptone or yeast extract

what is the purpose of the iron salt in TSIA

necessary to detect the production of hydrogen sulfide gas resulting from the utilization of sulfur sources

what is the method of inoculation in TSIA

stabbing inoculation needle into the bottom of the tube and then streaking down the surface of the slant (this method introduces bacteria deep in the tube, below the agar surface, limiting oxygen to encourage fermentation while also allowing aerobic respiration along the top of the slant)

what are the results of a TSIA

acid reaction (A/A): slant and butt of tube are yellow, organism can metabolize glucose and another sugar (lactose or sucrose)

partial alkaline reaction (A/K): slant portion of tube is red, butt is yellow, organism metabolizes only glucose

alkaline reaction (K/K): slant and butt of tube are red, organism does not metabolize sugars in agar, but instead peptone, increasing the pH

cracking/displacement of agar: organism produces gas

black iron sulfide is formed: production of hydrogen sulfide (hydrogen sulfide binds to the iron salt in the medium)

what is in litmus milk broth

sugar (lactose) and protein (casein) and a litmus pH indicator

what does litmus pH indicate

pink: acid reaction (lactose is broken down)

blue: alkaline reaction (casein is broken down)

what is reduction in a litmus test

medium turns white and remains fluid because oxygen has been removed

what is digestion in a litmus test

medium becomes less opaque or clear with a brownish tinge because protein is completely broken down and only small peptides and fragments are left

what are the results of a litmus milk test

broth turns pink: bacterium ferments lactose and acid is produced

broth turns blue: bacteria breaks down protein casein, an alkaline reaction

broth remains purple and turns white on the bottom: reduction occurs

broth is see-through (may turn a brownish color): protein was completely broken down, digestion has occurred

clot formation: coagulation of milk proteins in an acid environment

what does IMViC stand for

Indole, Methyl red, Voges-Proskauer, and citrate

what does SIM stand for

sulfide, indole, and motility

what does SIM test for

the production of tryptophanase, true motility of a bacterium, and the metabolism of sulfur compounds

what is in a SIM medium

semisolid agar containing iron salt and thiosulfate (for hydrogen sulfide production) and peptone (for tryptophanase production)

what is tryptophanase

an enzyme that hydrolyzes tryptophan to produce indole, pyruvic acid, and ammonia

what is added to a SIM medium to determine if a bacteria produces tryptophanase

Kovac’s reagent

how should you inoculate a SIM medium

stab with inoculating needle to the bottom of the tube and carefully retract the needle along the same streak line

what are the results of a SIM test

black precipitate: bacterium metabolizes sulfur compounds and produced hydrogen sulfide

cherry red color after addition of Kovac’s reagent: indole-positive, bacteria produce tryptophanase

pale yellow/no color change after addition of Kovac’s reagent: indole-negative, bacteria do no produce tryptophanase

bacteria growth in well-defined line along stab line: nonmotile bacteria

tube appears turbid with no visible stab line: motile bacteria

what does MRVP test for

carbohydrate metabolism, specifically pyruvate; differentiates between organisms that follow the mixed-acid and butylene glycol pathways (essentially if carbohydrate end products are acidic or neutral