Lab Exam 3 Flashcards
what are examples of simple stain?
methylene blue (blue)
safranin (pink)
what are examples of differential stains?
malachite green (green)
what are the four steps of differential staining?
- primary stain - colors the target cell
- mordant - helps color stick in target cells
- decolorizer - removes color from background and non-target cells
- counterstain - stains non-target cells
what are the four chemicals used for gram stain?
- primary stain: crystal violet
- mordant: iodine
- decolorizer: acetone/alcohol
- counterstain: safranin
what were the target and non-target cells used in gram stain? what color should they have been?
target: Staphylococcus aureus - purple
non-target: Escherichia coli - pink
what are the four chemicals used for acid fast stain?
- primary stain: basic fuchsin
- mordant: phenol + dimethyl sulfoxide
- decolorizer: acid-alcohol
- counterstain: methylene blue
what were the target and non-target cells used in acid fast stain? what color should they have been?
target: Mycobacterium smegmatis - pink
non-target: Staphylococcus aureus - blue
what are the four chemicals used for endospore stain?
- primary stain: malachite green
- mordant: steam
- decolorizer: distilled water
- counterstain: safranin
what were the target and non-target cells used in endospore stain? what color should they have been?
target: endospores - green
non-target: vegetative cells - pink
what is the primary stain in capsule staining?
congo red
what is the purpose of the acid-alcohol for the capsule stain? how is this different than how it was used for acid-fast?
In capsule staining, acid-alcohol chemically fixes the cells to the slide
In acid-fast, acid-alcohol is used as the decolorizer
what is the secondary stain used in capsule staining?
carbol fuchsin
How do capsules appear at the end of capsule staining?
background will be dark blue/purple and magenta
capsules will look like a clear halo around the pinkish color of the bacillus shaped cell
what was the target bacteria in capsule stain? what color?
Klebsiella pneumoniae - mostly colorless
what is the purpose of streak plating?
to produce pure, isolated colonies from a mixed sample
why do we flame the loop in between each zone?
to reduce the number of microbes as we progress steps
what antibiotic was most and least effective?
Most: Chloramphenicol
Least: Bacitracin
what antiseptic was most and least effective?
Most effective – 1% formaldehyde
Least effective – 70% isopropyl alcohol
Which bacterial type is the most and least susceptible to antibiotics?
most susceptible: Mycobacterium smegmatis
least susceptible: Escherichia coli
which bacterial type is the most and least susceptible to antiseptics?
most susceptible: Mycobacterium smegmatis
least susceptible: Escherichia coli
what does the death zone of a susceptible organism look like?
large death zone
what does the death zone of a resistant organism look like?
small/no death zone
what is the equation to convert Celsius to Fahrenheit
°F = (°C x 1.8) + 32
what is refrigerator temp in Celsius?
4 °C
what is room temp in Celsius?
25 °C
what is body temp in Celsius?
37 °C
what is the definition of selective media?
inhibits growth of unwanted microbes (only the selected microbes live)
what is the definition of differential media?
allows organisms to grow but the added chemicals make them look different (they change different colors and/or precipitate is visible)
what were the three specific plates that we used in our lab?
MSA: Mannitol salt agar
EMB: Eosin methylene blue
MAC: MacConkey agar
where were the colors of the MSA, EMB, and MAC plates before inoculation?
MSA: red
EMB: dark purple
MAC: raspberry
which plate used in lab this semester was not selective or differential?
TSA
which organisms used in the S&D exercise are Gram + ?
Staphylococcus aureus
Staphylococcus epidermidis
which organisms used in the S&D exercise are Gram - ?
