Isolation And Identification

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Most methods used to isolate and identify Salmonella are modifications of methods originally developed for clinical specimens. These modifications begin with preenrichment of test samples and follow through selective enrichments, selective plating, differential testing and plating, confirmatory biochemical determinations, and confirmatory serological testing (7,15).

A preenrichment culture is recommended for the recovery and identification of microorganisms that may have suffered injury or stress in some operational step of a process. The most common preenrichment broths are lactose and brilliant green, although lauryl tryptose broth, mannitol purple sugar broth, and nutrient broth are also used. These preenrichment broths allow for growth of most organisms present in the test sample. After this preenrichment step, selective enrichment is done to provide an opportunity for the small numbers of Salmonella to grow while the competing organisms are inhibited. Enrichment broths recommended for salmonellae include tetrathionate and sele-nite cystine broth. However, selenite cystine broth will quite likely be replaced with RV Rapport-Vassiliadis medium in the near future (15,16).

The use of selective enrichment broth, while allowing the growth of Salmonella, results in mixed cultures containing other organisms. Plating to highly selective media will allow the development of discrete salmonellae colonies while inhibiting the growth of other bacteria. Growth of discrete colonies on these media also permits recognition of colonies that are suspected to be Salmonella and transfer of these pure colonies to other media for confirmation of identity.

Selective and differential media used for salmonellae (Table 1) include Salmonella-Shigella (SS) agar, bismuth sulfite (BS) agar, and xylose lysine desoxycholate (XLD) agar. Others that are used include brilliant green agar and desoxycholate citrate agar. These media are highly selective and may inhibit growth of some salmonellae isolates. Several media that are less selective but primarily differential are MacConkey (MAC) agar, Hektoen Enteric (HE) agar, desoxycholate agar, and eosin-methylene blue (EMB) agar. The inclusion of the primarily differential media provides a better opportunity to obtain growth from all isolates, even those that may be inhibited by the highly selective media.

Suspected salmonellae isolates are subjected to a series of biochemical tests (Table 2) for further confirmation (7,15). These tests include glucose, lysine decarboxylase, hydrogen sulfide, urease, indole, Voges-Proskauer, citrate, methyl red, motility, failure to ferment sucrose or lactose, and growth in KCN broth. Once an isolate is confirmed as Salmonella, serological testing can be completed. Commercial preparations of Salmonella O, H, and Vi antibodies are available. Generally,

TABLE 1 Typical Growth Characteristics of Salmonella on Some Commonly Used Selective and Differential Media

Media

Colony appearance

Salmonella-Shigella (SS) agar

Colorless colonies on a pink background

Bismuth sulfite (BS) agar

Black colonies surrounded by a brown to black zone that casts a

metallic sheen

Brilliant green (BG) agar

Pink colonies surrounded by red zone

Xylose lysine desoxycholate (XLD)

Black-centered red colonies with H2S producers, red colonies

agar

with non-producers

MacConkey (MA) agar

Uncolored, transparent colonies

Hektoen Enteric (HE) agar

Blue to blue-green colonies, most with black centers (H2S pro-

ducers)

Eosin-methylene Blue (EMB) agar

Translucent amber to colorless colonies

Source: From Ref. 7.

Source: From Ref. 7.

serological testing for Salmonella begins with the O antisera, and H and Vi are reserved for later use in specific identifications. Salmonella serotypes can be further differentiated based on reactivity with a defined set of bacteriophage. This is generally used for epidemiological purposes and is performed in highly specialized laboratories.

The conventional culture methods described above require 4 days to complete, and this does not include serotyping and, if necessary, phage typing. Several rapid methods are currently being developed to identify Salmonella in general, while specific tests to identify serotypes are also under development. Among the rapid assays (17) presently available are culture methods that use selective and differential media, enzyme immunoassays, latex agglutination, immunodiffusion techniques, DNA colony hybridization (17), and the polymerase chain reaction (PCR) (18,19). These methods

TABLE 2 Biochemical Reactivity of Salmonella

Test or substrate

Positive reaction

Negative reaction

Salmonella reactivity3

Glucose

Yellow butt

Red butt

+

Lysine decarboxylase

Purple butt

Yellow butt

+

H2S

Blackening

No blackening

+

Urease

Purple-red color

No color change

-

Lysine decarboxylase broth

Purple color

Yellow color

+

Phenol red dulcitol broth

Yellow color and/or gas

No gas, no color change

+b

KCN broth

Growth

No growth

-

Malonate broth

Blue color

No color change

_c

Indole test

Violet color at surface

Yellow color at surface

-

Phenol red lactose broth

Yellow color and/or gas

No gas, no color change

_c

Phenol red sucrose broth

Yellow color and/or gas

No gas, no color change

_

Voges-Proskauer test

Pink-to-red color

No color change

_

Methyl red test

Diffuse red color

Diffuse yellow color

+

Simmons citrate

Growth, blue color

No growth, no color change

v

a +, 90% or more positive in 1 or 2 days; —, 90% or more negative in 1 or 2 days; v, variable. b Majority of S. arizonae cultures are negative. c Majority of S. arizonae cultures are positive. Source: From Refs. 7 and 15.

were designed to identify all Salmonella species. With the dramatic increase in outbreaks due to Enteritidis and the necessary surveillance of poultry and poultry products for this serotype, several methods have been developed for the rapid identification of Enteritidis. These assays include an enzyme-linked immunoabsorbent assay (ELISA) using two monoclonal antibodies (20), colony hybridization (21), and PCR (22). Although all of these methods are more rapid than traditional culture procedures, they currently are used only as a screening tool. Standard culture, serological, and biochemical methods are still used for definitive identification of Salmonella.

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