LAB Practical I Flashcards
What is selective media?
contains certain ingredients that enhance presence of desired organism
What media selects growth of Staphylococcus species?
mannitol salt agar
What is enrichment media?
contains specific nutrient additives to allow fastidious organisms to grow
What is an example of an enrichment media used?
chocolate agar plate
What are the C-agar lactose and glucose deeps used for?
determines the ability of an organism to utilize glucose/lactose
What is a “positive” result of C-agar lactose/glucose? What does this mean?
yellow color; demonstrates organism produced acid with/without gas
What is the pH indicator in the C-agar glucose/lactose deeps?
Phenol Red
What is the nitrate broth used for?
sees if organism can reduce NO3 (nitrate) into nitrite (NO2) or free nitrogen (N2) gas
What must be added into the nitrate broth tube in order to observe a color change?
sulfanilic acid (SA) and DMA
What is the Tryptone broth used for?
sees if the organism can breakdown tryptone into indole
What does the Tryptone broth test detect for?
tryptophanase
What reagent is added into the Tryptone broth to induce a color change and when do you add this reagent?
Kovac’s reagent; after incubation
What is a positive result in the tryptone broth test? What does this indicate?
red ring = indicates organism can break down tryptone into indole
What is the H2S agar plate?
contains thiosulfates and detects the production of H2S
What is a positive result on the H2S agar plate and what does this indicate?
colonies appear on plate with black centers; organism produces H2S gas
What is the Starch agar plate?
contains starch
What is the Starch agar plate used for?
detects presence of amylase
What must you add onto Starch agar plate (post incubation) to observe result?
Gram’s iodine
What is a positive result on the Starch agar plate and what does this indicate?
a clear zone/halo around colony represents organism produces amylase
What is the blood agar plate (BAP) what is it used for?
contains blood from animal (sheep); determines hemolytic reaction (pattern) of an organism
What was the dextrose tablet used for? What kind of test is this?
glucose sugar fermentation; rapid test
What was the indole tablet used for? What kind of test is this?
breakdown of tryptophan into indole; rapid test
What was the urease tablet used for? What kind of test is this?
determines ability of organism to hydrolyze urea; rapid test
What is the oxidase test?
tests for cytochrome oxidase
How was the oxidase test performed?
drop oxidase reagent on top of colonies
What is a positive result on the oxidase test? What does this indicate?
dark blue/purple color change on colonies within a few seconds; organism produces cytochrome c oxidase
What is the slide catalase test?
tests for catalase
How was the catalase test performed?
place drop of hydrogen peroxide on slide, scoop colony into it
What is a positive result on the oxidase test? What does it indicate?
formation of bubbles; indicates organism produces catalase
What must be added onto the nitrate broth if no color change is present after addition of Kovac’s reagent? What does a red color-change after this addition indicate? What does a non-red color-change indicate?
Zinc dust; if red = NO3 not reduced; if not red = nitrate reduced into something else
What does a gas bubble in the nitrate broth represent?
organism reduces nitrate to N2 gas
Why is there a clear halo as a positive result on the Starch agar plate?
organism produces amylase = breaks down starch; iodine sticks to starch
On an H2S plate, what does the black dot at a center of a colony indicate?
(positive result) H2S gas reacts with iron in media to form the black/brown precipitate
What is a beta hemolysis on a blood agar plate indicative of? How does it look like?
complete break down of red blood cell = clear zone of lysis
What is an alpha hemolysis on a blood agar plate indicative of? How does it look like?
partial breakdown of red blood cell = green/brown zone
What is a gamma hemolysis on a blood agar plate indicative of? How does it look like?
no breakdown of red blood cell = no zone of lysis
In the Dextrose (glucose) tablet rapid test, what is a positive result? What is it indicative of?
yellpwdsd
yellow color = organism ferments dextrose (glucose)
In the Indole tablet rapid test, what is a positive result? (or what must you add to observe a result?) What is it indicative of?
red ring after addition of Kovac’s reagent; breakdown of tryptophan into indole
In the Urease tablet rapid test, what is a positive result? What is it indicative of? What is a negative result, how does the media look like prior to inoculation?
positive = bright pink/magenta = organism hydrolyzes urea (via production of urease); negative = yellow; uninoculated = orange-ish
What is the motile nitrate agar deep used for?
determine if the organism is motile
What is a positive result on a motile nitrate agar deep?
bacteria spreads and fills tube thus turns media red in color
What is a negative result on a motile nitrate agar deep?
bacteria is non-motile and confined to place where it was originally inoculated
Which Staphylococcus species present an alpha hemolysis?
S. epidermidis
Which Staphylococcus species present a gamma hemolysis?
S. saprophyticus
Which Staphylococcus species present a beta hemolysis?
S. aureus
What kind of media is the Mannitol Salts Agar (MSA) plate?
selective and differential
What is the MSA plate used to identify? (staphylococcus)
S. aureus from other staphylococcus species
What does growth on the MSA plate indicate?
staphylococcus species
What is contained in the MSA plate?
