1. Enterobacteriaceae

2. The Enterics Family Enterobacteriaceae often referred to as “enterics”
Four major features:
All ferment glucose (dextrose)
All reduce nitrates to nitrites
All are oxidase negative
Many found in the intestines of human or other mammals
Varying pathogenicity:
Commensals, opportunists or pathogens

3. Laboratory Diagnosis of Enterics
Sugar fermentation
Phenol red broth
Triple Sugar Iron (TSI)
Methyl red - Vogues Proskauer
SIM
Break down of tryptophan and cysteine; motility
Citrate utilization
Urease production
Phenylalanine deaminase
Decarboxylase tests

4. Phenol red broth 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 - Fermentation
Orange - neutral pH - Oxidation
Red - alkaline pH - Oxidation

5. Phenol Red Broths Yellow (acid) + gas Yellow (acid) no 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

6. TSI — Three Sugars and Iron
Red slant/Red butt
No fermentation
Protein catabolism
Red slant/Yellow butt
Only glucose fermentation
Yellow slant/yellow butt
Lactose and/or sucrose fermentation

7. Methyl Red Test Test for fermentation with acid accumulation
Carbon Sources: Glucose and proteins
Glucose  pyruvate  lactic and/or acetic acid + CO2
Indicator -methyl red
MR +: red (pH < 5.2)
MR - : Yellow (pH > 5.2)
Neutral
Acid

8. - + Voges-Proskauer Test
Used to determine fermentation with the production of neutral end products
Glucose  pyruvate  acetoin  butanediol
Reagents
Acetoin + -naphthol + KOH + O2  Red color - +

9. Sim Test : Sulfide, Indole, Motility
Sulfide: Identifies bacteria capable of metabolizing cysteine
Cysteine  H2S; H2S + FeSO4  Black precipitate
Indole: Identifies bacteria capable of metabolizing tryptophan
Convert tryptophan to indole and pyruvic acid by using the enzyme tryptophanase
Kovac’s reagent reacts with indole to give a red color

10. Non-motile
Non inoculated
Indole
H2S and motile

11. Citrate Utilization Sodium citrate as a sole carbon source
pH indicator - bromothymol blue.
Sodium citrate → Pyruvic acid + Oxalacetic acid + CO2
Excess sodium from Sodium Citrate + CO2 + H2O
→ Na2CO3 (pH : green → blue)

12. Urease Test
Used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease
The pH indicator, phenol red, is used to detect the breakdown of urea and the production of ammonia
As the alkaline end products build, NH4(CO3)2, phenol red turns to pink

13. Phenylalanine Deaminase Test
Used to identify bacteria possessing the enzyme phenylalanine deaminase
Phenylalanine deaminase converts phenylalanine to phenylpyruvic acid + NH3
Ferric chloride (FeCl3) reacts with the phenylpyruvic acid and changes color from yellow to green

14. Decarboxylase Test Contains glucose, test amino acid and pH indicator
Fermentation of glucose generates acids (turns medium yellow) and activates decarboxylase
If decarboxylase is present it generates alkaline products and changes pH indicator to purple (positive).
If no decarboxylase present then media remains acid and yellow (negative)

15. Ornithine Decarboxylase Broth
Interpretation
Tubes 1 –aa & +aa: Acid reaction - glucose fermentation; no decarboxylation
Tube 2 -aa: Acid reaction - glucose fermentation
Tube 2 +aa: Acid reaction - glucose fermentation followed by decarboxylation (alkaline reaction)
- aa aa
-aa aa 1 2

16. Lysine Decarboxylase Lysine iron agar slants
Contain lysine, glucose, peptones, bromcresol purple (pH indicator), sodium thiosulfate
Lysine decarboxylation is an anaerobic process which occurs in the butt
Decarboxylation of lysine produces an amine end-product which reacts with the pH indicator to give a purple color in the butt
Negative decarboxylation: yellow butt

17. Lysine Deaminase Lysine iron agar slants
Lysine deamination is an aerobic process which occurs on the slant
Lysine deamination generates ammonia which reacts with pH indicator to produce a dark red color on the slant
Negative deamination: purple slant

18. Lysine Iron Agar slants
Purple slant & butt
Decarboxylase positive
Purple slant & yellow butt
Decarboxylase negative
Purple slant & black butt
Red slant & yellow butt
Deaminase positive

19. Nitrate Reductase Test
Used to detect the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) or some other nitrogenous compound, such as molecular nitrogen (N2) using the enzyme nitrate reductase

20. Nitrate Reductase Step 1: Test for nitrite
NO H+ + 2 e-  H2O + NO2- 
Nitrate
Nitrite
NH3 or N2
Step 1: Test for nitrite
NO2- + sulfanilic acid and alpha naphthylamine  HNO2
Red color indicates presence of Nitrite
Nitrate was reduced
Yellow color indicates absence of Nitrite
Nitrate was not reduced
Nitrate was reduced to Nitrite which was further reduced + ?

21. Nitrate Reductase (Cont’d)
Interpretation of ambiguous result after step 1
Step 2: Test for the presence of nitrate
NO3- + Zn (s)  NO2- + -
Red color indicates presence of Nitrate
Nitrate was not reduced by bacteria Nitrate was reduced by Zn
Yellow color indicates absence of Nitrate
Nitrate was reduced to Nitrite which was further reduced