1. Exercise 39: Oxidation and Fermentation Tests (Catalase)
Lab 11 Goals and Objectives:
Exercise 39: Oxidation and Fermentation Tests (Catalase)
Exercise 67: Staphylococci Identification (MSA & Coagulase)
Exercise 68: Streptococci & Enterococci Identification
(BAP Hemolysis & Bacitracin resistance)
Construct Dichotomous Key to solve for the 12 unknowns
Identification of Unknown Bacteria: individual assignment
Each person sign out one unknown culture
Streak for isolation on BHIA

2. Catalase Test
Discriminates aerobic organisms that produce catalase to degrade hydrogen peroxide into water and oxygen _ +
Figure 39.11

3. Don’t count a heavy streak only as growth!
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.
Results: Growth = salt tolerant to 7.5%
Yellow = positive for mannitol fermentation to acid
Pink = negative for mannitol fermentation to acid
No growth = negative for salt tolerance to 7.5%, inconclusive for mannitol fermentation
Growth = colonies
Don’t count a heavy streak only as growth!
Figure 67.2

4. _ + Rabbit Plasma: Coagulase Test Inoculation method: loop transfer
Contains: rabbit plasma (fibrinogen)
Discriminates organisms that can produce coagulase to catalyze the conversion of soluble fibrinogen into insoluble fibrin (clot)
Results: Solid = positive for fibrin formation, positive for coagulase production
Liquid = negative for coagulase production
Figure 67.4 _ +

5. 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)
Results:
Complete clearing of RBCs = Beta-hemolysis, positive for production of hemolysins
Partial clearing and greening of blood = Alpha-hemolysis, positive for degradation of hemoglobin pigment into biliverdin
No clearing with or without rusting = Gamma-hemolysis, negative for hemolysis
See Figure 68.2
Gamma
Alpha
Beta

6. Partial clearing and greening of blood = Alpha-hemolysis, positive for degradation of hemoglobin pigment into biliverdin
Complete clearing of RBCs =
Beta-hemolysis, positive for production of hemolysins
No clearing with or without rusting = Gamma-hemolysis, negative for hemolysis
Figure 68.2

7. Bacitracin Susceptibility or Resistance
Susceptible = organism killed bacitracin: zone of no growth around disk
Resistant = organism growth not affected by bacitracin: organism grows around and under disk

8. 1 2 3 4 5 6 7 8 9 10 11 12 Bacillus subtilis Enterobacter cloacae
Escherichia coli
Klebsiella pneumoniae 5 6 7 8
Proteus vulgaris
Pseudomonas aeruginosa
Salmonella typhimurium
Shigella flexneri 9 10 11 12
Staphylococcus aureus
Staphylococcus epidermidis
Enterococcus faecalis
Streptococcus pyogenes

9. The Dichotomous Key
-a visible map of logic flow
-divides a group of possible things into smaller and smaller groupings until it leads to the identification of just one
-tracing the pathway backwards shows all the characteristics that collectively are unique to only that one
-assesses only one characteristic per branch
-lines all the answers up across the bottom of the key

10. External covering: feathers External covering: hair
Example: explain with the least number of characteristics how to tell the difference between an old lady and her three pets: sort a human, a cat, a dog and a bird. Construct a Dichotomous Key to ascertain the identity of each from this small group:
Animals in the house
External covering: feathers
External covering: hair
No tail
Tail
Meow
Woof
Bird
Human
Cat
Dog

11. 12 Possible Unknowns

12. 1 2 3 4 5 6 7 8 9 10 11 12 Bacillus subtilis Enterobacter cloacae
Escherichia coli
Klebsiella pneumoniae 5 6 7 8
Proteus vulgaris
Pseudomonas aeruginosa
Salmonella typhimurium
Shigella flexneri 9 10 11 12
Staphylococcus aureus
Staphylococcus epidermidis
Enterococcus faecalis
Streptococcus pyogenes