NaCl, mannitol, and Phenol red
What is the purpose of each of the ingredients that make up the MSA plate?
NaCl = inhibits most organisms from growing except Staphylococcus species | Phenol Red = pH indicator | mannitol = differentiates S. aureus from other staphylococcus species
What does the red and yellow color changes on the MSA plate indicate?
red = pH 8.4 and yellow = pH 6.4
What is the coagulase test used for? Which of the three identifiable Staphylococcus species will it test positive for?
tests for bound coagulase on S. aureus only
What is a positive result on the Coagulase Test? What does this indicate?
clumping of the organism
How is the Coagulase test performed?
add drop of rabbit or human serum on glass slide and mix in organism
What is the DNase Agar? What does it contain?
differential media used to detect and differentiate Staphylococcus
What is a positive result on the DNase Agar plate and what does this indicate? What is a negative result?
red-purple color indicates organism hydrolyzed DNA via production of DNase | blue color = negative result (color change after 1min = false+)
What is the DNase Agar plate used for?
tests ability of organism to produce DNase
What is contained in the DNase Agar plate?
Tryptic soy agar supplemented with DNA
What must be added on the DNase Agar plate after incubation to observe a result?
1% toluidine blue
Which Staphylococcus organism tests positive for DNase Agar plate?
S. aureus
Which Staphylococcus species is resistant to Novobiocin?
S. saprophyticus
Are Streptococcus/Enterococcus species catalase positive or negative?
catalase negative
Which Streptococcus Group performs a beta hemolysis on BAP?
Groups A and B
Which Streptococcus Group performs an alpha hemolysis on BAP?
Group D
Which Streptococcus Group performs a gamma hemolysis on BAP?
the “No Group” Group
What is the Chocolate Agar Plate (CAP)?
lysed RBCs releases its growth factors contained within it = what some fastidious bacteria need to grow
Which Streptococcus species is sensitive to Optochin?
S. pneumoniae
What does the Optochin on the CAP test for? Which species does it differentiate?
sensitivity of alpha-hemolytic streptococcus; differentiates S. pneumoniae
What does the Bacitracin on the BAP test for? Which species does it differentiate and why?
sensitivity of beta-hemolytic streptococcus to bacitracin | S. pyogenes because they are highly sensitive to bacitracin
What does the 6.5% NaCl broth contain? Which species does it select for?
high concentrations of NaCl inhibit growth of most Streptococcus species | selects for E. faecalis
What is the positive result on the 6.5% NaCl broth? What does it indicate?
turbidity = positive = indicates growth of organism in high salt concentrations
What is the Bile Esculin Test? Which species does it select for?
contains bile = inhibits growth of most Streptococcus | selects for Enterococcus species (grows in presence of 40% bile)
What is the positive result on the Bile Esculin Test? What is the reaction indicator?
ferric citrate
What does a positive result on the Bile Esculin Test indicate?
organism hydrolyzes esculin to esculatin = forms complex iron in media = turns dark brown/black
What is the Sodium Hippurate test?
hippurate and sodium
What is the Sodium Hippurate test used to distinguish between?
Group A beta-hemolytic vs. Group B beta-hemolytic | Group B is able to hydrolyze hippurate to benzoic acid
What is the positive result on the Sodium Hippurate test? What does it indicate?
permanent precipitate after 10 minutes of adding ferric chloride = indicates presence of benzoic acid –» organism hydrolyzed hippurate to benzoic acid
What must be added into the Sodium Hippurate test after incubation to observe a result?
ferric chloride
What is the Bile Solubility test? Which species does it test for?
bile activates enzymes that lead to rapid autolysis of S. pneumoniae
What is the positive result on a Bile Solubility test? What does this indicate?
if the colony disappears due to the addition of the bile salts = indicates organism was led to rapid autolysis due to activation of autolytic enzymes
How was the Bile Solubility test performed?
dropped 10% of bile salts on colonies of an alpha-hemolytic streptococcus on CAP or BAP plates
What species does the StrepQuick Test identify?
Enterococcus and Group A streptococcus
What are the 3 tests on the StrepQuick Test? What do they stand for?
PYR (pyroglutamic aminopeptidase), LAP (leucine aminopeptidase), and ESC (esculin hydrolysis)
What is the Lancefield Test?
grouping based on the polysaccharide antigens on cell wall
What indicates a positive result on a Lancefield test?
agglutination in the circle = indicates which streptococcus species you have
What is inulin? Which test was it used for and what did it determine?
Inulin is a type of fiber (= carbohydrate) used as a C-agar to observe utilization of inulin as a carbon source in order to ferment it
What is Sporulating agar? What is it used for?
nutrient slant agar containing Mn and Ca | provides conditions to enhance spore formation = stresses bacteria into sporulating
What is the difference between C-agar and KL-agar?
