Bacteriology 102:
Outline of Enteric Isolation
Part 2

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Isolation Lecture Outline, Part 2
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Modified:
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As given in the Laboratory Manual for the Food Microbiology Laboratory (1998 edition, edited by John L. and published at the University) to go along with the food microbiology lab lectures given in the 1990's on the isolation of enterics in general and of coliforms and Salmonella in particular.


V.  SELECTIVE BROTH MEDIA FOR THE ENRICHMENT OF COLIFORMS AND SALMONELLA.

 COLIFORMSSALMONELLA
mediumLactose Lauryl
Sulfate Tryptose
Broth
Brilliant Green
Lactose Bile
Broth
EC BrothTetrathionate
Broth1
Selenite-Cystine
Broth2
selective agent(s)•sodium lauryl sulfate•bile salts (Oxgall)
•brilliant green
•bile salts
•(45.5°C incubation)
•bile salts•sodium acid selenite
fermentable sugar•lactose•lactose•lactose(none)•lactose
criteria for continuation•growth
•gas bubble in Durham tube
•growth
•gas bubble in Durham tube
•growth
•gas bubble in Durham tube
•growth•growth

1 TETRATHIONATE BROTH: In the preparation of the medium, sodium thiosulfate is converted to sodium tetrathionate by the action of iodine:

2Na2S2O3 + I2 —› Na2S4O6 +2NaI

Salmonella and some other enterics (especially Proteus and Citrobacter) possess tetrathionate reductase. Tetrathionate is used as a terminal electron acceptor in the respiratory catabolism of amino acids (in the peptone). This is anaerobic respiration of the amino acids which must occur for any anaerobic growth of these organisms in the medium, as there is no fermentable energy source present. Tetrathionate reductase catabolizes the conversion of tetrathionate back to thiosulfate. Acid (H2S2O3) which is generated is buffered by the calcium carbonate in the medium. Other enterics which do not possess this enzyme will not grow. Bile salts also aid in the selectivity of the medium.

2 SELENITE-CYSTINE BROTH: Salmonella (with few other enterics) is not inhibited by selenite. It will reduce selenite (SeO3–2; Se in the 4+ state) to elemental selenium (Se0) which accumulates in the periplasmic space and is not toxic to Salmonella. Most other enterics (including most lactose fermenters) are inhibited by selenite, as it ties up their sulfhydral enzymes.


VI.  SOME SELECTIVE-DIFFERENTIAL PLATING MEDIA FOR THE ISOLATION OF COLIFORMS, SALMONELLA AND OTHER ENTERICS.

"Permissive" media allow the growth of a variety of gram-negative organisms including enterics in general and organisms like Pseudomonas. Those permissive media specifically indicated in the table below are especially useful in detecting coliforms which are among those enterics which may be inhibited to a greater or lesser degree by the "restrictive" media.

The "moderately-restrictive" and "highly-restrictive" media in the table below are designed to enhance the isolation of the more highly-resistant enteric pathogens Salmonella, Shigella, and Edwardsiella. The detection of these organisms is also enhanced as they are bacteria which tend to produce alkaline colonies on these media (typical strains do not ferment lactose or sucrose).

The exceptional medium is Bismuth Sulfite Agar which is highly selective for Salmonella. For this medium, there is no pH-related reaction to look for. Rather, one looks for colonies with a black metallic sheen, highly indicative of Salmonella – including lactose-positive strains which would tend to appear uncharacteristic of Salmonella on the other media (and will therefore not be picked).

Further discussion about enteric isolation media (with photos and more tables) is here. The use of "screening media" to provide additional information about isolated colonies (to aid in choosing likely isolates to continue with) is covered here.

selectivityPERMISSIVEMODERATELY RESTRICTIVEHIGHLY RESTRICTIVE
mediumMacConkeyEMB (Levine's)XLDHektoen EntericBrilliant GreenBismuth Sulfite
selective agent(s)•bile salts
•crystal violet
•neutral red
•eosin-Y
•methylene blue
•Na desoxycholate•bile salts•brilliant green•brilliant green
•bismuth sulfite
source of amino acids which may be deaminated
(alkaline reaction)
•peptone
•proteose peptone
•peptone•yeast extract•proteose peptone
•yeast extract
•proteose peptone
•yeast extract
•beef extract
•peptone
amino acid added to note its decarboxylation
(alkaline reaction)
(none)(none)•lysine(none)(none)(none)
fermentable sugar(s)
(acidic reaction)
•lactose (1%)•lactose (1%)•lactose (0.75%)
•sucrose (0.75%)
•xylose (0.375%)
•lactose (1.2%)
•sucrose (1.2%)
•salacin (0.2%)
•lactose (1%)
•sucrose (1%)
•glucose (0.5%)
pH indicator•neutral red•eosin-Y
•methylene blue
•phenol red•brom-thymol blue
•acid fuchsin
•phenol red(none)
color of acidic colonyred or pinkdarkyellowyellow-orangeyellow(not applicable)
color of alkaline colonywhite or graylightredblue-greenred(not applicable)
source of hydrogen sulfide (H2S)(none)(none)•Na thiosulfate•Na thiosulfate(none)•bismuth sulfite
H2S indicator (producing black color)(none)(none)•ferric ammonium citrate•ferric ammonium citrate(none)•ferrous sulfate

Many of the media mentioned herein are
illustrated and explained here.
Go back to Part 1 of this outline.
Go to the Salmonella page.
Return to the general overview of enterics.
Page last modified on 9/14/00 at 10:00 AM, CDT.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison