Lab Practical 2

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Terms in this set (...)

essential elements
what a microbial cell is composed of

includes carbon, oxygen, nitrogen, hydrogen, phosphorus, sodium, sulfur, potassium, chlorine, magnesium, calcium, and iron
macromolecules
elements that serve as inorganic cellular ions and as cofactors in enzymatic reactions

include proteins, carbohydrates, nucleic acids, and lipids
trace elements
elements that do not need to be specifically added to growth media, but are present in media components or in water

their presence is necessary for cellular growth

elements include zinc, cobalt, copper, and molybdenum, which act as cofactors in cellular enzymatic reactions
fastidious microbes
organisms that have special nutritional requirements that go above trace elements

can be difficult to grow in lab without enriched medium to add nutrients they need to grow
heterotrophs
organisms that utilize organic carbon as a carbon source
autotrophs
organisms that obtain carbon by incorporating carbon present in atmospheric carbon dioxide
chemotrophs
organisms that obtain their energy from the oxidation of organic chemicals
lithotrophs
organisms that obtain energy from the oxidation of inorganic chemicals
phototrophs
organisms that use radiant energy in the form of sunlight as their source of energy
chemoheterotrophs
organisms that use organic chemicals as a source of both carbon and energy
chemoautotrophs
organisms that obtain their energy from organic sources and obtain carbon from carbon dioxide
photoheterotrophs
organisms that utilize sunlight as an energy source and organic chemicals as a carbon source
photoautotrophs
organisms that obtain energy from sunlight and carbon from carbon dioxide
lithoautotrophs
organisms that obtain energy from inorganic compounds and carbon from carbon dioxide
lithoheterotrophs
organisms that utilize organic chemicals for carbon, while utilizing inorganic compounds as a source of energy
obligate anaerobes
cannot grow in the presence of oxygen
obligate aerobes
must have oxygen present or they will die
facultative anaerobes
may survive in either aerobic or anaerobic environments
selective media
used by researchers when they may want to grow only one particular group of microorganisms that is present in a mixed population of organisms

contain compounds that inhibit the growth of some organisms while allowing the growth of other organisms

ex: the dye crystal violet used in MAC, which inhibits the growth of most gm+ bacteria while the growth of certain gm- organisms are permitted
differential media
used by researchers when they want to differentiate between organisms growing in a culture

contain substances that are utilized differently by microorganisms and allow the differentiation between organisms based on their particular metabolic capabilities

ex: glucose phenol red broth- contains glucose that expresses an enzyme that ferments glucose from those that do not (pH will change color)
selective and differential media
select for specific organisms and differentiate between the organisms capable of growing under those selective conditions
undefined all-purpose growth medium
or complex medium
used when a microbiologist may want to grow all the organisms in a mixed population of microbes

do not contain selective agents and allow for the growth of most microbes under standard incubation conditions
tryptic soy agar (TSA)
complex medium which can be used for the isolation of most microbes

an excellent growth medium that contains digests of casein (milk protein) and soybean meal

allows for the growth of a wide variety of microorganisms from both clinical and nonclinical specimens
fermentation
series of enzymatic reactions that breakdown (oxidize) carbohydrates to produce ATP and bio-precursors for microbial metabolism

does not require oxygen

mechanism that bacteria use to generate 2 ATP and bio-precursors for microbial metabolism

glucose is the most common carbohydrate oxidized
glycolysis
most microorganisms rely on this pathway which results in 2 molecules of the end-product pyruvic acid and 2 ATP molecules

generates electrons that must be sequestered by NAD+ being regenerated by transferring these electrons from NADH to an organic final electron acceptor, pyruvic acid to form lactic acid
phenol red broth
contains a particular carbohydrate (glucose, lactose, sucrose or mannitol) combined with nutrient broth (containing peptone) and a pH indicator

neutral pH of 7.2-7.4 (pink/peach color)

if no fermentation-no change in pH nor color; deamination- has enzymes that can hydrolyze the peptone amino acids in the media to release ammonia raising pH to 8.2 and bright pink color; if ferment, acid lowers pH to 6.9 and turns yellow
deamination
non-fermenting organisms possess enzymes that can hydrolyze the peptone amino acids in a media, releasing ammonia, causing a rise in pH to above 8.2 and color change to a bright pink
reversion
if the organism expresses the enzyme for catabolizing peptones (found in proteins), it can continue to produce ATP and will liberate ammonia, causing a rise in pH and the phenol red indicator turns bright pink

