EnteroScreen 4™

Cat. no. L225 EnteroScreen 4™, 16x125mm Tube, 4 Layer Slant, 12ml 20 tubes/box

INTENDED USE

Hardy Diagnostics EnteroScreen 4™ is recommended for use as a single tube screen for non-lactose-fermenting, oxidase-negative, enteric pathogens isolated from stool specimens. It is specifically designed to aid in the detection of Yersinia enterocolitica, Edwardsiella, Salmonella and Shigella species.

SUMMARY

Three media, Triple Sugar Iron (TSI) Agar, Lysine Iron Agar (LIA), and Urea Agar, are used to screen non-lactose-fermentative cultures isolated from stool specimens. This battery of tests is helpful in differentiating Salmonella and Shigella from other members of the Enterobacteriaceae family.(2,3,5) Hardy Diagnostics EnteroScreen 4™ contains a unique media combination, which has been demonstrated to be less redundant and labor intensive, yet equivalent in performance, to the traditional three tube set. It is recommended that EnteroScreen 4™ be used in conjunction with the developed algorithm (see Figure 2 or Figure 3) for organism identification. The EnteroScreen 4™ system consists of four layers in a single tube; three agar layers and a petrolatum layer (see Figure 1). The tube contains, in order from bottom to top, Urea Agar, Petrolatum Plug, Modified Lysine Iron Agar, and Lysine Iron Agar. With this method, urease activity, lysine-decarboxylase, lysine-deaminase, gas production, and hydrogen sulfide production reactions can be simultaneously evaluated.(6,7)

Urea Agar, the bottom agar layer, contains urea and phenol red as a pH indicator. Organisms capable of hydrolyzing urea form ammonia as a by-product, thus turning the medium alkaline. The pH indicator turns from pale orange to pink-red in color under these conditions. The reduced buffer content and peptone in this medium promote more rapid growth and reaction time for many members of the Enterobacteriaceae. Dextrose is included in the formulation to stimulate urease activity in organisms that hydrolyze urea slowly, and to exclude false-negative reactions.

Urea Agar is added to the tube to differentiate other non-lactose-fermenters, i.e. Proteus, Morganella and Providencia species, from Yersinia enterocolitica, lactose-negative E. coli, Edwardsiella, Salmonella, and Shigella species. These latter organisms, all potential stool pathogens, can be separated from non-pathogenic organisms by the inability to hydrolyze urea. However, some strains of Yersinia enterocolitica may hydrolyze urea and can be differentiated from non-pathogenic organisms by the inability to deaminate lysine, lack of H2S production, and the absence of gas. The Urea Agar, is separated from the next agar layer by the addition of a sterile, hydrophobic petrolatum plug.(2,3,5-7)

Modified Lysine Agar comprises the agar layer above the petrolatum plug and is used to detect by-products of dextrose-fermentation and the presence of lysine-decarboxylase. The media formulation is similar to traditional Lysine Iron Agar (LIA), however, hydrogen sulfide indicators are absent, to prevent excessive hydrogen sulfide production from masking the lysine-decarboxylase reaction.(6,7) The media contains 0.1% percent dextrose as a carbohydrate source. Enteric organisms that are capable of fermenting dextrose will produce acid and sometimes gas, seen as cracks and bubbles in the medium.

Modified Lysine Iron Agar is especially useful for differentiating organisms from the genus Salmonella and Edwardsiella from Shigella spp. and Yersinia enterocolitica, as the lysine-decarboxylase reaction is significantly easier to interpret than the traditional LIA reaction. Salmonella and Edwardsiella species possess lysine-decarboxylase enzymes, which remove a molecule of carbon dioxide from lysine to form an alkaline reacting amine. The pH indicator in the media, bromcresol purple, turns the agar purple in the presence of these alkaline compounds. A yellow color, the result of dextrose-fermentation, is observed in the agar when lysine-decarboxylation does not occur, as is the case with Shigella and Yersinia enterocolitica species.(6,7)

The top layer (the slanted portion) of EnteroScreen 4™ contains Lysine Iron Agar (LIA) which is used to detect hydrogen sulfide production as well as the deamination of lysine. LIA contains sodium thiosulfate and ferrous sulfate, which are added to detect organisms that have the ability to liberate sulfur from sulfur containing compounds in the form of hydrogen sulfide. Production of hydrogen sulfide generates an insoluble, heavy metal sulfide, which produces a black precipitate in the medium. Another advantage of LIA is that certain enteric organisms, like Proteus and Providencia species, possess enzymes responsible for the oxidative-deamination of lysine.(5-7) If lysine is deaminated, in the presence of oxygen, a red color is observed on the slant. A negative lysine-deamination reaction, observed when the slant is purple in color, is indicative of Salmonella, Edwardsiella, Yersinia enterocolitica, and Shigella species. Furthermore, organisms can be isolated from the slant, and used to perform additional biochemical and serologic testing.(6,7)

FORMULA(6)

Formulations are listed in order from the bottom (butt) of the tube to the top (slant).

