*Save old
plates in case
Exercise 41: Multiple Test Media: Read and record results
you need to
Exercise 71: Gram Negative Intestinal Pathogens
buy back or
Read and record results, observe PA provided controls
repeat any
Exercise 69: Staphylococci Identification - Read lab
observations
Perform according to supplemental directions (packet pg. 61) next class
Lab 13 Goals and Objectives:
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 tubes
5 coagulase (rabbit serum) tubes 5 Bile Esculin tubes
Control broth cultures: Staphylococcus aureus, Staphylococcus
epidermidis, Enterococcus faecalis, Streptococcus pyogenes
Exercise 22: Mold Cultures
Set 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
page 289
cloacae
Klebsiella pneumoniae -
+
-
+
SIM Medium
Inoculation 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 production
I
Red with Kovac’s = cleavage of tryptophan into indole, positive
for tryptophanase production
Colorless = no indole present, negative for tryptophanase
production
M 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)
cloacae
Klebsiella pneumoniae -
+
-
+
MacConkey Agar
Inoculation method: surface streak with loop
Contains: 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 negative
Bright pink = positive for lactose fermentation
Pale pink/colorless = negative for lactose fermentation
No growth = Gram positive, inconclusive for lactose
fermentation
lactose +
lactose No growth = Gram positive
*Dead organisms cannot be
scored for lactose
fermentation!*
Growth = Gram negative
Russell’s Double Sugar Agar
Inoculation method: streak and stab slant with needle
Contains: glucose (0.1%), lactose (1%), peptone, Phenol red pH
indicator: alkaline pH = red, acidic pH = yellow
Discriminates 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 acid
Red slant / yellow butt = positive for fermentation of glucose
only to acid
Red slant / red butt = negative for fermentation of either
glucose or lactose
Russell’s Double Sugar Agar
Results: Yellow slant / yellow butt = positive for fermentation of either
lactose alone or both glucose and lactose to acid
Red slant / yellow butt = positive for fermentation of glucose
only to acid
Red slant / red butt = negative for fermentation of either
glucose or lactose
Alkaline/acid
glucose only
Acid/acid
lactose +
glucose unknown
Alkaline/no change
lactose glucose -
Fig 71.1
H2S+
(Confirm answer with IMViC)
12 Possible Unknowns
Gram Negative
Gram Positive
Bacillus subtilis
Catalase +
Gelatinase -
Gelatinase +
Lactose +
Catalase -
Pseudomonas aeruginosa
Gelatinase +
Gelatinase -
Lactose -
Fig 71.1
H2S+
(Confirm answer with IMViC)
Gram Negative
Gelatinase -
Gelatinase +
Lactose -
Lactose +
H2S+
Pseudomonas
aeruginosa
Escherichia
coli
Klebsiella
pneumoniae
Enterobacter
cloacae
Proteus
vulgaris
Salmonella
typhimurium
Shigella
flexnari
Exercise 22
*Save old
plates in case
Exercise 41: Multiple Test Media: Read and record results
you need to
Exercise 71: Gram Negative Intestinal Pathogens
buy back or
Read and record results, observe PA provided controls
repeat any
Exercise 69: Staphylococci Identification - Read lab
observations
Perform according to supplemental directions (packet pg. 61) next class
Lab 13 Goals and Objectives:
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 tubes
5 coagulase (rabbit serum) tubes 5 Bile Esculin tubes
Control broth cultures: Staphylococcus aureus, Staphylococcus
epidermidis, Enterococcus faecalis, Streptococcus pyogenes
Exercise 22: Mold Cultures
Set 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