Phenylalanine deaminase Test:
Phenylalanine will undergo oxidative deamination and convert to phenylpyrovic acid which is detected by ferric chloride. Requires an 18-24 hour incubation at 35C. The test will be positive if the bacteria has the enzyme deaminase. Green is positive, no color change is negative.

Ornithine decarboxylate Test (ODC):
The ODC determines if the bacteria can decarboxylate ornithine.

Mannitol Fermentation Test:
This test determines the ability of the bacteria to ferment mannitol.

ONPG Test:
The ONPG test determines if the bacteria has beta-galactosidase which will convert o-nitrophenyl-beta-D-galactopyranoside (ONPG) to galactose and o-nitrophenyl. A color change will be observed.

Indole Test:
There are two types of indole tests, the rapid spot indole test and the overnight incubation test. The spot test will detect rapid indole producing bacteria, and the overnight will detect weak indole producing organisms along with strong indole producers. Both tests detect the presence of tyrptophanase which breaks tryptophan into multiple components including indole.

Study Tip
You might have noticed the indole test is not listed on the flow chart, and that’s because I found it much easier to omit than writing it in multiple places. I use a simple mnemonic for any encounter with an indole question. “Morgan the vulgar ox ate E. coli on the indole pasture with Ed.” Whuuut? This sentence helps to remember important bacteria that are indole positive. Morgan refers to Morganella, vulgar refers to Proteus vulgaris, ox refers to Klebsiella oxytoca, E. coli is obviously E. coli, pasture refers to Pasteurella (not Enterobacteriaceae), and Ed refers to Edwardsiella.

Other than the flow chart for G- bacilli, the Triple Sugar Iron Agar test (TSI) is one of the most fundamental and important tests to understand. The test differentiates G- bacteria based on fermentation of glucose, lactose, and sucrose, and also production of H₂S and gas.

The test is run in a two chambered tube with an aerobic slant and an anaerobic butt. The slant contains sucrose and lactose, and the butt contains glucose (insert picture). It is stab inoculated to the bottom and then the slant is streaked. The tube is then incubated for 18-24 hours at 35C in O₂ with the cap loose before determining results.

There is a short hand to reading the TSI results and they are as follows:

K = alkaline (red)
A = acid (yellow)
NC = no change
⃝     = gas production
H₂S = dark brown to black color
/ = separates the slant result from the butt results (eg K/A H₂S)

Each bacteria will have a combination of the above letters or symbols depending on its ability to ferment glucose (butt), ferment lactose or sucrose (slant), and cause and produce H₂S or gas. I’ve developed a shorthand tool to quickly be able to remember these reactions.

Slant Butt   Bacteria A Ⓐ   KEE A Ⓐ H₂S PC K Ⓐ H₂S PEd C Sal K A   ProvSS K K   PsAlc

Bacteria abbreviations: K = Klebsiella EE = E. coli/Enterobacter P = Proteus C = Citrobacter Ed = Edwardsiella Sal = Salmonella Prov = Providencia SS = Serratia/Shigella Ps = Pseudomonas Alc = Alcaligenes

I write the TSI reactions out like this because it’s easy to recall and can quickly be jotted down on scratch paper during a test for quick reference. The less time you have to spend doing mental gymnastics and remembering if a bacteria produces gas or not the better. Practice writing this out like the flow charts above until you’ve got it down no problem.

Other notes about Enterobacteriaceae:

XLD agar:
G- GI Enterobacteriaceae, differential for Salmonella and Shigella, Shigella = translucent, E. coli = yellow, Salmonella and Edwardsiella are red with black centers, Proteus are yellow or translucent with black centers, Citrobacter are yellow with black centers.