1. BIOCHEMICAL TESTING

2. Distinguishing Enterobacteriaceae
Large family of bacteria
Gram negative rods
Capable of fermenting various sugars
Many found in the intestines of human or other mammals
Varrying pathogenicity:
Commensals, opportunists or pathogens
Some found in the environment
Example species:
Escherichia coli
Klebsiella pneumoniae
Citrobacter freundii
Enterobacter aerogenes
Proteus mirabilis
Salmonella typhi
Shigella dysenteriae
Yersinia enterocolitica

3. Previous Experience with Enterobacteriaceae: EMB Media
Large amounts of acid from lactose fermentation cause the dyes to precipitate on the colony surface, producing a black center or a “green metallic sheen” (E. coli)
Smaller amounts of acid production result in pink coloration of the growth (E. aerogenes)
Nonfermenting enterics do not produce acid so their colonies remain colorless or take on the color of the media (P. vulgaris)

4. Biochemical Testing
Triple Sugar Iron (TSI): fermentation of sugars, sulfur reduction
IMViC:
Indole: Break down the amino acid Tryptophan
Methyl Red: Glucose oxidation
Voges-Proskauer: Production of neutral end products
Citrate: Citrate fermentation
Urease: Hydrolyzation of Urea
Phenylalanine Deaminase: converts the amino acid phenylalanine to phenylpyruvic acid
Nitrate Reductase: Reduction of nitrate (NO3) to nitrite (NO2)

5. TRIPLE SUGAR IRON TEST (TSI)
Used to differentiate among the different groups of Enterobacteriaceae
based on their ability to ferment glucose, lactose and/or sucrose
Also differentiates between groups capable of reducing sulfur to hydrogen sulfide
(Sodium Thiosulfate -> Hydrogen sulfide)

6. TSI Results: Red slant/Red butt = no fermentation
Red slant/Yellow butt = only glucose fermentation
Yellow slant/yellow butt = lactose and/or sucrose fermentation
Dark color: Hydrogen Sulfide produced
Sodium thiosulfate reduced
P 190

7. IMViC TESTS A series of four tests consisting of:
Used to differentiate the Enterobacteriaceae
We will look at each test individually
Indole: Break down the amino acid Tryptophan
Methyl Red: Glucose oxidation
Voges-Proskauer: Production of neutral end products
Citrate: Citrate fermentation

8. Indole Test (SIM: Sulfide,Indole,Motility)
Identifies bacteria capable of producing indole
Some bacteria are capable of converting tryptophan (an amino acid) to indole and pyruvic acid by using the enzyme tryptophanase
Pyruvic acid can be converted to energy or used to synthesize other compounds required by the cell
Tryptophan Indole Ring Pyruvic Acid Ammonia

9. Procedure: Obtain 4 SIM Deep tubes
Inoculate by the stab method with the following organisms:
E.coli, P.vulgaris,
E. aerogenes K. pneumoniae

10. Indole Test Results:
If indole is produced, upon addition of Kovac’s Reagent (10 drops), a “cherry-red” band forms on the surface of the media
Motility (if present) can be seen as growth of the bacteria away from the stab line
Sulfur in the media may be reduced to hydrogen sulfide (H2S); this appears as a “blackening” within the media

11. Methyl Red Test
Used to determine the ability of a bacteria to oxidize glucose and produce stable acid end products
Methyl red is a pH indicator (red at pH less than 4.4 and yellow at a pH greater than 6)
The combination medium used for this test is the MR-VP (methyl red/Voges-Proskauer) broth
Acid production: positive methyl red
End products of neutral pH : positive Voges-Proskauer

12. Procedure: Obtain 3 MR-VP broth tubes
Inoculate (using a loop) with the following organisms:
E.coli K.pneumoniae E.aerogenes

13. Results:
From the 3 MR-VP broths that you inoculated, transfer 2 mLs from each and place into 3 separate clean tubes (set aside these aliquots for the VP test)
To the remaining, original tubes that you inoculated add 5 drops of methyl red indicator
A red color indicates that glucose has been oxidized

14. Methyl Red Test Results:
A red color indicates that glucose has been oxidized.
Methyl red positive
tube on the right
Methyl red negative
tube on the left

15. Voges-Proskauer Test
Used to determine the ability of microbes to produce nonacidic or neutral end products
Remember that the MR-VP broth is a combined medium used for two tests—
Methyl Red and Voges-Proskauer
You have already inoculated the 3 MR-VP broth tubes from the previous procedure (Methyl Red Test) with
E.coli K.pneumoniae E.aerogenes

