quiz microbio Flashcards
The action of catalase results in
the decomposition of hydrogen peroxide to water and oxygen.
H2O2→ H2O + ½ O
catalyse procedure
Using a sterile loop, pick up some bacterial cells and smear onto a small area of a slide
Place a drop of hydrogen peroxide on the bacterial cells. and observe for (gas/bubbles) this indicates the presence of catalase.
hemolysis
Blood agar is a differential media, differentiating between those that are able to lyse red bloodcells and those that cannot.
.
hemolysis procedure
inoculate a blood plate. Perform a T streak.
2. Inoculate organism onto the appropriate section of the plate. Incubate the plate for 24 hours in
a candle jar (decreased oxygen) at 37oC.
amylase
catalyzes the breakdown of starch (a branched polymer of glucose) to maltose (a disaccharide).
amylase procedure
Divide a Starch agar plate into sections.
2. Inoculate organism using the loop to draw a straight line onto the plate.
3. Incubate at 37oC for 24-48 hours.
4. Flood the plate with iodine. Starch, in the presence of iodine turns the media blue-black.
Organisms able to produce amylase will show a clear zone of no color around the culture, due
to breakdown of the starch. Organisms which do not hydrolyze starch will have a blue-black
color to the medium up to the edge of the growth.
coagulase
Coagulase is a cell surface associated virulence factor of certain organisms, in particular certain
Staphylococci. In infected tissues coagulase may protect a bacterium from the normal defenses of the
host by coating itself with host proteins. Coagulase catalyzes the conversion of fibrinogen to fibrin
forming an insoluble clot. The clot formation assay, indicating the action of coagulase, is indicative of
pathogenic strains of Staphylococci.
coagulase procedure
Procedure
1. Inoculate 0.5ml of diluted (1:4) rabbit plasma with a loopful of culture of from a fresh plate
2. Incubate at 37oC; check after 2-4 hours, but continue incubation for up to 24 hours.
3. Check for coagulation by tilting the tubes; coagulated plasma will stay as a clot in the bottom of
the tube
BACITRACIN SUSCEPTIBILITY
Initial presumptive identification is done by growth on a blood agar plate, looking for characteristic -hemolysis.
Furtheridentification can be done by determining the sensitivity of the -hemolytic organism to the antibiotic
bacitracin. Although most group A streptococci are sensitive, some strains (2-7%) may give false
negative results (resistant to bacitracin); similarly other streptococci (5-15%) may be susceptible to
bacitracin. However, a -hemolytic, bacitracin sensitive organism is characteristic of Streptococcus
pyogenes.
Bacterian susceptibility procedure
Procedure
1. Perform a T streak on a Blood Agar.
2. Carefully place a bacitracin disc, on Section I. Press gently using sterile forceps to ensure the
discs are on the medium.
3. Incubate for 24 hours at 37oC in candle jar.
4. Record your results for zone of inhibition
OPTOCHIN SUSCEPTIBILITY
. -hemolytic organisms suspected as being S. pneumoniae can
be differentiated from other -hemolytic organisms (normal flora) by their sensitivity to optochin
(ethylhydrocupreine hydrochloride).
OPTOCHIN SUSCEPTIBILITY procedure
Procedure
1. Perform a T streak on a Blood Agar.
2. Carefully place a optochin disc, on Section I. Press gently using sterile forceps to ensure the
discs are on the medium.
3. Incubate for 24 hours at 37oC in candle jar.
4. Record your results for zone of inhibition.
FURAZOLIDONE SUSCEPTIBILITY
some coagulase-negative staphylococci (i.e. not Staphylococcus aureus) are resistant to furazolidone. The result for this test is based on the zone of inhibition therefore, it is important to measure the zone of inhibition.
Resistant microorganisms can still show a zone of clearing between
6mm to 9mm.
In order to identify Staphylococcus spp. correctly, a zone of inhibition has to be greater than 15mm.
FURAZOLIDONE SUSCEPTIBILITY procedure
- Inoculate a TSA plate with an unknown isolate by the T-streak method.
- Carefully place a furazolidone disc in the first section. Press gently using sterile forceps to ensure
the discs are on the medium. - Incubate for 24 hours at 37oC.