13. 12 Possible Unknowns
Gram Positive
Gram Negative

14. 12 Possible Unknowns
Gram Positive
Gram Negative

15. 12 Possible Unknowns Gram Positive Gram Negative Gelatinase +
Pseudomonas aeruginosa
Bacillus subtilis

16. Go to Exercise 69 12 Possible Unknowns Gram Positive Gram Negative
Gelatinase +
Gelatinase -
Gelatinase +
Gelatinase -
Go to Exercise 69
Pseudomonas aeruginosa
Bacillus subtilis

17. Figure 69.1

18. 12 Possible Unknowns Gram Positive Gram Negative Gelatinase +
Lactose +
Lactose -
Pseudomonas aeruginosa
Bacillus subtilis

19. Figure 69.1

20. EXAMPLE!!!! Gram Negative Gelatinase + Gelatinase - Lactose +
H2S+
H2S-
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumoniae
Enterobacter cloacae
Proteus vulgaris
Salmonella typhimurium
Shigella flexneri

21. ? 12 Possible Unknowns Gram Positive Gram Negative Gelatinase +
Bacillus subtilis

22. Streptococcus and Enterococcus
12 Possible Unknowns
Gram Positive
Gram Negative
Gelatinase +
Gelatinase -
Catalase +
Catalase -
Staphylococcus
Streptococcus and Enterococcus
Bacillus subtilis

23. -Mannitol fermentation -Coagulase Strep/Entero assays: Choose one
Gram Positive
Gelatinase +
Gelatinase -
Catalase +
Catalase -
Staph assays:
Choose one
-Mannitol fermentation
-Coagulase
Strep/Entero assays: Choose one
-Hemolysis type
-Bacitracin sensitivity
Bacillus subtilis

24. EXAMPLE!!!! Gram Positive Gelatinase + Gelatinase - Catalase +
Coagulase +
Coagulase -
Bacitracin R
Bacitracin S
Bacillus subtilis
Staphylococcus aureus
Staphylococcus epidermidis
Enterococcus faecalis
Streptococcus pyogenes

25. All 12 Possible Unknowns
Gram Positive
Gram Negative
Gelatinase +
Gelatinase -
Gelatinase -
Gelatinase +
Catalase +
Catalase -
Lactose +
Lactose -
Mannitol + OR
Coagulase +
Mannitol - OR
Coagulase -
 Hemolytic OR
Bacitracin R
 Hemolytic OR
Bacitracin S
Indole + OR
Citrate -
Indole - OR
Citrate +
H2S + OR
Motile
H2S - OR
Nonmotile
Urea + OR
MR +
VP -
Urea - OR
MR -
VP +
Urea +
Urea -
Staphylococcus epidermidis
Pseudomonas aeruginosa
Staphylococcus aureus
Enterococcus faecalis
Streptococcus pyogenes
Klebsiella pneumoniae
Salmonella typhimurium
Enterobacter cloacae
Bacillus subtilis
Escherichia coli
Proteus vulgaris
Shigella flexneri

26. Unknown Identification
Day 1: Each person sign out one unknown
Make streak for isolation on BHIA
Put your name on original slant and return
Day 2: Gram stain
Catalase test
Inoculate MacConkey Agar
Inoculate Gelatin Deep
Day 3: Based on Gram, Gelatinase, Catalase,
Lactose, set up assays to solve:
A. If Staph: MSA and Coagulase
B. If Strep/Entero: BAP and Bacitracin
C. If Gram Neg: SIM, Citrate, MR, Urea

27. Exercise 39: Oxidation and Fermentation Tests (Catalase)
Lab 11 Goals and Objectives:
Exercise 39: Oxidation and Fermentation Tests (Catalase)
Exercise 67: Staphylococci Identification (MSA & Coagulase)
Exercise 68: Streptococci & Enterococci Identification
(BAP Hemolysis & Bacitracin resistance)
Construct Dichotomous Key to solve for the 12 unknowns
Identification of Unknown Bacteria: individual assignment
Each person sign out one unknown culture
Streak for isolation on BHIA
Return slant for storage