16. Voges-Proskauer Procedure:
To the aliquots of each broth culture separated during the methyl red test, add:
10 drops of Barritt’s Reagent A; shake
10 drops of Barritt’s Reagent B; shake
Reshake the culture every 3 to 4 min.
It can take as long as 15 min. for a color change to occur

17. Voges-Proskauer Results:
The presence of a deep rose color after 15 minutes is indicative of non-acidic / neutral metabolic end products and a positive VP test result.
Voges-Proskauer
positive on the right
negative on the left

18. Citrate Utilization Test
Used to determine if an organism is capable of fermenting citrate and using that citrate as its sole carbon source
The ability of an organism to utilize citrate occurs via the enzyme citrase

19. Procedure: Obtain 3 Simmons Citrate agar slants
Inoculate these slants using the stab and streak method (the same way you inoculated the TSI media using a needle) with the following organisms:
E.coli K.pneumoniae E.aerogenes

20. Citrate Test Results:
Simmon’s Citrate agar utilizes sodium citrate as its sole carbon source
Bromthymol blue is included as a pH indicator; the medium initially is green
Organisms capable of using citrate as a carbon source turn the media “Prussian blue”.

21. Summary of IMViC Reactions
Page 199

22. 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 which is used by bacteria to produce amino acids and nucleotides

23. Procedure: Obtain 2 urea broth tubes
Inoculate with the following organisms:
E.coli P.vulgaris

24. Urease Test Results:
Urinary tract pathogens from the genus Proteus may be distinguished from other enterics
urease
Urea + H2O CO2 + H20 + NH3
As the alkaline end products build, phenol red turns from yellowish gold to pink—a positive result

25. Urease Test Results
As the alkaline end products build, phenol red turns from yellowish gold to pink—a positive result
Urease positive organism on the right
Urease negative organism on the left

26. Phenylalanine Deaminase Test
Used to identify bacteria possessing the enzyme phenylalanine deaminase
Phenylalanine deaminase converts the amino acid phenylalanine to phenylpyruvic acid + NH3

27. Procedure: Obtain 2 phenylalanine agar slants
Inoculate (with a loop on the surface) with the following organisms:
E.coli P.vulgaris

28. Results Phenylpyruvic acid produced by some organisms is colorless
After inoculation and incubation, 10% ferric chloride, an oxidizing agent, is added to the surface of the slants
Ferric chloride (FeCl3) reacts with the phenylpyruvic acid (if present) and changes color from yellow to green—a positive result

29. Phenylalanine Deaminase Results:
Positive Negative
Ferric chloride (FeCl3) reacts with the phenylpyruvic acid (if present) and changes color from yellow to green — a positive result
Positive Negative

30. 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 (N3) using the enzyme nitrate reductase
NO3 NO2
Nitrate Reductase -
Forms red color once solutions A and B are added.

31. Procedure: Obtain 3 Nitrate broth tubes
Inoculate (with a loop) those tubes with the following organisms:
E.coli A.faecalis P.aeruginosa

32. Results: (Pay close attention to this test; its one of the hardest test to read)
After inoculation and incubation, the ability of an organism to reduce nitrate to nitrite (or molecular nitrogen) is detected by adding two reagents:
Solution A (sulfanilic acid)
Solution B (α-naphthylamine)
If a red color appears after addition of solution A and B, this is considered a positive result
NO3 NO2
Nitrate Reductase -
Forms red color once solutions A and B are added.

33. Results continued:
If there is no color change occurs after additions of solutions A & B, two possibilities must be considered:
nitrates were not reduced by the organism
the organism possessed such potent nitrate reductase enzymes that nitrates were reduced beyond nitrites to ammonia or even molecular nitrogen
NO3 NO3 -
NO3 NO2
Nitrate Reductase -
NH3+ (Ammonia)
N2 (Nitrogen Gas)
P 219

34. Results Continued:
Next, solution C (zinc) is added to the tubes that showed no color change after addition of Nitrate A & B
Zinc, a reducing agent, is capable of converting nitrate to nitrite; the development of a red color after addition of Nitrate C means that nitrates were present and reduced by the zinc not the organism; this is a negative result
If the addition of zinc does not produce a color change, the nitrates were reduced beyond nitrites to ammonia or nitrogen gas; this is a positive reaction
NO3 NO3 -
NO3 NO2
Nitrate Reductase -
NH3+ (Ammonia)
N2 (Nitrogen Gas)

35. Solutions A and B have been added to these tubes
Solution C has been added to these tubes
C. xerosis - Reductase Negative
NO3 NO3 -
Unreactive
tubes
P. aeruginosa
Reductase Positive
E. coli - Reductase Positive
NO3 NO2
Nitrate
Reductase -
NH3+ (Ammonia)
N2 (Nitrogen Gas)
NO3 NO2
Nitrate Reductase -