Escherichia coli
Enterobacter aerogenes
Proteus vulgaris
How is MSA selective?
selects for gram +
selects against gram -
how is EMB selective?
selects for gram -
selects against gram +
how is MAC selective?
selects for gram -
selects against gram +
How is MSA differential? (fermentation?)
differential for mannitol fermentation
how is EMB differential? (fermentation?)
differential for lactose fermentation
how is MAC differential? (fermentation?)
differential for lactose fermentation
what are the possible bacteria and colors produced by inoculating MSA plate?
fermenter: growth turns yellow (Staphylococcus aureus)
nonfermenter: growth remains red (Staphylococcus epidermidis)
what are the possible bacteria and colors produced by inoculating EMB plate?
strong fermenter: growth turns shiny metallic green (Escherichia coli)
weak fermenter: growth turns pink around the edges of colonies (Enterobacter aerogenes)
nonfermenter: growth remains dark purple (Proteus vulgaris)
what are the possible bacteria and colors produced by inoculating MAC plate?
strong fermenter: entire plate turns bright pink (Escherichia coli)
weak fermenter: growth turns light pink with precipitate and background turns light brown (Enterobacter aerogenes)
nonfermenter: entire plate turns brown (Proteus vulgaris)
what genus is used for the gram + sphere project?
Staphylococcus
what are the potential species used within the gram + sphere project?
Staphylococcus aureus
Staphylococcus capitis
Staphylococcus epidermidis
Staphylococcus haemolyticus
Staphylococcus lentus
Staphylococcus saprophyticus
What type of media was used to test for growth at elevated temperature?
TSB
what was a positive/negative result for the test for growth at elevated temperature?
positive: turbid = indicated that growth was present
negative: clear = growth not present
what tool did we use to inoculate the 45 degree experiment?
cotton swab
what does the urea agar slant test for?
urease production
what tool was used to inoculate the urea agar slant?
loop
what is the original media color of urea agar slant?
peach color
what is the weak acid/base pair that is produced by urease activity?
carbon dioxide = weak acid
ammonia = weak base
what gives a pos/neg result in the urea agar slant?
pos: hot pink color change = ^ pH
neg: yellow color change = v pH
what was the reaction for the substrate and products of the urea agar slant?
what does the Vogel Johnson Agar test for?
- mannitol fermentation
- coagulase production
what chemicals are in Vogel Johnson Agar? what do they serve as?
tellurite and lithium chloride
selective chemicals
what tool and what technique is used within the Vogel Johnson Agar experiment?
cotton swab
zag plating technique
what shows a pos/neg result for mannitol fermentation within the Vogel Johnson Agar?
pos: yellow color change
neg: remains red
what shows a pos/neg result for coagulase production within the Vogel Johnson Agar?
pos: black colonies (tellurite gets reduced to tellurium)
neg: no black colonies
what is the enzyme associated with the Vogel Johnson Agar?
coagulase
What does the Blood agar plate test for?
hemolysin production
What color is the blood agar plate before inoculating?
red
What does the blood agar plate contain to give it its color?
5% sheep’s blood
What tools did we use to inoculate the blood agar plate? what technique?
Cotton swab
Zag plating technique
What is the indication of a positive result for the blood agar plate?
What does this indicate?
What is the pattern of growth called?
Red color disappears under colonies
Indicates that hemolysin production occurred
Beta pattern = clearance zones
What is the indication of a negative result for the blood agar plate?
What does this indicate?
What is the pattern of growth called?
No color change (growth remains red)
Indicates no hemolysin was produced
Gamma pattern = no clearance zone
What kind of molecule is trehalose?
plant carbohydrate
Which tool is used to inoculate an agar stab?
needle
What is the name of the toxin that destroys red blood cells?
hemolysin
What does the trehalose stab test for?
trehalose fermentation
what color was the trehalose stab before inoculating?
red
what was the pH indicator of the trehalose stab?
phenol red
what was pos/neg result for trehalose stab?
pos: yellow color change = v pH
neg: color remains red = neutral
What did the sodium chloride broth test for?
Growth in high salinity media
What was the color of the sodium chloride broth before incoluation?
purple
what was the pH indicator of the sodium chloride broth?
bromocresol purple
What tools did we use for the sodium chloride broth?
loop
What did a pos/neg result look like and indicate for the sodium chloride broth?
Pos: yellow color change = decrease in pH/ growth was present
Neg: remains purple = neutral/ no growth
What type of agar is used to screen for antibiotic sensitivity of our unknown? what is the test called? what plating technique was used?
TSA
Novobiocin resistance test
Lawn plate technique
How does novobiocin inhibit bacterial growth?
