1. Bacteria identification : Media

2. What you have to know about the media
What are the sources of C,H,N,O,P,S?
What type of media is it?
What are the indicators?
What are the selective agents?
They allow the growth of what bacteria?
What are the possible reactions?

3. Ex. MacConkey Agar Peptone - 17 g Proteose peptone - 3 g
Lactose - 10 g
Bile salts g
Sodium chloride - 5 g
Neutral red g
Crystal Violet g
Agar g
Sources of C,H,N,O,P,S?
Type of media?
Indicators?
Selective agents?
Allow growth of what bacteria?
Possible reactions?

4. Identification : Complex Carbon Sources

5. Complex Carbon Utilization
Too large to be transported inside
Requires exocellular enzymes for the external degradation into smaller units
Polysaccharides (starch)
Lipids (triglycerides, etc…)
Proteins (casein)
Polynucleotide chain (DNA)

6. Complex Carbon Sources: Starch
Media used: Starch Agar
Detected Enzyme: α-amylase
cleaves α-1,4 bound between glucose monomers
Identification: Iodine (halo = starch digestion)

7. Complex Carbon Sources: Starch
Before iodine addition
After iodine addition

8. Complex Carbon Source: Protein
Media used: Milk agar
Detected Enzyme: Caseinase (protease)
cleaves peptide bounds joining amino acids in the casein protein
-Identification: clear area (halo) under and surrounding growth

9. Complex Carbon Source: Protein

10. Complex Carbon Sources: Fatty Acids
Media used: Spirit Blue
Detected Enzyme: Lipase
can degrades complex fats (triglycerides) into individual fatty acids
Identification: Spirit Blue (clear area (halo) under and surrounding growth)

11. Complex Carbon Sources: Fatty Acids

12. Complex Carbon Sources: DNA
Media Used: DNA agar
Detected Enzyme: DNase
catalyzes the hydrolytic cleavage of phosphodiester bounds in the DNA backbone
Identification: Precipitates of polymerized DNA are opaque, clearing represents digestion of DNA

13. Complex Carbon Sources: DNA

14. Identification: Metabolic Tests
Phenol red broth
Allows determination of carbon source preferred and metabolism (Oxidation or fermentation)
Contains simple carbon sources:
Peptone (protein  amino acids)
Desired sugar added
Contains a pH indicator
Phenol red
Yellow - acid pH
Orange - neutral pH
Red - alkaline pH

15. Phenol Red Broths - Interpretation
Yellow (acid) + gas = Fermentation of sugar
Yellow (acid) no gas = Fermentaion of sugar
Orange (neutral) no gas = Oxidation of sugar
Red (alkaline) no gas = Oxidation of proteins
Uninoculated

16. TSI — Three Sugars and Iron
Glucose (limiting)
Sucrose
Lactose
Proteins
Cysteine
Indicator
Phenol red

17. IMViC Tests Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) :
These four tests include an important series of determinations which are collectively called the IMViC reaction series
The IMViC reaction series allows the discrimination of bacteria of the Enterobacteriaceae family

18. IMViC Test Methyl Red-Voges Proskauer
Methyl Red Test :
Fermentation with accumulation of acids:
Glucose  pyruvate  lactic and/or acetic acid + CO2
Voges Proskauer Test
Fermentation with accumulation of butanediol
Glucose  pyruvate  acetoine  2 butanediol + CO2 - + - +

19. Methyl Red Test Test for acid accumulation
Carbon Sources: Glucose and proteins
Indicator -methyl red; Added after growth
MR +: red (pH < 5.2)
MR - : Yellow (pH > 5.2)
Neutral
Acid

20. - + Voges-Proskauer Test Reagents VP: Usual results of MR/VP:
butanediol + -naphthol + KOH + O2  acetoin
VP + = red
VP - = Yellow - +
Usual results of MR/VP:
MR+/VP-; MR-/VP+ MR-/VP-
Neutral
Acid
Acid
produced No
acetoin
Neutral
Acetoin

21. IMViC: Indole Test Principal
Some microorganisms can metabolize tryptophane by the tryptophanase
Tryptophanase
Tryptophane
Indole + acide Pyurvic + NH3
Kovac’s reagent
Red color

22. IMViC Test : Citrate Utilization
Unique carbon source
Citrate
Indicator
Bromthymol blue
Citrate utilization generates alkaline end products
Changes from green to blue
Positive
Klebsiella, Enterobacter
Negative
E. coli

23. Urea Utilization Enzyme tested pH Indicator Urease
Phenol red (turns pink)
Positive
Negative
C O H2O  CO2 + H2O + 2 NH3  (NH4)2CO3
H2N
Urea
ammonium
carbonate
(alkaline)
Amino acids