Bottom Agar Layer:
Urea Agar, 2ml/tube
Urea 20.0gm
Sodium Chloride 5.0gm
Monopotassium Phosphate 2.0gm
Peptone 1.0gm
Yeast Extract 1.0gm
Dextrose 1.0gm
Phenol Red 12.0mg
Agar 12.0gm

Hydrophobic Layer:
Sterilized Petrolatum Plug, 2.5ml/tube

Middle Agar Layer:
Modified Lysine Iron Agar (LIA), 2.5ml/tube
L-Lysine 10.0gm
Sodium Chloride 5.0gm
Peptone 5.0gm
Yeast Extract 3.0gm
Dextrose 1.0gm
Bromcresol Purple 20.0mg
Agar 12.0gm

Top Agar Layer:
Lysine Iron Agar (LIA), 5ml/tube (slant)
L-Lysine 10.0gm
Sodium Chloride 5.0gm
Peptone 5.0gm
Yeast Extract 3.0gm
Dextrose 1.0gm
Ferrous Sulfate 0.3gm
Sodium Thiosulfate 0.3gm
Bromcresol Purple 20.0mg
Agar 12.0gm

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE(6,7)

Specimen Collection: This product is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organism. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism.

The EnteroScreen 4™ algorithm is recommended for use only with non-lactose-fermenting, oxidase-negative organisms; the detection of oxidase-positive organisms will require the use of another test system (OxiStrips™, Cat. no. Z93).

Method of Use:

1. Allow the EnteroScreen 4™ tubes to warm to room temperature prior to use. In addition, it is recommended that a Blood Agar plate (Cat. no. A10) be run in parallel as a check of pure culture, as well as additional serology and biochemical testing.

2. Using an inoculation needle, obtain a well-isolated, lactose-negative colony from a differential media such as; MacConkey Agar (Cat. no. G35), HE Agar (Cat. no. G63), XLD Agar (Cat. no. G65), or SS Agar (Cat. no. G50).

3. Inoculate by stabbing to the bottom of the tube. Streak the surface of the slant while withdrawing the needle.

4. Incubate tubes aerobically with loose caps at 35-37ºC. Examine agar layers for reactions at 18-24 hours and again at 48 hours. Urea should not be read after 24 hours.

Note: An oxidase test (OxiStrips™, Cat. no. Z93) must be performed prior to interpreting the results of EnteroScreen 4™. See "Interpretation of Results" section for more information.

INTERPRETATION OF RESULTS(6)

Refer to Figure 2 and Figure 3 for algorithms describing expected reactions. Oxidase reactions obtained from the Blood Agar plate are useful in the EnteroScreen 4™ algorithm. Reactions are listed in order from bottom of tube to slant. See Table 1 (below) for more information.

Bottom Agar Layer - Urea Agar:
Urease Production
Positive:     pink color (alkaline reaction)
Negative: light orange (acid reaction)


Hydrophobic Layer:
No reaction observed


Middle Agar Layer - Modified Lysine Iron Agar (LIA):
Lysine-Decarboxylase
Positive:     purple (alkaline reaction)*
                    *   Only a true purple color should be considered a positive reaction for lysine-                                 decarboxylase. A purple-yellow color is a negative reaction.
Negative:   yellow (acid reaction)


Gas Production as a Result of Dextrose-Fermentation
Positive:     presence of bubbles or cracks
Negative:   absence of bubbles or cracks


Top Agar Layer - Lysine Iron Agar (LIA):
Lysine-Deamination
Positive:     red slant
Negative:   purple or yellow


Hydrogen Sulfide Production
Positive:     blackened medium, concentrated at the bottom of slant
Negative:   purple bottom of slant



Typical Reactions of Lactose-Negative Enterobacteriaceae in EnteroScreen 4™
(Table 1)
Organism Urease(Bottom Layer) Lysine-Decarboxylase(Middle Layer) Lysine-Deaminase(Slant) H2S Gas PYR Notes
Salmonella spp. orange
(-)
purple/yellow
(v)
purple
(-)
black
(+)
yes
(+)
negative Some S. choleraesuis and most S. paratyphi will be H2S negative. All Salmonella spp. are lysine-decarboxylase positive except for S. paratyphi
E. coli (lactose-negative) orange
(-)
purple/yellow
(v)
purple
(-)
purple
(-)
no
(-)
negative Similar to H2S negative strains of Edwardsiella spp. but E. coli is ONPG and MUG positive.
Edwardsiella spp. orange
(-)
purple
(+)
purple
(-)
black/purple
(v)
yes
(+)
negative H2S negative strains similar to lactose-negative E. coli but Edwardsiella spp. is ONPG and MUG negative.

