Gram-Negative Bacilli Approach
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Direct answer
Oxidase first, lactose second: that is how the Gram-negative bacilli fall into place. Oxidase-positive rods such as Pseudomonas, Vibrio and Aeromonas are distinguished from the oxidase-negative Enterobacterales, after which lactose fermentation on MacConkey agar splits the Enterobacterales into rapid fermenters (Escherichia coli, Klebsiella), late or non-fermenters (Citrobacter, Serratia) and non-fermenters (Salmonella, Shigella, Proteus). Triple sugar iron slopes, IMViC reactions and a few rapid enzymes (urease, phenylalanine deaminase) then take most isolates to a species name, and non-lactose-fermenting coliforms on a stool plate always demand exclusion of Salmonella.
What you must remember
- Oxidase test: Pseudomonas aeruginosa is positive, all Enterobacterales negative; oxidase therefore decides whether a non-lactose-fermenter is Salmonella or Pseudomonas within 30 seconds.
- IMViC patterns: E. coli is positive-positive-negative-negative; Klebsiella pneumoniae and Enterobacter are negative-negative-positive-positive; Proteus is indole variable but phenylalanine deaminase positive — a test almost specific for Proteus, Providencia and Morganella.
- Triple sugar iron reading: acid slant with acid butt and gas means glucose plus lactose fermented (E. coli, Klebsiella); alkaline slant with acid butt means glucose only (Salmonella, Shigella); blackening adds hydrogen sulphide.
- Salmonella signatures: S. Typhi gives little or no hydrogen sulphide and no gas; S. Typhimurium and S. Paratyphi B give abundant hydrogen sulphide with gas; S. Paratyphi A gives gas without hydrogen sulphide — a classic Ananthanarayan table.
- Shigella: non-motile (sharing non-motility only with Klebsiella among common Enterobacterales), hydrogen sulphide negative; S. flexneri is the commonest Indian serogroup, S. dysenteriae type 1 carries the Shiga toxin of epidemic dysentery.
- Pseudomonas aeruginosa: pyocyanin (blue-green) is produced by this species alone, plus fluorescein, a grape-like odour, growth at 42 but not 4 degrees Celsius, and multidrug resistance by nature.
- Vibrio cholerae: darting shooting-star motility on dark field, enrichment in alkaline peptone water pH 8.6, yellow colonies on thiosulphate citrate bile salts sucrose agar, and a positive string test.
How to read a triple sugar iron slope step by step
Begin with the slant, because lactose and sucrose are present in high concentration and only vigorous fermenters acidify the aerobic slant. An acid (yellow) slant with acid butt, gas bubbles and no blackening is E. coli or Klebsiella; add a negative indole and mucoid, lactose-fermenting colonies with a positive capsule string test and Klebsiella is confirmed. An alkaline (red) slant with acid butt says glucose alone was fermented, which is Salmonella or Shigella territory.
Now read the butt. Blackening means hydrogen sulphide from thiosulphate, so an alkaline slant, acid butt, gas and heavy blackening points to S. Typhimurium. If the butt is acid without gas and blackening is minimal, S. Typhi is the pick, and slide agglutination with O and H antisera plus enrichment history confirms it. Urea splitting within minutes of touching a Christensen urea slope would instead announce Proteus, whose swarming growth you already saw overnight across the blood plate — a habit CLED agar deliberately abolishes.
The lesson is that these cheap tests form a decision tree; running them in the right order names most Indian clinical isolates of Gram-negative bacilli within 24 hours, long before an automated machine is needed.
Where marks are lost
Candidates report "Salmonella isolated" without asking which week of illness the sample came from, yet specimen choice in enteric fever is week-dependent: blood culture positivity peaks in week 1, stool and urine culture do better in weeks 2 and 3, and the Widal test is worth interpreting only from week 2, with TO titres of 1:160 and TH titres of 1:320 or more taken as provisional cutoffs in many Indian laboratories against local baseline titres. A second recurring loss: calling every non-lactose-fermenter a Salmonella when Proteus, Pseudomonas and Acinetobacter (an oxidase-negative, non-motile coccobacillus that is neither) sit in the same colonial territory.
Frequently asked questions
Which enzyme test identifies Proteus among Enterobacterales?
Phenylalanine deaminase, positive in Proteus, Providencia and Morganella, which deaminate phenylalanine to phenylpyruvic acid; rapid urease positivity and swarming motility support the identification.
What distinguishes S. Typhi biochemically?
On triple sugar iron, S. Typhi gives an alkaline slant, acid butt, no gas and only scanty hydrogen sulphide, unlike S. Typhimurium's abundant blackening and gas.
What single pigment identifies Pseudomonas aeruginosa?
Pyocyanin, the blue-green phenazine pigment produced by no other human pathogen; fluorescein is a fluorescent yellow-green adjunct also seen in other species.
What colony count and medium handle Vibrio isolation?
Enrich stools in alkaline peptone water at pH 8.6 for 6-8 hours, then subculture to thiosulphate citrate bile salts sucrose agar where sucrose-fermenting Vibrio cholerae appears as yellow colonies.
Which common Enterobacterales are non-motile?
Shigella and Klebsyella — remembering this pair quickly distinguishes a non-motile, lactose-negative stool isolate (Shigella) from everything else that swims.