this reaction is quite common in bacterial metabolism
Durham tube
small glass tube that phenol red broth is prepared in that is inverted in the bottom

collect gases produced during fermentation
aerobic cellular respiration
oxygen serves as the final electron acceptor

the enzyme cytochrome c oxidase, the last component of the electron transport chain, facilitates the transfer of low-energy electrons to oxygen; these electrons have negative charge and oxygen binds with two positively charged protons to form water
anaerobic cellular respiration
an inorganic molecule, for example nitrate, nitrite or sulfate serves as an alternate final electron acceptor, rather than oxygen

less ATP generated, but ability to undergo respiration using alternate final electron acceptors allows microorganisms to produce more ATP than fermentation would provide and can survive in a greater diversity of environments that experience low oxygen concentrations
nitrogen cycle
begins when atmospheric nitrogen gas is fixed by plants and bacteria and is eventually converted into organic material

nitrogen in organic material (proteins and nucleic acids) will end up in soils and sediments where they are decomposed by bacteria into nitrate

nitrification- decomposition process, begins with ammonia that is sequentially oxidized into nitrite then into nitrate

nitrate is then converted by a series of reduction reactions, by many different types of bacteria, that produce intermediate nitrogen molecules, NO2-, N2O, and ultimately ends in the liberation of N2, thus completing the nitrogen cycle
nitrification
the nitrogen in organic material such as proteins and nucleic acids, will eventually end up in soils and sediments where they are decomposed by bacteria into nitrate (NO3-)

begins with ammonia, a reduced form of nitrogen, that is sequentially oxidized into nitrite then into nitrate
denitrification
series of reduction reaction in which nitrate (NO3-) is converted to N2

NO3- and the intermediate products are reduced (gain electrons) when bacteria use these molecules as alternate electron acceptors during anaerobic respiration and subsequent ATP production
nitrate reduction test
can provide microbiologists information to differentiate bacteria since there is much variation amongst bacteria and their ability to use different alternate electron acceptors
positive nitrate reduction test
red precipitate has formed in the tube

if the bacteria in the culture have reduced NO3- to NO2- the nitrate reagent A converts NO2- into a diazonium salt, which reacts with nitrate reagent B to form a red precipitate

indication of positive for nitrate reduction and anaerobic respiration

finished with test
inconclusive nitrate reduction test
no color change occurred in the tube because either the bacteria in the culture tube could not utilize nitrate as an alternate electron acceptor or bacteria in the culture tube completed denitrification, producing N2 gas which left the tubes via the holes in the cap

must add zinc now
negative nitrate reduction test after zinc added
red precipitate forms after addition of zinc

zinc catalyzes the reduction of any remaining NO3- to NO2-. If NO2- is formed after adding the zinc powder (because NO3- was not reduced), the newly formed NO2- will combine with nitrate reagent A that was added before and will form red precipitate

negative for nitrate reduction and anaerobic respiration because zinc catalyzed the reduction of nitrate, not the bacteria
positive nitrate reduction test after zinc added
no red precipitate forms after addition of zinc

zinc catalyzes the reduction of any remaining NO3- to NO2-; if after adding zinc powder, NO2- is not formed indicating that denitrification took place in the tube and all forms of nitrogen where converted to N2 and left the tube as gas

positive for nitrate reduction and denitrification and anaerobic respiration
motility test
allows the differentiation between two species

gives info about its ability to move through a watery medium and the respiratory status of the isolate- whether the tested bacteria are producing ATP by cellular respiration

its medium is a semi-soft agar that allows motile bacteria to move through the agar

bacteria that are inoculated into the tube will digest all nutrients along the line of inoculation. as the nutrient level drops, non-motile bacteria will remain in place, but the motile bacteria will move away from the stab line in search of nutrients

if a sharp delineated stab line is observed, bacteria have not moved away from original inoculation

if fuzzy ling is radiating out from the stab in agar, bacteria are motile

if growth spread on the top of media only, inconclusive motility and are unreliable- occurs when bacteria are obligate aerobic bacteria that must stay on the surface of the media to survive
flagella
long filamentous, whip-like appendages that consist of three parts; basal body, hook, and filament (protein flagellin)
basal body
part of the flagella