KL-agar contains additional ingredients top enhance growth of bacillus
What is KL-agar used for?
determine sugar fermentation pattern of bacillus, Corynebacteria and other strict anaerobes
Which KL-agars were used and what are positive results?
salicin and glucose; yellow = acid fermentation w/ or w.o gas
How will bacillus grow on BAP plates?
unique white fuzzy colonies
What is the Nutrient Gelatin deep?
contains gelatin that solidified at 30ºC or below
What does the Nutrient Gelatin deep test for?
presence of gelatinase
What is a positive result on the Nutrient Gelatin deep? What does this indicate?
the media does not solidify after placed in freezer (almost liquidy) | organism produces gelatinase that hydrolyzes gelatin
What is the Lowenstein-Jensen Agar?
contains inhibitors to prevent normal flora growth allowing slow-growing mycobacteria to multiply = selective media
What is the Middlebrook-Cohn 7H10 agar?
contains inhibitors to prevent contaminants from growing
What is the positive result on the Middlebrook-Cohn 7H10 agar? What does it indicate?
growth = organism is not susceptible to antimicrobials present
What is the purpose of the Middlebrook-Cohn 7H10 agar? Which species does it select for?
determines antimicrobial susceptibility | selects for mycobacteria
What does the Middlebrook 7H9 broth test for?
tellurite reduction test
What must you add to the Middlebrook 7H9 broth to observe a positive result?
0.2% potassium tellurite
What is a positive result on the Middlebrook 7H9 broth? What does this indicate?
jet black precipitate = organism could reduce tellurite
What is the Arylsulfatase test broth? What must you add in order to observe a result?
contains tripotassium phenolphthalein sulfate | must add 0.3mL of 1M of sodium carbonate
What is the purpose of the Arylsulfatase test?
detect arylsulfatase activity
What is a positive result on the Arylsulfatase test? What does it indicate?
red/pink color = organism can produce arylsulfatase = can split phenolphthalein from tripotassium phenolphthalein sulfate
What is the Tween 80 hydrolysis broth? What does it test for?
contains Tween 80 and neutral red = forms an amber color | tests for the presence of lipase that can hydrolyze Tween 80
What is the positive result on the Tween 80 hydrolysis broth test? What does this indicate?
red color = no Tween 80 | indicates organism produced lipase and hydrolyzed Tween 80 thus no amber color observed
What does the Christensen’s urea slant test for?
production of urease
What is the positive result on Christensen’s urea slant test? What does this indicate?
red color = organism can produce urease that led to the hydrolysis of urea
What is the MacConkey Agar plate? What does it differentiate or select for?
a selective/differential media that is not specific to mycobacteria but selects for gram– bacilli and coliforms
What is a positive result on the MacConkey Agar plate? What does this indicate?
pink colonies indicate organism can ferment lactose = coliform; growth indicates gram– bacilli and other enterics such as Salmonella and Shigekka
What is the Hektoen-enteric agar plate (HE)? What does it select for? Which species does it differentiate?
selects for gram– bacteria; differentiates between coliforms and Shigella/Salmonella species
What do salmon-orange colored colonies indicate on an HE plate?
coliforms as they ferment sugar
What do bluish-green colonies indicate on an HE plate?
Shigella/Salmonella since they don’t ferment sugars
What do colonies with a black center indicate on an HE plate?
H2S production is detected = formation of an iron sulfide precipitate
What is an ornithine decarboxylase plate (ODC)?
contains a phenol red pH indicator = detects production of ornithine decarboxylase
What is a positive result on a ODC plate?
media will turn from yellowish-orange to red due to the production of the enzyme that caused the media pH to go from 5.6 to 7
What is the Kliger’s Iron Agar slant (KIA) used for?
differentiates between certain members of the Enterobacteriaceae via hydrogen sulfide production and fermentation of glucose and lactose
What does yellow butt indicate on the KIA slant?
glucose fermentation
What does a complete yellow color change indicate on the KIA slant?
glucose and lactose fermentation throughout
What does a black color change indicate on the KIA slant?
H2S production
What does a no-color change indicate on the KIA slant?
no glucose or lactose fermentation or H2S production
What is the purpose of the Lysine Iron Agar (LIA) slant?
detects the production of lysine decarboxylase (LDC) or lysine deaminase (LDA) and H2S production
What does a purple butt indicate on the LIA slant?
Lysine decarboxylase positive
What does a yellow butt indicate on the LIA slant?
lysine decarboxylase negative; glucose fermentation occurred
What does a red slant (top part) indicate on the LIA slant?
lysine deaminase positive
What does a black color change on the LIA slant indicate?
H2S production
What is the Simmon’s Citrate Agar slant?
contains citrate
What does the Simmon’s Citrate Agar slant test for?
use of citrate as the only source of carbon
What is a positive result on the Simmon’s Citrate Agar slant? What does this indicate?
color change from green to blue = organism utilized citrate to produce NH3