- Record your results. A positive test for Staphylococcus is indicated by a zone of no growth around the furazolidone disc (≥15mm
UREASE
Urease catalyzes the breakdown of urea by the removal of two ammonia molecules from each urea
molecule.
urease procedure
inoculate the surface of a Urea agar slant with your organism in a zig-zag pattern.
2. Incubate the slants for 24 hours at 37oC. Continue incubation for up to 4 days.
3. A positive result will be seen by a deep-pink to red color in the slant
Catalase is an important enzyme for organisms growing aerobically by
prevents the accumulation of
toxic metabolites (H2O2) that form during oxidation-reduction reactions of the respiratory chain.High intracellular levels of such compounds could kill the cell.
Some bacteria are able to _____ red blood cells, which results in a zone of clearing around a bacterial
colony
lys
gamma hemolysis
no lysis or significant change in the appearance of the medium surrounding
the cells
alpha hemolysis
a zone of partial clearing due to incomplete lysis if the red blood cells. A
greenish halo around the bacteria due to reduction of hemoglobin to methemoglobin, suggested to
be due at least in part to the action of hydrogen peroxide.
beta
a clear zone of hemolysis is present around the colony. There is complete
destruction of the cell and hemoglobin
Bacteria are not able to transport starch into the cell. how do they overcome this,
organisms that are able to hydrolyze starch do so by hydrolyzing sugar to maltose. Maltose is then
able to enter the cell and be utilized by the bacterium
urea enzyme does what
detoxification of waste products,
supplying a source of nitrogen (via ammonia) for amination reactions producing amino acids and
nucleotides.
the assay medium for urease activity contains a high concentration of ____ and a pH indicator.
urea
The ammonia released by the urease reacts in solution to form _____ which results in an increase in the pH of the medium
ammonium carbonate
Staphylococci are susceptible to _________while micrococci are resistant.
furazolidone
_______ is a quinine derivative that has a detergent-like action
capable of causing lysis of S. pneumoniae
Optochin
identification of pathogenic -hemolytic streptococci, in particular ______________, is important in the diagnosis of disease
Streptococcus pneumoniae
Identification of ________ (Streptococcus pyogenes) is required for the prompt therapy of
patients.
group A streptococci
Not all -hemolytic organisms are
group A streptococci.
Denitrification is the process in which
nitrate is reduced to molecular nitrogen
Some organisms are able to use nitrates, instead of oxygen as the terminal electron acceptor, in a
process known as
anaerobic respiration
he reduction of nitrate to nitrite is detected by
assaying for the presence of the product (nitrite)
he agar inhibits diffusion of oxygen, favoring ___________
therefore utilization of anaerobic respiration
anaerobic growth,
in nitrate reduction and denitrification after incubation, reagents A (________) and B (________) are added to the nitrate broth medium.
sulfanilic acid , a-naphthylamine
The presence of a color reaction (red)
indicates the presence of _____, due to ___
nitrite, formation of nitrous acid HNO2 from nitrite which reacts with sulfanilic acid and a-naphthylamine
procedure for nitrite reduction and denitrification
inoculate broth, incubate for 24-48 hours at 37 degrees
examine dirham tube for evidence of gas.
add 3-5 drops of solution A and 3-5 drops of solution B to all nitrate broths
development of red color means precense of nitrite. so tis positive.
add small amount of zinc dust to tubes that are negative.
A red color obtained by addition of zinc is a true negative for nitrate reduction. If no red color develops after addition of zinc, then the nitrite has been further reduced to ammonia,
molecular nitrogen or other reduced nitrogenous compounds. This is therefore a positive test
for nitrate reduction
zince does what to nitrate
reduces nitrate to nitrite
how long should it take color to turn red in nitratre reduction and denitrification
30 sec
Some organisms ferment pyruvate resulting in production of 2,3 _____
butanediol
voges proskauer test an intermediate in this pathway is the compound ______
acetoin
voges proskauer test Organisms that
utilize this pathway produce smaller amounts of
mixed acids, generally insufficient to result in a
positive methyl red test.