attaches to the cell wall and cell membrane
peritrichous flagella
bacteria cells that are completely covered by flagella
lophotrichous flagella
bacteria cells that have polar flagella that protrude from one end
amphitrichous flagella
bacteria cells with flagella that extend from both ends
monotrichous flagella
bacteria cells with only one flagellum that extends from one pole of the cell
endoflagella
bundles of fibrils that protrude from a pole and wrap around the cell

cell and fibrils encased by an outer sheath

move bacteria in a spiral motion
pili
long protein structures found on gram-negative bacteria

may be involved in either a jerking type of movement (twitching motility) or a smooth movement (gliding motility)

some involve adhesion of bacteria to surfaces and DNA transfer through conjugation
TTC
colorless molecule that competes with the electron transport chain for the electrons donated by the coenzyme NADH

competes with the electron transport chain for the electrons released when NADH is oxidized

steals the electrons used when cellular respiration makes ATP in the motility agar, forming a red-colored molecule

allows us to determine the types of metabolism used by bacteria- if bacteria is moving, the red-color will spread through the media and determines if the organisms is motile
catalase
converts H2O2 (peroxide) into H2O (water) and O2 (oxygen)

present in aerobic and facultative anaerobic bacteria and all cells of plants and animals

one of the most potent catalysts known and is capable of neutralizing over 40 million molecules of hydrogen peroxide per second

presence is detected by adding a drop of substrate H2O2 (peroxide) to bacterial cells and observing the mixture for bubbling which indicates the liberation of molecular oxygen as gas; if no bubble, organism is either an obligate anaerobe or breaks down hydrogen peroxide with peroxidase

euk organisms- sequester potentially harmful chemical reactions involving oxygen in membrane-bound organelles that contain this enzyme to detoxify these radicals

prok organelles- aerobic and facultative anaerobic bacteria can transport these toxic by-products of aerobic respiration to the periplasmic space where this enzyme is stored and detoxifies these super radicals- cytoplasmic membrane protects internal cell from these toxic substances; also secrete this enzyme into the external environment to protect organisms from destruction by human WBCs
peroxidase
converts H2O2 (peroxide) into H20 (water) and a harmless non-oxygen end product
halophilic
salt-loving
mannitol salt agar (MSA)
pink colored medium that selects for growth of halophilic bacteria

its selective quality can be used in medical micro to assist in identifying bacteria that are known to be pathogenic and salt tolerant

promotes only scant growth (does not grow past initial quadrant of streak pattern) of gm+ catalase positive Micrococcus and Kocuria species

inhibits growth of gm+ catalase negative bacteria in Streptococcaceae family except Enterococcus faecalis

contains mannitol- monosaccharide; differential ingredient that allows observation of whether the gm+ halophilic bacteria growing on the medium can or cannot ferment the carbohydrate

results in the production of acid that diffuses into the medium causing the phenol red pH indicator in medium to change to yellow
coagulase
most identifying trait produced by S. aureus

will clot plasma
fibrinogen
causes clot formation when chemically converted into fibrin

soluble protein found in blood and body tissues
fibrin
stringy, sticky protein that when synthesized, precipitates out of solution

in blood clots, it forms a sticky mesh that catches platelets and forms a clot
Staphylococcus aureus
produces the coagulase enzyme which can also catalyze the conversion of fibrinogen to fibrin and bounds to the cell membrane and secreted unbound from the cell

part of the normal human microbiota found on the mucous membranes of nasal cavity, intestinal tract and urinary tract and skin

most common pathogen and opportunistic organism- carries more virulence factors (coagulase)

if passes skin (epidermis and dermis), trigger an immune response from neutrophils and macrophages
coagulase test
tests for presence of bound coagulase ("clumping factor") on surface of S. aureus cells

if coagulase is present, it will react with prothrombin in rabbit plasma to catalyze precipitation of fibrin from fibrinogen- this fibrin on surface causes cells to stick together and form clumps

bacteria that are coagulase negative will not clump in plasma and rabbit plasma will appear "milky"
hemolysins
toxins produced by certain bacterial organisms that destroy RBCs and damage human tissue

can also be a useful method of differentiating gram positive cocci
blood agar plate (BAP)
tryptic soy agar supplemented with 5% ovine or bovine blood