H2S positive strains similar to Salmonella spp. Confirm negative agglutination with Salmonella antisera.

Most Edwardsiella are indole-positive and Salmonella spp. are indole-negative.
Yersinia enterocolitica pink/orange
(v)
yellow
(-)
purple
(-)
purple
(-)
no
(-)
positive
Shigella spp. orange
(-)
yellow
(-)
purple
(-)
purple
(-)
no
(-)
negative
Hafnia alvei orange
(-)
purple
(+)
purple
(-)
purple
(-)
yes/no
(v)
negative
Providencia spp. pink/orange
(v)
yellow
(-)
red
(+)
red
(-)
yes/no
(v)
negative
Proteus spp. pink
(+)
purple/yellow
(v)
red
(+)
black/red
(v)
yes
(+)
negative Most P. penneri will be H2S negative.
Morganella spp. pink
(+)
purple/yellow
(v)
red
(+)
red/black
(v)
yes
(+)
negative Most will be H2S negative.
Citrobacter spp. pink/orange
(v)
yellow
(-)
purple/yellow
(-)
black/purple
(v)
yes
(+)
positive Usually lactose-positive but rare lactose-negative exists.
Enterobacter spp. pink/orange
(v)
purple/yellow
(v)
purple
(-)
purple
(-)
yes
(+)
positive Usually lactose-positive but rare lactose-negative exists.
Klebsiella spp. pink/orange
(v)
purple
(+)
purple
(-)
purple
(-)
yes
(+)
positive Usually lactose-positive but rare lactose-negative exists.
Serratia spp. orange
(-)
purple/yellow
(v)
purple
(-)
purple
(-)
yes/no
(v)
positive Usually lactose-positive but rare lactose-negative exists.

Color Codes:

Note: Some reactions are variable. See the EnteroScreen 4™ algorithm (Figure 2) for accurate interpretations.

FIGURES

Figure 1: Diagram illustrating components of Hardy Diagnostics EnteroScreen 4™.

Figure 2: Algorithm for Hardy Diagnostics EnteroScreen 4™.(6)
The EnteroScreen 4™ algorithm is recommended for use with non-lactose-fermenting, oxidase-negative organisms only.

Figure 3: Abbreviated algorithm for Hardy Diagnostics EnteroScreen 4™.(7)
The EnteroScreen 4™ abbreviated algorithm is recommended for use with non-lactose-fermenting, oxidase-negative organisms only.

LIMITATIONS

It is important to stab the butt of the medium. Failure to stab all the way to the bottom (butt) of the media can produce weak or erroneous urease results.

Caps must be loosened during incubation or erroneous results will occur.

Certain species or strains may give delayed reactions or completely fail to ferment the carbohydrate in the stated manner. However, in most cases if the organism fails to ferment dextrose within 48 hours, and growth is definitely present, the organism is most likely not in the Enterobacteriaceae family and cannot be differentiated with the EnteroScreen 4™ algorithm. It is recommended that an oxidase test be performed prior to use with the EnteroScreen 4™ algorithm as a negative oxidase reaction will confirm that an organism is a member of Enterobacteriaceae.

After prolonged incubation a false-positive alkaline reaction may be seen in the Urea Agar. To rule out this occurrence, check the test with a control (an uninoculated tube of EnteroScreen 4™) along with the inoculated tube during prolonged incubation.

Do not heat EnteroScreen 4™ above 37ºC., as urea decomposes very readily when heated.

Gastroenteritis has also been associated with Edwardsiella tarda, an organism that resembles Salmonella species in its pathogenic mechanisms, morphologically, as well as biochemically.(3) In addition to serology, an indole test (Cat. no. Z65) is useful in differentiating Edwardsiella (indole-positive) from Salmonella species (indole-negative). See EnteroScreen 4™ algorithm (Figure 2).