24. Urea Utilization – Phenol Red

25. Ornithine Decarboxylase Assay
Detects ornithine decarboxylase
Catalyzes the decarboxylation of ornithine
Produces diamine putrescine and carbon dioxide (causes alkaline change)
Indicator: Brom Cresol purple
Purple when alkaline or neutral
Yellow when acid

26. Ornithine Decarboxylase Assay
Left: Alkaline with and without ornithine
Center: Alkaline with ornithine, acidic without ornithine
Right: Acidic with and without ornithine

27. Phenylalanine Slants Detects phenylalanine deaminase
Phenylpyruvic acid reacts with ferric chloride to produce a green colour

28. Phenylalanine Slants A B A: Positive for phenylpyruvic acid
B: Negative for phenylpyruvic acid

29. Lysine Agar Slants Detects lysine decarboxylase
Primarily used to detect bacteria in the Enterobacteriaceae group (for example, salmonella)
Indicator: Brom cresol purple

30. Lysine Agar Slants : Brom Cresol Purple

31. Lysine Agar Slants Purple butt : lysine decarboxylase positive
Purple slant: lysine deamination negative
Yellow butt: glucose fermentation
Red slant: lysine deamination positive
Black precipitate: sulfur reduction

32. SIM — H2S, Indole and Motility
Semi-solid medium
Allows to visualize motility
Cystein metabolism
CysteineH2S; H2S+ FeSO4 Black precipitate
Tryptophan metabolism
(A) Tryptophan Indole + NH4 + Pyruvate
(B) Indole + Kovac reagent Red

33. Non-motile
Non inoculated
Indole
H2S and motile

34. Anaerobic Respiration : Nitrate Reductase
2 H+
3 H+ + 3 OH -
3 H2O
NO2- + H2O (N = +3) nitrite
NO H+ (N = +5) nitrate
2 e- Fp
Fe-S Q
Cyt b
NADH + H+
FADH2
Nitrate reductase
Interior
Exterior
Final e- acceptor

35. Anaerobic Respiration : Nitrate Reductase (con’t)
NO H+ + 2 e-  H2O NO  NO, N2O,
NH2OH,
NH3, N2
nitrate
nitrite
Step 1: Test for nitrite
NO2- + sulfanilic acid and alpha naphthylamine  HNO2
Nitrate is not reduced
No Nitrite
Yellow
Nitrate is reduced
Production of Nitrite
Red
Nitrate is reduced to nitrite
Nitrite is reduced
No Nitrite

36. Anaerobic Respiration : Nitrate Reductase (con’t)
NO H+ + 2 e-  H2O NO  NO, N2O,
NH2OH,
NH3, N2
nitrate
nitrite
Step 2: Test for the presence of nitrate
NO3- + Zn (s)  NO2-
Nitrate is present
Reduction to Nitrite
Red
Nitrate is absent
Nitrite was reduced
Yellow

37. Oxidase Test : Aerobic Respiration Electron Transport Chain
3 H2O H+
2 H+
3 H+ + 3 OH-
H2O
3 H+ + 1/2 O2
2 e- Fp
Fe-S Q
Cyt b
Cyt o
NADH + H+
FADH2
interior
exterior

38. Oxidase Test : Aerobic Respiration
phenylenediamine
Cytochrome oxidase catalyzes the reduction of a final electron acceptor, oxygen
An artifcial e- donor, phenylenediamine, is used to reduce the cytochrome oxidase
If the enzyme is present, the colorless reagent (reduced state) will turn blue (oxidized state)

39. Differential Tests for the Identification of Gram Positive Cocci

40. Blood Hemolysis Media used: Blood agar Detected Enzyme: hemolysins
Identification:
α-hemolysis: greenish hue, partial breakdown of red blood cells
β-hemolysis: clearing, breaks down red blood cells and hemoglobin completely
γ-hemolysis: no hemolysins

41. Blood Hemolysis β α γ

42. Catalase
Enzyme found in most organisms living in the presence of oxygen
Reduces peroxide, which can be damaging to a cell (free radical)
First step in the discrimination between:
Micrococcaceae (catalase positive)
Streptocaccaceae (catalase negative)

43. Catalase 2H2O2 2H2O + O2  catalase Product of respiration
Does bacteria make this?
Detect bubbles.
2H2O2 2H2O + O2 
catalase
Product of respiration
Damaging for DNA
We add this.
Add 3% H2O2 to bacterial growth
bubbles
(O2)
Aerobic metabolism requires catalase

44. Other Gram Positice Cocci Identification Tests
Bile-Esculin
Bacitracin, optochin, and Novobiocin sensitivity
Mannitol + Salts Agar
Tellurite Agar or Baird Parker Agar
Pyr Test