Transcript Lab 13

*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 70: Staphylococci Identification - Read lab
observations
Perform according to supplemental directions (packet pg. 61) next class
Lab 13 Goals and Objectives:
Exercise 71: 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
Tests: Gram cell wall and Lactose fermentation
Media: MacConkey Agar
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
Tests: Glucose and Lactose fermentation
Media: Russell’s Double Sugar Agar
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.
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
Alkaline/no change
Acid/acid
lactose lactose +
glucose glucose unknown
Tests: Hydrogen sulfide production; Thryptophanase production;
Motility
Medium: SIM
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
Fig 71.1
H2S+
(Confirm answer with IMViC)
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 -
+
-
+
12 Possible Unknowns
Gram Negative
Gram Positive
Bacillus subtilis
Catalase +
Gelatinase -
Gelatinase +
Lactose +
Catalase -
Pseudomonas aeruginosa
Gelatinase +
Gelatinase -
Lactose -
Gram Negative
Gelatinase -
Gelatinase +
Lactose -
Lactose +
H2S+
Pseudomonas
aeruginosa
Escherichia
coli
Klebsiella
pneumoniae
Enterobacter
cloacae
Proteus
vulgaris
Salmonella
typhimurium
Shigella
flexnari
Mannitol fermentation and salt tolerance
Mannitol Salt Agar
Inoculation method: surface streak with loop
Contains: 7.5% NaCl, mannitol, Phenol red pH indicator: alkaline
pH = red/pink, acidic pH = yellow
Selective and differential medium: selects for growth of
organisms salt tolerant to 7.5% (usually Staphylococci). Of
those that grow, differentiates organisms that have the ability to
ferment mannitol to acid.
1. Blood Agar hemolysis
Blood Agar
Inoculation method: surface streak and stab with loop
Contains: BHIA, sheep blood
Discriminates organisms that have the ability to hemolyse red blood
cells completely through production of hemolysins (streptolysins or
alpha- toxin) or partially through ability to degrade hemoglobin pigment
into green products (biliverdin)
Exercise 70: Staphylococci Identification—Read lab
Perform according to supplemental directions (packet pg. 61)
Exercise 71: Streptococci Identification – Read lab
Perform according to supplemental directions (packet pg. 62)
2. Bacitracin Susceptibility or Resistance
Two
Coagulase Test
Rabbit Serum:
Inoculation method: loop transfer
Contains: rabbit serum (fibrinogen)
Discriminates organisms that can produce coagulase to catalyze the
conversion of soluble fibrinogen into insoluble fibrin (clot)
Bile Esculin Hydrolysis Slant
Inoculation method: surface streak with loop
Contains: esculin, ferric citrate (reacts with hydrolyzed esculin
(esculetin) to produce black precipitate)
Discriminates organisms that can hydrolyze esculin into esculetin and
dextrose
Salt Tolerance Broth
Inoculation method: loop transfer
Contains: 6.5% NaCl, Bromcresol purple pH indicator: alkaline
_ pH =+
purple, acidic pH = yellow (any growth should cause acid
accumulation)
Discriminates organisms that display salt tolerance to 6.5%
Exercise 24: 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 24.1, 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
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
Controls
• Blood agar
– Staphylococcus aureus – Staphylococus epidermidis
• Blood agar+Bacitracin
– Streptococcus pyogenes,
– Enterococcus faecalis
• Manitol salt agar
– Staphylococcus aureus,
– Staphylococcus epidermidis
• Coagulase tubes
– Staphylococcus aureus
• BE slants
– Enterococcus faecalis
• Salt tolerance broth
– Enterococcus faecalis