Novobiocin is an antibiotic that acts as DNA synthesis inhibitor to bacteria
What would a pos/neg test result for novobiocin resistance look like?
pos: no death zone = resistant
neg: large death zone >4 mm = susceptible
Which form of metabolism utilizes nitrate salt as a terminal electron acceptor?
anaerobic respirator
Is nitrate an oxidized or a reduced salt? what does this mean?
oxidized salt
it can accept electrons
what is the equation for nitrate being successfully oxidized in the nitrate test?
Which of these reduced products are gases?
nitrous oxide (N2O)
nitrogen gas (N2)
What is the function of a Durham tube?
to trap any potential gasses
Is the presence of bubbles in the Durham tube positive or negative for nitrate reduction?
positive
what is a pos/neg result for the nitrate broth test?
pos: color change to red and possibly gas bubbles formed inside Durham tube
Neg: color remains clear and no bubbles
what did the nitrate broth test for?
tests for nitrate respiration
Gelatin is what type of biological molecule?
protein
What is the name of the enzyme that hydrolyzes gelatin?
protease
How did we inoculate the gelatin agar so that it could be incubated?
used needle
If we were to analyze the tubes for gelatin hydrolysis directly after removing the cultures from the incubator our results would be incorrect. What do we need to do before analyzing the results of our hydrolysis test?
allow them to chill
Briefly describe the appearance of a tube that is positive for gelatin hydrolysis.
pos: liquid after chilled = break down into amino acids occurred
neg: solid after chilled = break down did not occur
how do black colonies form in the Vogel Johnson Agar test?
tellurite gets reduced to tellurium when coagulase is present
What concentration of NaCl is used in our hypertonic broth?
6.5%
what are a few of the potential genera used in the gram - rod unknown experiment?
Citrobacter freundii
Enterobacter aerogenes
Escherichia coli
Klebsiella pneumoniae
Proteus vulgaris
Pseudomonas aeruginosa
What type of media was used for the catalase test?
TSA
(In a slant)
what is the function of catalase?
detoxify hydrogen peroxide
Which substrate is broken down by catalase?
Hydrogen peroxide (H2O2)
what does the catalase broth test for?
catalase production
write out the complete catalase reaction, including the names and molecular formulas of all substrates and products.
what does a pos/neg test look like for the catalase test?
pos: bubble formation after H2O2 is added
neg: no bubbles formed after H2O2 is added
Which product of catalase activity is responsible for the visible reaction?
oxygen gas
what is a pos/neg result for the simmons’ slant?
pos: blue color change
Neg: color remains green
What color is the Simmons’ slant before it is inoculated?
green
what does the Simmons’ slant test for?
citrate utilization
ammonium utilization
what is the pH indicator of the Simmons’ slant?
bromothymol blue
What is the carbon source in the Simmons’ slant?
citrate
What is the nitrogen source in the Simmons’ slant?
ammonium
How does nutrient utilization affect the pH of the Simmons’ slant? what happens to the ammonium as well?
citrate utilization removes citrate (acidic) from media which increases pH
ammonium (NH4+) gets reduced to ammonia (NH3)
What does the abbreviation ‘SIM’ stand for?
S = sulfide
I = Indole
M = motility
Which of the three tests did we not record data for? why?
motility
because some results would be black in color and would be too hard to see microbes being motile
Which amino acid is degraded to produce ‘S’?
cysteine
What is the enzyme that is responsible for ‘S’?
cysteine desulferase
What are the three products that are produced as part of ‘S’? (show entire reaction including enzyme)
Which specific product of the three is detected by ‘S’?
hydrogen sulfoxide
what is a pos/neg result for ‘S’ test?
pos: black color change
neg: no color change (remains plain agar)
Which amino acid is degraded to produce ‘I’?
tryptophan
What is the enzyme that is responsible for ‘I’?
tryptophanase
What are the three products that are produced as part of ‘I’? (show entire reaction including enzyme)
Which specific product of the three is detected by ‘I’?
indole
What reagent is used to test for ‘I’?