its hemolytic patterns can be determined by culturing the organisms on this agar

enriched medium

differential media
beta hemolysis
complete hemolysis- complete destruction of the RBCs in BAP

produces a lightened area on the blood agar around and underneath the colonies that will appear slightly yellow and transparent

this is especially evident around the stab marks make with a loop into the agar
alpha hemolysis
incomplete destruction of RBCs in BAP

partial or incomplete hemolysis- result of partial breakdown of hemoglobin in the RBCs

its patter can be distinguished by observing a green discoloration around or under the colonies

this is especially evident around the stab marks made with a loop into the agar
gamma hemolysis
no change occurs in the appearance of BAP

nonhemolytic

any discoloration that is observed in the media is due to the presence and buildup of bacterial waste products
enriched medium
some organisms that require enriched media to support their growth in the laboratory

an example of BAP
purpose of stabbing the BAP
stabbing the organism into the agar after streaking checks for the presence of oxygen-labile hemolysins

3 stabs in initial sector and in between the final sector
streptolysins
hemolysins produced by streptococcus species
streptolysin O
hemolysins produced by streptococcus species that is unstable and breaks down in the presence of oxygen

can be detected by inoculating the organisms beneath the surface of the agar, away from oxygen
streptolysin S
hemolysins produced by streptococcus species that is oxygen stable
streptococci
this bacteria are among the most important and frequent of human pathogens

gram-positive cocci arranged in chains or pairs

classified on the basis of their hemolytic patterns and surface carbohydrates
Lancefield grouping
differentiate the streptococci into Groups A-T based on the specific carbohydrate composition of antigens on the bacterial cell surface

the three medically important groups are Group A, Group B, and Group N
Group A
Lancefield group that includes Streptococcus pyogenes

normal inhabitant of the human upper respiratory tract and skin and most commonly cause streptococcal pharyngitis (strep throat) and skin infections

if left untreated, streptococcal pharyngtits can progress to scarlet fever, rheumatic fever, and glomerulonephritis

beta hemolytic and pathogenic
Group B
Lancefield group that includes Streptococcus agalactiae and Group D streptococci and enterococci

normally colonizes the lower digestive and genitourinary tracts

infection and disease in adults usually results from would or invasion, or postpartum infection in women

more commonly associated with causing neonatal septicemia, pneumonia and meningitis in newborns whose mothers do not possess maternal antibodies against S. agalactiae

beta hemolytic and pathogenic
Group N
Lancefield group that includes Lactococcus lactis, a non-pathogen that is important in the production of buttermilk and cheese
viridans
group of streptococci that are not classified by the Lancefield groupings because they lack the carbohydrate antigens on their cell wall surface
Bacitracin Test
can differentiate the two beta hemolytic streptococci, S. pyogenes and S. agalactiae, through culture methods using antimicrobial tests

Group A S. pyogenes is sensitive to low concentrations of the antibiotic bacitracin- demonstrated by a zone of inhibition with a diameter of 10 mm or more. hemolysis can also be seen on the BAP where bacteria grew

Group B S. agalactiae are not sensitive to bacitracin- demonstrated by no zone of inhibition, where the bacteria grown right up to the antibiotic disc, or the zone has a diameter less than 10 mm. hemolysis can be seen on the BAP where the bacteria grew, especially at stab marks
Group D
Lancefield group of streptococci and enterococci display a wide variety of hemolytic patterns, but most pathogens in this group are hemolytic

mostly found in the intestines of humans and other animals and occasionally contaminate food, usually those of animal origin

enterococci- much more commonly associated with human disease usually acquired through fecal contamination; can cause UTI and opportunistic infections and display resistance to penicillin

can be differentiated by BEA Test
vancomycin resistant enterococci (VRE)
nosocomial infections caused by enterococci that are of rising concern
bile esculin agar (BEA)
can differentiate Group D streptococci and enterococci from other streptococci

is a selective medium- contains bile which is inhibitory to gm+ organisms but not to enteric streptococci and enterococci

also differential medium- contains esculin, which is a glycoside (sugar molecule bound to another chemical functional group or molecule (esculetin) by a glycosidic bond)

only Group D streptococci and enterococci can hydrolyze the glycosidic bond to further break down the glucose during glycosis in the presence of the selective ingredient bile

