1. PHT 381 Lab# 6
Biochemical
reactions

2. Enzyme production

3. 1- Catalase Test Principle: Procedure: 3 1 2 Catalase enzyme H2O2
H2o O2
Air bubbles
Procedure: 3
H2O2 1 2

4. Results: Positive test: rapid appearance of gas bubbles. Catalase +ve

5. 2-. Coagulase Test: Principle: Procedure: 2 1 3 Fibrinogen Plasma
Coagulase enzyme
Fibrin
Visible Clot
Procedure: 2
1 ml rabbit plasma 1 3
Place at water bath at 37oC, observe for formation of visible clot for up to 4 hrs.

6. Results: Positive test: formation of visible clot. Coagulase -ve
S.epidermidis
Coagulase +ve
S.aureus

7. Tetramethyl p-phenylene diamine
3- Oxidase Test:
Principle:
Tetramethyl p-phenylene diamine
(oxidase reagent)
colourless
Cytochrome oxidase enzyme
Indophenol
(Purple colour)

8. Oxidase test: Results:
+ve Test: Appearance of purple colour within 1-2 min.
No colour
purple colour
-ve test
+ve test

9. Oxidase test: Results:
+ve Test: Appearance of purple colour within 1-2 min.
No colour
purple colour
-ve test
+ve test

10. 4- Urease Test: Principle: Urease enzyme Urea NH3 ( alkaline)
Phenol red indicator
Pink colour

11. Results:
+ve
-ve

12. 5- Deoxyribonuclease (DNase) Test:
Principle:
DNA
DNase enzyme
Nucleotides
Insoluble
In acid
soluble
In acid
Procedure:
1. Inoculate DNase agar plate with the test organism.
2. Incubate the plate at 35oC for 24 hrs.
3. Flood the plate with 1M HCl.

13. Results:
DNase activity is indicated by a clear zone around the growth after addition of Hcl
Clear zone around the growth while the rest of the plate appears cloudy
Cloudiness in all the plate
S.aureus
S.epidermidis

14. Sugar
Fermentation

15. 1. Sugar fermentation:
Sugar fermentation can be indicated by change of color of the medium from red to yellow.
Glucose
Maltose
Sucrose
Glucose
Maltose
Sucrose
+ve
+ve
+ve
+ve
+ve
- ve

16. (glucose 0.1%, lactose 1%, sucrose 1%).
2- Triple Sugar Iron (TSI) agar:
Principle:
(glucose 0.1%, lactose 1%, sucrose 1%).
butt
slant

17. 1. No Fermentation:
Butt: alkaline (red)
Slant: alkaline (red)

18. a) Initial reaction: 2.Glucose Fermentation: (after 10 – 12 hrs)
Dextrose
acid
Butt: acidic (yellow)
Slant: acidic (yellow)

19. b) Delayed reaction: 2. glucose Fermentation: (after 24 hrs) O2
Peptone O2
Small amount of acid
Alkaline amines
Butt: acidic (yellow)
Slant: alkaline (red)

20. 3. Lactose/ sucrose Fermentation:
Peptone O2
Large amount of acid
Alkaline amines
Butt: acidic (yellow)
Slant: acidic (yellow)

21. This medium contains also ferrous sulfate as an indicator for H2S production.
H2S + FeSo4
FeS
Black ppt. of ferrous sulfid

22. Results: acidic (yellow) acidic (yellow) acidic (yellow) Butt: Slant:
H2S Production:
acidic (yellow)
alkaline (red)
alkaline (red)
-ve
+ve
-ve

23. 3-O/F Test (Oxidation Fermentation Test)
Principle:
Saccharolytic bacteria attack carbohydrates either:
fermentatively ( in absence of oxygen) to yield relatively strong acids, or
oxidatively ( in presence of oxygen) to yield weak acids.
Oxidation process is much more easier than Fermentation process.

24. Procedure:
1 ml liquid paraffin O F

25. O-/F+ O-/F- O+/F+ O+/F- Results: Positive Test: Non Saccharolytic
Fermentative
Oxidative

26. sensitive O/F Test (Oxidation Fermentation Test)
Principle:
sensitive O/F medium is a specifically formulated medium to detect weak acids produced from saccharolytic Gram’s –ve bacteria.

27. To be more sensitive this medium contains:
Higher conc. Of sugar to increase amount of acid produced.
Lower amount of peptone to reduce formation of alkaline amines which neutralize weak acids formed.
Lower conc. Of agar making the medium semisold to facilitate diffusion of acid throughout the medium.

28. Oxidation Fermentation (O/F) Test:
Procedure:
1 ml liquid paraffin O F

29. Oxidation Fermentation (O/F) Test:
Positive Test:
Results:
O-/F+
O-/F-
O+/F+
O+/F-
Non Saccharolytic
Fermentative
Oxidative

30. 4- MR-VP Test: Principle: Dextrose Acidic pathway Neutral pathwayOR
Neutral pathway
Mixed acids
 pH less than 4.4
Acetyl methyl carbinol
α-naphthol
KOH
MR indicator
Brick red colour
Red colour
MR +ve
VP +ve

31. Amino acid
Metabolism

32. Purple red color in the upper organic layer
4- Indole test
Principle:
Tryptophanase
enzyme
Tryptophane
Indole
Kovac’s reagent
Purple red color in the upper organic layer

33. Results:
+ve
-ve

34. Thank you