Hafnia alvei is a rare opportunistic stool pathogen which is biochemically similar to Salmonella species and agglutinates with Salmonella and Shigella antisera. Expected reactions for Hafnia alvei include urease-negative, lysine-decarboxylase-positive, and lysine-deaminase-negative. It can be differentiated from Salmonella by a positive VP reaction at 25ºC.

There are rare strains of Citrobacter that will show a reaction similar to Salmonella paratyphi (lactose-negative, urease-negative, H2S-positive, gas-positive). These strains can be differentiated by a rapid PYR test (Citrobacter PYR-positive, Salmonella PYR-negative).

Some strains of Yersinia enterocolitica are incapable of hydrolyzing urea and are biochemically similar to Shigella species which may not be differentiated by this algorithm. Most strains (75%) will be urease-positive but may take longer to develop (> 48 hours). In addition to serology, Pyroglutamate aminopeptidase (PYR) activity is effective for differentiating Yersinia (PYR-positive) from Shigella species (PYR-negative). Yersinia enterocolitica colonies will be smaller (< 1mm) than Shigella. See EnteroScreen 4™ algorithm (Figure 2).

Strong urease producers, including Proteus spp., may rarely produce a false-positive decarboxylase reaction.

Quality Control results are for the EnteroScreen 4™ combination of tests only and not to be confused with the results expected with single biochemical tests.

Gas reaction should be read strictly from the lysine-decarboxylase layer. Bubbles in the urea layer should not be interpretated as gas production.

Only a red color reaction should be considered as positive for Lysine-Deamination (top layer). Usually the negative reaction is characterized by a purple color, but a yellow color may develop due to a strong pH reaction from the middle layer in isolates that are negative for Lysine-Decarboxylase.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as inoculating loops and needles, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents (OxiStrips™, Cat. no. Z93; Indole, DMACA Spot, Cat. no. Z65; PYR Test Kit, Cat. no. Z75), are not provided.

QUALITY CONTROL

Note: Refer to "Procedure" section, above, for information concerning inoculation protocol, incubation temperature, and incubation atmosphere.

Test Organisms
Results
Urea Agar
Modified Lysine Iron Agar
Lysine Iron Agar
Salmonella enterica
ATCC® 14028
Urease-negative
(orange color)
Lysine-decarboxylase-positive
(purple color)
Gas production positive
(bubbles in media)
Growth on slant
Lysine-deamination-negative
(purple slant)
H2S-positive
(blackening at base of slant)
Shigella flexneri
ATCC® 12022
Urease-negative
(orange color)
Lysine-decarboxylase-negative
(yellow color)
Gas production negative
(no bubbles in media)
Growth on slant
Lysine-deamination-negative
(purple slant)
H2S-negative
(purple color at base of slant)
Proteus mirabilis
ATCC® 12453
Urease-positive
(pink-red color)
Lysine-decarboxylase-negative
(yellow color)
Gas production variable
Growth on slant
Lysine-deamination-positive
(red slant)
H2S-positive/negative
(purple or slight black color at base of slant)

User Quality Control

Physical Appearance

EnteroScreen 4™ should consist of four layers within a single tube, which includes three media layers and a hydrophobic layer. The bottom layer should appear opalescent and pale yellow with a slight orange tinge in color. The subsequent layer is a hydrophobic layer, which should appear opaque and white to off-white in color. The next layer and the top slant should appear slightly opalescent and purple in color.

PERFORMANCE CHARACTERISTICS(6,7)

EnteroScreen 4™ was evaluated using 218 clinical isolates. Included among these isolates were 106 isolates Salmonella species (23 S. typhi and 83 non-typhi species of Salmonella from 20 serotypes); 56 Shigella isolates (representing all four species); and 56 other gram-negative bacteria (representing many lactose-negative P. aeruginosa as well as other members of the Enterobacteriaceae family). In this study, Hardy Diagnostics EnteroScreen 4™ correctly identified 100% of all Salmonella and Shigella isolates. Of the other bacteria, there was a 94.6% agreement between EnteroScreen 4™ and the result obtained from TSI, LIA, and Urea agars. The results of this evaluation demonstrated that Hardy Diagnostics EnteroScreen 4™, when used in conjunction with the algorithm, is an acceptable method for screening of lactose-negative stool isolates that may represent Salmonella or Shigella species.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

6. Procop, G.W., et al. 2002. Introducing the Single Tube Screen for Salmonella and Shigella. Cleveland Clinic Foundation, Cleveland, OH.

7. Procop, G.W., et al. 2008. Am. J. Clin. Pathol.; 130:284-289.


ATCC is a registered trademark of the American Type Culture Collection.
Patent Pending no. 60/333,400

022516gr