when esculetin is formed, it will bind with the color indicator (ferric citrate) present in BEA to produce dark brown-black iron precipitate

positive reaction- at least half of BEA is darkened

negative reaction- no growth or color change in agar
6.5% salt tryptic soy broth
selectively determines the separation of Group D into enterococci and non-enterococci

high concentrations of sodium chloride (NaCl) are inhibitory to non-enterococci such as streptococcus bovis but selective for enterococci such as Enterococcus faecalis

positive result- turbid or sedimentary growth in broth; contains Enterococcus faecalis

negative result- broth remains clear with no growth
viridans streptococci
large group of streptococci that lack carbohydrate antigens

many are normal flora in the respiratory tract and oral cavity as S. mutans, mitis, and salivarius
Streptococcus mutans
number one cause of dental caries

synthesizes a dextran capsule from the breakdown of dietary sucrose

its capsule contributes to the formation of a biofilm allowing it to attach to the tooth surface- will then ferment the fructose from sucrose hydrolysis into lactic acid, which can destroy tooth enamel
Streptococcus pneumoniae
non-Lancefield classifiable streptococcus

leading cause of bacterial pneumonia, otits media, and meningitis

possess a prominent polysaccharide capsule which results in a cultural characteristic of mucoid growth on agar

considered an opportunistic pathogen existing as normal flora in heathy individuals

distinguished from other viridans streptococci by its sensitivity to optochin
optochin test
optochin- surface reactant that will lyse cells S. pneumoniae but not another alpha-hemolytic streptococci

S. pneumoniae is sensitive to low concentrations of optochin, which is demonstrated by a zone diameter of 10 mm or more and mucoid growth

resistance to optochin- demonstrated by no zone of inhibition- bacteria grew right up to disc or zone was less than 10 mm in diameter
complex IV
a large protein complex in the electron transport chain that spans the membrane of the mitochondria in eukaryotic cells or the cell membrane in bacterial cells

consists of several iron-containing cytochrome subunits that are capable of being oxidized and reduced; can vary among organisms that can differentiate (cytochrome c oxidase)

functions as an enzyme in the final transfer of electrons to oxygen, reducing it to water
oxidase test
makes use of the different protein subunits for complex IV between major groups of bacteria to categorize and identify them by specifically detecting cytochrome c oxidase

oxidase positive- organisms that have cytochrome c oxidase

oxidase negative- organisms that do not have cytochrome c oxidase as part of electron transport chain; doesn't imply that the bacteria are not capable of aerobic respiration, could have another cytochrome complex instead

employs the use of a redox indicator- phenylenediamine- purple if oxidized

rapid way to differentiate into major groups and is first biochemical test performed when identifying gram neg

family enterobacteriaceae- gram negative bacteria that are all oxidase negative; found in intestinal mucosa and generas Escherichia, Citrobacter, and Enterobacter; oxidase positive include Alcaligenes and Neisseria
phenylenediamine
redox indicator used in oxidase test that changes colors from colorless to dark purple when it becomes oxidized

acts as an artificial electron donor of electrons which then becomes oxidized while cytochrome c oxidase becomes reduced
MacConkey agar (MAC)
a selective and differential medium with the active ingredients lactose, bile salts, crystal violet, peptone, and pH indicator neutral red

used to isolate and differentiate members of the family Enterobacteriaceae (consists of gm- enteric bacteria)

bile salts- key ingredient bc it inhibits any non-enteric gm- bacteria; gm- bacteria that do not colonize intestinal tracts of animals cannot survive with bile (ex: ones in upper resp tract)

crystal violet- second selective ingredient; inhibits growth of most gm+ bacteria

after gm- has been isolate in MAC, lactose is supplied along with neutral red, a pH indicator (pH>6.8=basic, colorless; pH<6.8=acidic, red)
coliform
member of Enterobacteriaceae

bacteria that are typically isolated from the colons of humans and other animals and often indicators of fecal contamination if they are isolated from water, food, or soil

usually not associated with causing disease except in immunocompromised patients

produce acid from lactose fermentation- will accumulate, cause pH to drop, colonies will turn red

bile salts precipitate around colonies producing hazy red appearance- Escherichia and Citrobacter

some will produce a capsule by utilizing the lactose, producing heavily mucoid colonies on MAC; encapsulated bacteria will not ferment all lactose to make ATP, so only outer edge of colony will be red
lactose nonfermenters
member of Enterobacteriacieae

not able to ferment lactose

hydrolyze the peptones in medium and ammonia builds up as a waste product, which raises pH, remains colorless (no red)

pathogens include Salmonella and Shigella

MAC can be used as a rapid way to selectively isolate these pathogens from a patient's stool sample
triple sugar iron agar (TSIA)
important differential medium that distinguishes the members of family Enterobacteriaceae by differences in their ability to ferment glucose, lactose, and sucrose, produce gas while fermenting these sugars, and produce hydrogen sulfide (H2O)

surface area of the slant; large amount of media in the buttt providing an anaerobic environment, therefore only aerobic organisms will not be able to grow in butt

two color indicators:
-phenol red: pink at pH 7.4, yellow at pH <6.8, and orange between
-ferrous ammonium sulfate: turns black in presence of hydrogen sulfide (H2O)

can differentiate bacteria on their ability to reduce medium ingredient thiosulfate to hydrogen sulfide (H2S); if reduced, ferrous sulfate turns black in buttt; assume buttt is yellow due to acidic environment
why is glucose exhausted first by the bacterium before the other carbohydrates can be used?
all member of Enterobacteriaceae ferment glucose, but not all ferment lactose and sucrose

therefore, glucose is added to the medium at a concentration ten times less than concentration of lactose and sucrose

glucose will be exhausted first, especially in the slant which has less media than the buttt
yellow slant/yellow buttt
A/A

glucose fermentation

lactose and/or sucrose fermentation
red slant/yellow buttt
K/A

glucose fermentation only

(exhausted supply in slant after 12 hours, bacterium switched to peptone catabolization)
red slant/red buttt or no change
K/K or K/NC

no fermentation, peptone catabolized

not in family Enterobactreriaceae

no change in butt indicates organism cannot grow anaerobically
cracks or lifting of agar
+G

gas produced during fermentation of carbohydrates
black precipitate in buttt
+H2S

sulfur reduction (always assume butt is yellow beneath precipitate)
urea
waste product of protein degradation in most vertebrates and is excreted in the urine

many gam negative bacilli are able to utilize this waste product as a source of nitrogen
urease
produced by gram-negative bacilli as a source of nitrogen by hydrolyzing the carbon-nitrogen bond in urea, resulting in production of carbon and ammonia
urease test
can be a rapid diagnostic tool for determination of the presence of pathogen (H. pylori, Proteus species- UTIs)
ammonia
basic- some bacteria synthesize this enzyme allowing them to survive in environments where the pH is acidic

ex: H. pylori- causes gastric ulcer disease, uses mechanism to neutralize acidic conditions of the stomach

ex: proteus species with UTIs- neutralize acidic condition that exist when urine is present
urea broth
contains mostly urea, two different buffers to neutralize reaction, and small amount of yeast extract as an additional source of nutrients

contains phenol red- pH indicator: neutral=pale orange; pH rises if organism produces urease and hydrolyzes urea, ph>8.4=hot pink; remains neutral if urea is not hydrolyzed and ammonia is not produced=no color change

can also be used to distinguish rapid urease positive organisms from slow or weak urease positive organisms: proteus- pos result in 4 hours (rapid); incubate longer than 48 hours for a reaction (slow)
IMViC
Indole - Methyl Red - Voges Proskauer - Citrate

series of four tests that determine if the enteric organism in question produces specific enzymes, can utilize alternate carbon sources for energy , or produce characteristic end products from glucose fermentation

useful for differentiating coliforms- ferment lactose with production of acid and gas; enterics- sources of fecal contamination

run all four tests in the series when you have a suspected coliform as sometimes a single test can produce a false positive or negative
Indole test
some enterics synthesize tryptophanase- hydrolyzes substrate tryptophan into indole, pyruvic acid, and ammonia

pyruvic acid- precursor molecule in fermentation or respiration

ammonia- fulfills nutritional needs

indole- waste product

the test indirectly detects presence of tryptophanase by detecting indole as an end-product

performed by inoculating bacteria into tryptone broth (contains tryptophan); addition of Indole Kovac's reagent- allows detection of indole by appearance of a red ring on top of the culture broth (positive), and no color change is negative result
Methyl Red-Voges Proskauer Test
all member of Enterobacteriaceae have ability to ferment glucose to obtain energy, but end-products vary greatly from organism to organism

