1. Exercise 41: Multiple Test Media: Read and record results
Lab 13 Goals and Objectives:
Exercise 41: Multiple Test Media: Read and record results
Exercise 71: Gram Negative Intestinal Pathogens
Read and record results, observe PA provided controls
Exercise 70: Staphylococci Identification - Read lab
Perform according to supplemental directions (packet pg. 61)
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
*Save old plates in case you need to buy back or repeat any observations next class

2. 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

3. 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.

4. 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/no change
lactose -
glucose -
Acid/acid
lactose +
glucose unknown
Alkaline/acid
glucose only

5. 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

6. 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

7. (Confirm answer with IMViC)
Fig 71.1
H2S+
(Confirm answer with IMViC)

8. 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

9. 12 Possible Unknowns Gram Positive Gram Negative Gelatinase +
Catalase +
Catalase -
Lactose +
Lactose -
Pseudomonas aeruginosa
Bacillus subtilis

10. Gram Negative Gelatinase + Gelatinase - Lactose + Lactose - H2S+
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumoniae
Enterobacter cloacae
Proteus vulgaris
Salmonella typhimurium
Shigella flexnari

11. 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.

12. Inoculation method: surface streak and stab with loop
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

13. _ + 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
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% _ +

14. 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

15. 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

16. 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
Salt tolerance broth