Kovac’s reagent
what does a pos/neg look like for ‘I’ test?
pos: red ring on top of stab
neg: no color change but drops of reagent remain yellow
what does the oxidase slant test for?
cytochrome oxidase utilization
what tools did we use for the oxidase test? what type of media is it?
loop
TSA slant
Where specifically is cytochrome oxidase located? What is the protein cytochrome oxidase normally used for?
used in ETC (electron transport chain)
transfers electrons from cytochrome C (protein) into oxygen
what is the reaction associated with oxidase test?
what type of reagent is used to interpret oxidase test? what color is it initially?
oxidrops (a reducing agent) - light purple
what is a pos/neg result for oxidase slant test?
pos: dark purple color change w/in 20 seconds
neg: no color change w/in 20 seconds (drops remains plain light purple)
What kills Gram + & some Gram – in the Brilliant Green Agar?
brilliant green dye
what tools did we use to inoculate the Brilliant Green agar? what plating technique?
cotton swab
zag method
what does the brilliant green agar test for?
lactose and sucrose fermentation
what is the original color of the brilliant green agar?
orange
What color is brilliant green agar that is positive for fermentation (Acidic)?
- What color is brilliant green agar that is negative for fermentation (Basic)?
- What color is brilliant green agar when the bacteria is dead (Also negative)?
acidic: fermenter = v pH and lime green color change
alkaline: non-fermenter = ^ pH and bright pink color change
death: inhibited by green dye/ plate remains organge + no growth
what does the Hektoen Agar test for?
tests for lactose, sucrose, and salicin fermentation
what tools did we use to inoculate the Hektoen agar? what plating technique?
cotton swab
zag method
what is the initial color of Hektoen agar?
green
where does salicin come from?
willow bark
What is in the Hektoen agar that makes it selective? what does it select against?
bile
inhibits some gram -
how many pH indicators are in the Hektoen agar?
2
what are the possible results of the Hektoen agar test, and what do they mean?
acidic: fermenter = v pH and orange color change + growth
alkaline: nonfermenter = ^ pH and blue-green color change + growth
death: inhibited by bile/ plate remains green + no growth
what are the two key ingredients of litmus broth?
skim milk
litmus powder
what are the two functions of the litmus powder in the litmus milk broth?
- pH indicator = change color based on lactose fermentation
- electron acceptor = can be reduced
what are the three reactions that can occur with the litmus powder?
acidic result: pos for lactose fermentation = pink color change
alkaline result: neg for lactose fermentation = color remains purple
reduction result: pos for reducing litmus powder = white band that sinks to bottom of tube
what is the milk protein within the litmus milk broth?
casein
describe peptonization?
when a protein is utilized
what are the two possible reactions that can occur with the milk protein in the litmus milk broth?
- Peptonizaton - Cloudiness is removed, media becomes transparent
- Coagulation - Solid formation due to casein in the milk coagulating w/ acid
describe peptonization?
when a protein is utilized
what are the two possible reactions that can occur with the milk protein in the litmus broth?
- peptonization occurred = cloudiness is removed & media becomes transparent
- coagulation occurred = solid formation due to casein in the milk coagulating w/acid
how will coagulation effect the broth?
media will become solid
what causes the broth to coagulate?
casein not being used, and instead clumping with the acids
what does MRVP stand for?
Methyl Red
Vogues Proskauer
what does the MR in MRVP test for? what reagent is testing for this?
methyl red reagent (pH indicator) - tests for mixed acids (lactic, formic, etc.)
what are the potential results of the MR test within the MRVP test?
pos: acidic result = red color change
neg: alkaline/neutral result = yellow color change
what does the VP in MRVP test for?
test for alcohol fermentation
how many days does the 2,3-butanediol test using MRVP tub incubate for?
5 days
Why doesn’t 2,3-butanediol react with a pH indicator, like phenol red?
b/c it is neutral? (pH of 7)
What is the exact procedure for performing the VP test in lab?
What is the purpose of shaking the small tube?
Which final chemical product will be created?
diacetyl (wat we detect)
How long does the VP test sit before the color change is complete?
What color is a positive VP test? What color is a negative test result?
pos: deep red color change
neg: brown color change
What does VP test for?
Alcohol fermentation
What is the specific alcohol the VP tests for?
2,3-butanediol
What is the intermediate molecule involved in the VP test?
Acetoin
What is the final product after adding the reagent to the VP test?
Diacetyl
