Bacteriology 102:
Outline of Enteric Isolation
Part 1

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Isolation Lecture Outline, Part 1
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Modified:
9/14/00

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.


I.  GENERAL PLAN FOR THE ENRICHMENT, ISOLATION AND IDENTIFICATION OF BACTERIA.

This "flow chart" summarizes the main elements of bacterial isolation as taught in our Bacteriology 102 course and applied in our enrichment and isolation exercises.

ENRICHMENT AND ISOLATIONPURE CULTURE WORK
SOURCE
MATERIAL
BROTH
ENRICHMENT
PLATING FOR
ISOLATION
STOCK
CULTURES
CHARACTERIZATION & IDENTIFICATION
•Consider inoculum: what organisms may or may not be present.
•May pre-treat inoculum, e.g., by heat-shocking.
•Usually is selective.
•May be skipped altogether.
•Usually selective or selective-differential – but not always!! 
Note: Throughout procedure, appropriate media and incubation conditions must be considered. 

II.  SUMMARY OF PROCEDURES FOR THE DETECTION AND ISOLATION OF COLIFORMS AND SALMONELLA.

This summary generally follows the basic outline above and indicates the objectives for the various steps of these procedures.

PROCEDURE and SOURCEBROTH ENRICHMENTSPLATING/ISOLATIONCHARACTERIZATION and IDENTIFICATION
COLIFORMS with use of water or food sample•Select against gram-positive bacteria.
•Look for gas from lactose fermentation.
•A two-step enrichment1 is performed, utilizing the MPN procedure.
•EMB Agar is used traditionally.
•Select against gram-positive bacteria.
•Pick colonies which show lactose fermentation.
•Check gram reaction and lactose fermentation.
•Run IMViC tests.2
•Do other tests to identify to the desired level. (MIO Medium is useful.)
SALMONELLA with use of food or fecal sample•Encourage growth of Salmonella.
•Inhibit most other organisms.
•MPN series may be set up.
•May begin with a non-selective pre-enrichment followed by the traditional, selective enrichment.
•Select against gram-positive (and many gram-negative) bacteria.
•Pick colonies which show no lactose (or no lactose and sucrose) fermentation.
•May utilize Bismuth Sulfite Agar which will show the special colonial appearance of Salmonella.
•Initially inoculate screening media (e.g., LIA,TSI) to screen out as many non-salmonellae as possible.
•Serological identification.
•Perform other tests as may be required for further, complete identification.

1 Two-step Enrichment for Coliforms:

2 IMViC Tests:

I = indole production from tryptophan
M = methyl red test
V = Voges-Proskauer test
C = citrate utilization

 IMVC
Most strains of E.coli++
Most strains of
Klebsiella and Enterobacter
++
Most lactose-positive strains of
Citrobacter
+ or –++

III.  COMMONLY-USED SELECTIVE AGENTS AND pH INDICATORS IN ENTERIC MEDIA.


IV.  PRODUCTION AND DETECTION OF HYDROGEN SULFIDE.


Go on to Part 2 of this outline.
Many of the media mentioned herein are
illustrated and explained here.
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