2,3-Butanediol is not a very reactive compound so in the VP test the presence of the ______ is assayed.
intermediate
acetoin
In the presence of atmospheric oxygen and potassium hydroxide, acetoin is
converted to ______- with a-naphthol serving as a catalyst
diacetyl,
in VOGES-PROSKAUER TEST what color do you get if positive
pink-red color
in VOGES-PROSKAUER TEST how long do you have to wait
24-48 hours
in VOGES-PROSKAUER TEST what happenes if there is a short incubation times
will not allow for
sufficient to accumulate acetoin
VOGES-PROSKAUER TEST procedure
- Inoculate 3 ml of MR/VP broth medium.
- Incubate at 37oC for 24-48 hours.
- Add 30 drops of a-naphthol. Mix thoroughly.
- Add 10 drops of 40% KOH. Mix well.
- Incubate the tubes in a slanted position (increases surface area) for up to 60 minutes with a
slightly loose cap. Do not exceed 60 minutes, as a false positive test may be obtained. (Do not
keep shaking tube during incubation.) - Development of a pink-rose color is a positive VP tes
BILE ESCULIN TEST procedure
inoculate the slant using an inoculating loop and inoculating needle.
2. Make a zig-zag pattern on the surface of the slant, and stab the butt of the slant using an
inoculating needle.
3. If you are working off a blood agar plate, then incubate slant in candle jar, if not then incubate
in aerobic conditions at 37oC
Fermentation of glucose can occur via a number of pathways, one of which, the mixed acid ____________, results in the formation of a very low pH due to production of strong acids
fermentation pathway,
glucose fermentation via the mixed acid fermentation results in the formation of stable acid end
products such as
acetic, lactic, succinic and formic acids.
Production of strong acids is detected by addition of
methyl red
methyl red has a ph range of
6.0 (yellow)-4.4(red)
o maintain the red color on addition of methyl red, large quantities of strong,
stable acids must be produced from the _________
carbohydrate source
Organisms not producing stable acids in methyl red testing result in formation of a change in color of the pH indicator to _____
yellow
Mythel red procedure
inoculate 3 ml of MR/VP broth medium.
2. Incubate at 37oC for 48-96 hours.
3. Add 5 drops of methyl red pH indicator.
4. Development of a stable red color at the surface of the medium indicates the presence of
sufficient acids to lower the pH to 4.4. This is a positive methyl red (mixed acid fermentation)
test. (Do not keep shaking tube during incubation.)
Most microorganisms obtain their energy by either
respiration (aerobic or anaerobic) or fermentation
CARBOHYDRATE FERMENTATION (ACID & GAS PRODUCTION procedure
1.Collect tubes of phenol red broth base + sugar (mannitol, glucose). Make sure the Durhamtube does not contain any air bubbles.
- Inoculate each organism in to each sugar.
- Incubate at 37oC for 24-48 hours.
- Determine the production of acid or acid and gas
Organisms are able to use carbohydrates differently depending on the _____ at its disposal
complement of enzymes the
cell has
In fermentation, the substrate acts as the ___________ such that there is no net change in the oxidation state of the product(s) relative to the starting substrate.
electron acceptor
In general, fermentation pathways are named for __________ , a property which is widely used in the identification of bacteria.
characteristic end products
Examples of fermentation pathways are:
ethanol fermentation, lactic acid, fermentation, mixed acid fermentation, butanol, fermentation and butanediol fermentation
Fermentations are assayed in a __________ broth tube containing an inverted Durham tube to detect
production of gas (CO2).
phenol red
The growth tube contains:
fermentation broth-which has all the nutrients required for growth of the microorganism
a specific carbohydrate for fermentation
a pH indictor
which changes color at an acidic pH.
Fermentation is indicated by the presence of ______ , i
acid (media will
turn yellow)
CARBOHYDRATE FERMENTATION in some cases there may also be evolution of _____ which will be collected in the _______
gas; durham tube
Lack of fermentation does not _________; some microorganism can use othernutrients (such as peptides) as energy sources.
indicate absence of growth
in carbohydration femrentation peptides are broken down into amino acids which can be converted to _______ by deamination which are then metabolized.
ketoamino acids
CARBOHYDRATE FERMENTATION reactions result in release of ammonia which accumulates as _____________.
(alkaline) ammonium hydroxide
release of ammpnia which makes ammonium hydroxide may also occur if the tubes are
incubated too long and all the fermentable sugar is
used up
CARBOHYDRATE FERMENTATION it is important to read the results in ________ otherwise some of the broths may revert in color due to the presence of alkaline products
24hr-48hr
