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Review of Features Which May be Noted for Many Enteric Differential Media (in Isolation and Differentiation)

Aerobic or anaerobic	Substrate	Microbial activity	Reaction noted	Some Examples
aerobic	various amino acids in peptones	deamination*	alkaline (with pH indicator)	KIA (and probably most media)
	specific amino acid present in large amount	decarboxylation	alkaline (with pH indicator)	XLD Agar, MIO, Lysine Broth
anaerobic	one or more specific sugars	fermentation	acid (with pH indicator)	MacC. Agar, KIA, Lactose Broth, etc.
	thiosulfate	reduction with formation of H2S	black color (with Fe)	Modified MacC. Agar, KIA

^* All enterics (like most common chemoheterotrophs) will deaminate amino acids in peptones, yeast extract, and similar materials. (Deamination of specific amino acids may be tested for as in Phenyl-alanine Agar; see Period 4.

Review of Selective-Differential Media For Initial Isolation

	MacConkey Agar (modified)	Brilliant Green Agar 3	XLD Agar 3
selective agent(s)	bile salts, crystal violet, neutral red	brilliant green	sodium desoxycholate
source of amino acids which may be deaminated 1	peptone, proteose peptone	proteose peptone, yeast extract	yeast extract
amino acid added for detection of decarboxylation 1	none	none	lysine
fermentable sugar(s) 2	lactose (1%)	lactose (1%), sucrose (1%)	lactose (0.75%), sucrose (0.75%), xylose (0.375%)
pH indicator	neutral red: net acid = red, net alkaline = white	phenol red: net acid = yellow net alkaline = red	phenol red: net acid = yellow net alkaline = red
source of H2S	sodium thiosulfate	none	sodium thiosulfate
indicator of H2S production	ferric ammonium citrate	none	ferric ammonium citrate

¹ alkaline reactions.

² acid reactions.

³ optional media; may be used in Exp. 14.

How Acid And Alkaline Reactions Influence The Net pH Observed For Colonies On Various Selective-Differential Media

In the isolation of the enteric pathogens Salmonella and Shigella from food or clinical samples, one usually takes advantage of their gram-negativity (hence the media are selective against gram-positive bacteria) and the fact that they usually react negative for lactose and sucrose fermentation. Therefore, one tends to pick alkaline (non-acid-producing) colonies, ignoring the acid colonies which are produced by coliforms and certain other enterics.

Colonies of usual strains of Citrobacter (a commonly-occurring, non-pathogenic enteric) look identical to those of Salmonella on most isolation media. To assist in differentiating these organisms, XLD Agar is useful.

Shown below is a comparison of Salmonella to selected nonpathogenic enterics, showing the bases for the net pH reactions (and consequently the colony color) one may see on these media, the components of which were given in the preceding section. H₂S production is not pH-related but its detection (with a black color) is a feature of many selective-differential media. (MMAC=Modified MacConkey Agar; BGA=Brilliant Green Agar; XLD=XLD Agar.)

biochemical process	typical Salmonella	typical Citrobacter	typical coliform	process applicable to
Amino Acid Deam.	+ (alk)	+ (alk)	+ (alk)	X	X	X
Lactose Ferm.	-	-	+ (acid)	X	X	X
Sucrose Ferm.	-	-	+ (acid)		X	X
Xylose Ferm.	+ (acid)	+ (acid)	+ (acid)			X
Lysine Decarbox.	+ (alk)	-	+ or -			X
H2S Production	+	+	-	X		X

appearance in	net reaction of typical Salmonella	net reaction of typical Citrobacter	net reaction of typical coliform
MMAC	alkaline(white)+black	alkaline(white)+black	acidic (red or pink)
BGA	alkaline (red)	alkaline (red)	acidic (yellow)
XLD	alkaline (red)+black	acidic (yellow)	acidic (yellow)

Review of Differential Media Used For Preliminary Biochemical Screening

	Kligler Iron Agar (KIA)	Lysine Iron Agar (LIA) 3
source of amino acids which may be deaminated 1	peptone, proteose peptone, beef extract, yeast extract	peptone, yeast extract, lysine
amino acid added for detection of decarboxylation 1	none	lysine
fermentable sugar(s) 2	lactose (1%), glucose (0.1%)	glucose (0.1%)
pH indicator	phenol red: net acid = yellow net alkaline = red	brom-cresol purple: net acid = yellow net alkaline = purple
source from which H2S may be produced	sodium thiosulfate	sodium thiosulfate
indicator of H2S production	ferrous sulfate	ferric ammonium citrate

¹ alkaline reactions.

² acid reactions.

³ optional medium; may be used in Exp. 14.

Explanation Of Reactions Seen in KIA

(First four tubes are demonstrated)

examples	Pseudomonas (a non-enteric)	Morganella Providencia Serratia Shigella	Citrobacter Proteus Salmonella	Escherichia Enterobacter Klebsiella	occasional strains (none for Exp. 14)
glucose fermentation (acid rx.)	-	+	+	+	+
lactose fermentation (acid rx.)	-	-	-	+	+
H2S production (black color of FeS)	-	-	+	-	+
deamination of amino acids (alkaline rx.)	+	+	+	+	+

^* A slight pink or peach color may be seen in the upper part of the slant. Also, much gas (not depicted in this figure) is usually present, indicated by gaps in the butt of the medium.

Key for Enteric Unknowns

	Principal Differentiating Tests				Additional Differentiating Tests
	Phenylalanine Deam.	H2S Production	Lactose Ferm.	Motility	Indole Prod.	Methyl Red	Simmons Citrate	Lysine Decarbox.	Ornithine Decarbox.
Escherichia coli	-	-	+ (w/gas)	+	+	+	-	+	+ or -
Enterobacter cloacae	-	-	+ (w/gas)	+	-	-	+	-	+
Enterobacter aerogenes	-	-	+ (w/gas)	+	-	-	+	+	+
Klebsiella pneumoniae	-	-	+ (w/gas)	-	-	- or ±3	+	+	-
Klebsiella oxytoca	-	-	+ (w/gas)	-	+	- or ±3	+	+	-
Typical Shigella 1	-	-	-	-	+ or -	+	-	-	+ or -
Typical Salmonella 1	-	+	-	+	-	+	+	+	+
Citrobacter freundii	-	+	+2 or -	+	-	+	+	-	+ or -
Proteus vulgaris	+	+	-	+	+	+	-	-	-
Proteus mirabilis	+	+	-	+	-	+	+ or -	-	+
Morganella morganii	+	-	-	+	+	+	-	-	+
Providencia rettgeri	+	-	-	+	+	+	+	-	-

¹ Included on table for comparison only. (Enteric pathogens are not included in your unknown.)

² Lactose fermentation should not be used to differentiate Citrobacter from other organisms. Many

strains of Citrobacter (including the one we generally use) ferment lactose weakly (just a trace of yellow in Lactose Fermentation Broth after 1 or 2 days); such organisms appear lactose-negative on MacConkey Agar and in Kligler Iron Agar.

³ ± = equivocal (orange) reaction in MR-VP Broth.

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