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