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Lab 13 Goals and Objectives:
Exercise 41: Multiple Test Media: Read and record resultsExercise 71: Gram Negative Intestinal Pathogens
Read and record results, observe PA provided controlsExercise 69: Staphylococci Identification - Read lab
Perform according to supplemental directions (packet pg. 61) Exercise 70: Streptococci & Enterococci Identification – Read lab
Perform according to supplemental directions (packet pg. 62) Each pair will need: 6 blood agar plates
5 Mannitol Salt Agar plates 5 salt tolerance tubes5 coagulase (rabbit serum) tubes 5 Bile Esculin tubes
Control broth cultures: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pyogenes
Exercise 22: Mold CulturesSet up one set of mold cultures (2 cultures total) per pair for use next
week (needs 7 days to grow) as per Fig 22.1, but inoculate sides of block after placing on slide instead of top and bottom while holding
One culture of each from spore suspensions:Rhizopus stolonifer Penicillium notatum
*Save old plates in case you need to buy back or repeat any
observations next class
- + - +Klebsiella pneumoniae
cloacae
page 289
SIM MediumInoculation method: stab deep with needle
Contains: casein (source of tryptophan and cysteine), ferrous salts (reacts with H2S to produce black ferrous sulfide), 0.7% agar (semisolid)
Additional reagents added: Kovac’s reagent, reacts with indole to produce a red product
Discriminates three characteristics:S = “sulfide”, discriminates organisms that can produce cysteine
desulfurase to hydrolyze the amino acid cysteine into pyruvic acid, ammonia and hydrogen sulfide
I = “indole”, discriminates organisms that can produce tryptophanase to hydrolyze the amino acid tryptophan into indole, ammonia and pyruvic acid
M = “motility” discriminates motility (presence of flagella), ability to “swim” through media
SIM Medium Results:S Black = formation of ferrous sulfide, hydrolysis of cysteine into
hydrogen sulfide, positive for cysteine desulfurase production
Colorless = negative for cysteine desulfurase productionI Red with Kovac’s = cleavage of tryptophan into indole, positive
for tryptophanase productionColorless = no indole present, negative for tryptophanase
productionM Organism growing only in line of inoculation = non-motile
Organism appears as haze beyond line of inoculation = motile
IMViC: tests to differentiate lactose +, gram -, enterics
I = Indole (tryptophan degradation)
M = Methyl Red (mixed acid fermentation of glucose)
V = Voges Proskauer (butanediol fermentation of glucose)
C = Citrate (use of citrate as carbon source)
- + - +Klebsiella pneumoniae
cloacae
MacConkey AgarInoculation method: surface streak with loopContains: bile salts, crystal violet and sodium desoxycholate to inhibit
Gram positive growth, lactose, Neutral Red pH indicator: neutral pH = red, acidic pH = bright hot pink
Selective and differential medium: selects for growth of Gram negative organisms by inhibiting growth of Gram positives. Of those that grow, differentiates ability to ferment lactose
Results: Growth = Gram negativeBright pink = positive for lactose fermentationPale pink/colorless = negative for lactose fermentation
No growth = Gram positive, inconclusive for lactose fermentation
Growth = Gram negative
lactose + lactose -No growth = Gram positive*Dead organisms cannot be
scored for lactose fermentation!*
Russell’s Double Sugar AgarInoculation method: streak and stab slant with needleContains: glucose (0.1%), lactose (1%), peptone, Phenol red pH
indicator: alkaline pH = red, acidic pH = yellowDiscriminates organisms that can ferment only glucose to acid from
those that can ferment both glucose and lactose or lactose alone to acid. Organisms that ferment only glucose will show alkaline reversion of the slant when the glucose is exhausted.Results: Yellow slant / yellow butt = positive for fermentation of either
lactose alone or both glucose and lactose to acidRed slant / yellow butt = positive for fermentation of glucose
only to acidRed slant / red butt = negative for fermentation of either
glucose or lactose
Russell’s Double Sugar AgarResults: Yellow slant / yellow butt = positive for fermentation of either
lactose alone or both glucose and lactose to acidRed slant / yellow butt = positive for fermentation of glucose
only to acidRed slant / red butt = negative for fermentation of either
glucose or lactose
Alkaline/acidglucose only
Acid/acidlactose +
glucose unknown
Alkaline/no changelactose -glucose -
Fig 71.1
H2S+
(Confirm answer with IMViC)
12 Possible Unknowns
Gram Positive Gram Negative
Gelatinase + Gelatinase - Gelatinase + Gelatinase -
Bac
illu
s su
btil
is
Pse
udom
onas
aer
ugin
osa
Catalase + Catalase - Lactose -Lactose +
Fig 71.1
H2S+
(Confirm answer with IMViC)
Gram Negative
Gelatinase + Gelatinase -
Pseudomonas aeruginosa
Lactose -Lactose +
Escherichia coli
Klebsiella pneumoniae
Enterobacter cloacae
H2S+
Proteus vulgaris
Salmonella typhimurium
Shigella flexnari
Exercise 22
Lab 13 Goals and Objectives:
Exercise 41: Multiple Test Media: Read and record resultsExercise 71: Gram Negative Intestinal Pathogens
Read and record results, observe PA provided controlsExercise 69: Staphylococci Identification - Read lab
Perform according to supplemental directions (packet pg. 61) Exercise 70: Streptococci & Enterococci Identification – Read lab
Perform according to supplemental directions (packet pg. 62) Each pair will need: 6 blood agar plates
5 Mannitol Salt Agar plates 5 salt tolerance tubes5 coagulase (rabbit serum) tubes 5 Bile Esculin tubes
Control broth cultures: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pyogenes
Exercise 22: Mold CulturesSet up one set of mold cultures (2 cultures total) per pair for use next
week (needs 7 days to grow) as per Fig 22.1, but inoculate sides of block after placing on slide instead of top and bottom while holding
One culture of each from spore suspensions:Rhizopus stolonifer Penicillium notatum
*Save old plates in case you need to buy back or repeat any
observations next class