two broad classes of fermentation patterns for Enterobacteriaceae- mixed acid fermentation (methyl red test) and 2, 3-butanediol fermentation- both performed with MR-VP broth which contains glucose, peptones, and buffers

differentiate between coliforms by detecting fermentative end products
methyl red test
coliforms that utilize the mixed acid fermentation pathway will continue to produce acids with prolonged incubation- the acids will overcome the buffers in the media until pH drops to 4

methyl red, pH indicator, is added after incubation, color will change to red

organisms not capable of mixed acid fermentation will metabolize and acids produced through decarboxylation, resulting in build up of basic end products after incubation

test detects presence of long-term stable acid produce- minimum 72 hrs incubation required
Voges-Proskauer Test
enteric bacteria further break down the pyruvate that was produced during glucose fermentation into other compounds, such as 2, 3-butanediol

bacteria in 2, 3-butanediol fermentation are negative for mixed acid fermentation and vice versa

does not detect 2, 3-butanediol, but detects an intermediate in the fermentation pathway acetoin

acetoin is oxidized to a red diacetyl compound in the presence of oxygen when the two reagents alpha-naphthol (reagent A) and 40% potassium hydroxide (reagent B) are added after incubation

positive reaction- rose color in upper third of broth
citrate test
uses Simmon's citrate agar

tests the ability of organisms to utilize citrate as the sole source of carbon

organisms must have the enzyme citrate permease to facilitate the transport of citrate into the cell

once citrate is inside the cell, organism must have another enzyme, citrase, to oxidize this substrate

during oxidation of citrate, sodium carbonate, an alkaline end product, forms causing the pH of the medium to increase

pH indicator, bromthymol blue, is included in medium to detect change: green=neutral; blue=basic

citrate utilization is detected by more than half of the slant turning blue

if organism cannot metabolize citrate, the slant remains green
Paul Ehrlich
In 1908, won the Nobel Prize for Physiology or Medicine

contributed to immunology which led to the emerging field of chemotherapy

found chemical treatments using various dyes and arsenic compounds against Treponema pallidum (syphilis) and Trypanosoma brucei gambiense (Trypanosomiasis)

had serious side effects (blindness), but made less toxic forms of treatment
Alexander Fleming
In 1928- recognized importance of the fungi Penicillium and a toxin (dubbed penicillin) it produced to control the growth of bacteria

further developed field of antibiotic chemotherapeutics
antibiotic
a compound produced by a living microorganism that kills or inhibits the growth of another

were produced and isolated from other organisms, which most originate for soil-inhabitating microbes

almost exclusively target bacteria- prokaryotes have metabolic processes that must properly function for them to reproduce, grow and survive in any environment; antibiotics have a selective toxicity to prok structures (may cause serious effects)
semisynthetic antibiotics
chemist manipulate the chemical formula of an antibiotic to produce many different derivatives of original antibiotic
MOA of antibacterial drugs:
disruption of cell wall synthesis
specifically interfere with peptidoglycan synthesis, exploiting selective toxicity since eukaryotes lack cell walls and peptidoglycan

ex: large class of beta-lactam drugs comprising the penicillins and their derivatives as well as the cephalosporins
MOA of antibacterial drugs:
interference with enzymatic activity and metabolism
drugs interfere with metabolism which prevent the production of metabolites such as amino acids, nucleotides, and organic acids

ex: include all of the sulfanilamide based antimicrobials and trimethoprim
MOA of antibacterial drugs:
disruption of the cytoplasmic membrane
cause leakage of cellular content and failure to maintain the correct osmotic balance

ex: polymyxin and daptomycin
MOA of antibacterial drugs:
interference with translation
causes inhibition of protein synthesis

because of the difference between prokaryote (70s) and eukaryote (80s) ribosome structure, this is another large class of antimicrobial drugs

ex: tetracyclines, chloramphenicol, erythromycin, and streptomycin
MOA of antibacterial drugs:
inhibition of nucleic acid synethesis
interfere with DNA replication and reproduction of the cell or RNA synthesis and transcription, which will ultimately lead to inhibition of protein synthesis

ex of DNA synthesis inhibitors: quinolones
ex of RNA synthesis inhibitors: rifampin3e