BD BBL Enterotube

INSTRUCTIONS FOR USE ? BBLTM Enterotube II

IA-273176.01

Rev.: Jan. 2007

BDTM BBLTM EnterotubeTM II

INTENDED USE

BBL Enterotube II is a ready-to-use identification system for the identification of Enterobacteriaceae and a variety of other oxidase negative Gram negative rods.

PRINCIPLES AND EXPLANATION OF THE PROCEDURE

Microbiological method. Enterobacteriaceae play a major role as infectious agents. This group of bacteria utilizes carbohydrates principally by fermentation and by oxidation. They are catalase positive and nearly all of them are oxidase negative [It has been proposed recently to include Plesiomonas shigelloides into the Enterobacteriaceae based on 16S rRNA sequencing and because it contains the enterobacterial common antigen.1,2 Previously, this organism which is is oxidase positive, was included into the family Vibrionaceae]. The identification of Enterobacteriaceae is based on biochemical reactions as described by Ewing and by Farmer. For details, consult the references.1-11 While a huge variety of genera and species has been described over the years, the organisms that are isolated from clinical specimens belong to 20 to 25 species that are well-known for many years.1 BBL Enterotube II is a self-contained, compartmented plastic tube containing twelve different media that allow the determination of 15 biochemical reactions (glucose, gas production from glucose, lysine decarboxylase, ornithine decarboxylase, hydrogen sulfide (H2S), indole, adonitol, lactose, arabinose, sorbitol, Voges-Proskauer (VP), dulcitol, phenylalanine deaminase (PA), urea and citrate). The enclosed inoculating wire allows inoculation of all compartments in one step from one or a few single colonies of an isolate. The resulting combination of reactions, together with the Interpretation Guide (codebook), allow identification of clinically significant Enterobacteriaceae. The Interpretation Guide (codebook) for BBL Enterotube II was constructed using the percentage data contained in the references for the biochemical tests performed by BBL Enterotube II.3-11 The Results Pad and Color Reaction Chart permit a rapid check of the positive reactions obtained with BBL Enterotube II. The checked positive numbers are totaled, and the composite number is then located in the Interpretation Guide to identify the organisms. Where two or more organisms are listed, the confirmatory tests required to further identify them are also given. The following flow diagram demonstrates how the oxidase test may used to differentiate members of the family Enterobacteriaceae from nonfermentative, oxidase positive or negative, and from fermentative, oxidase positive Gram negative bacteria and when BBL Enterotube II or BBL Oxi/FermTM Tube II are used:

Pure culture on nonselective medium

Oxidase test

positive

negative

Inoculate BBL Oxi/Ferm Tube II

Inoculate BBL Enterotube II

Anaerobic glucose

Glucose

Positive

Negative

Positive

Negative*

Aeromonas spp.,

Achromobacter spp.,

Enterobacteriaceae Acinetobacter spp.,

Plesiomonas

Alcaligenes spp., Bordetella

Stenotrophomonas

shigelloides,

bronchiseptica, Moraxella

maltophilia, etc.

Vibrio spp.

spp., Pasteurella spp., etc.

* These species may be identified with the BBL Enterotube II system, but use of BBL Oxi/Ferm Tube II may be necessary for

confirmation.

REAGENTS BBL Enterotube II

Substrates, other active ingredients, and coloration of the uninoculated media:

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? Medium 1 (Glucose): Glucose (20.0 g/l) contained in an appropriate base medium, with cresol red as a pH indicator. The medium is covered with wax to provide anaerobic conditions and to allow detection of gas formation. Uninoculated: red.

? Medium 2 (Lysine): Lysine (10.0 g/l) contained in an appropriate base medium, with bromcresol purple as a pH indicator. The medium is covered with wax to provide anaerobic conditions. Uninoculated: yellow.

? Medium 3 (Ornithine): Ornithine (10.0 g/l) contained in an appropriate base medium, with bromcresol purple as a pH indicator. The medium is covered with wax to provide anaerobic conditions. Uninoculated: yellow.

? Medium 4 (H2S/Indole): Sodium thiosulfate (4.7 g/l) , ferric ammonium citrate (0.6 g/l) , and tryptophan (1.2 g/l) in an appropriate base medium. Uninoculated: Beige to light amber.

? Medium 5 (Adonitol): Adonitol (20.0 g/l) contained in an appropriate base medium, with cresol red as a pH indicator. Uninoculated: red.

? Medium 6 (Lactose): Lactose (20.0 g/l) contained in an appropriate base medium, with cresol red as a pH indicator. Uninoculated: red.

? Medium 7 (Arabinose): Arabinose (20.0 g/l) contained in an appropriate base medium, with cresol red as a pH indicator. Uninoculated: red.

? Medium 8 (Sorbitol): Sorbitol (20 g/l) contained in an appropriate base medium, with cresol red as a pH indicator. Uninoculated: red.

? Medium 9 (Voges-Proskauer): Glucose (20.0 g/l) contained in an appropriate base medium. Uninoculated: colorless to light amber.

? Medium 10 (Dulcitol/PA): Dulcitol (20.0 g/l) , phenylalanine (7.0 g/l), and ferric ammonium citrate (0.5 g/l) contained in an appropriate base medium, with bromthymol blue as a pH indicator. Uninoculated: green.

? Medium 11 (Urea): Urea (20.0 g/l) contained in an appropriate base medium, with phenol red as a pH indicator. Uninoculated: beige to light amber.

? Medium 12 (Citrate): Sodium citrate (2.0 g/l) contained in an appropriate base medium, with bromthymol blue as a pH indicator. Uninoculated: green.

PRECAUTIONS

. For professional use only Do not use tubes if they show evidence of microbial contamination, discoloration, wax detachment from the respective media, drying, cracking or other signs of deterioration. Any of the following conditions may interfere with the accuracy of BBL Enterotube II: dehydration or liquefaction, lifting of wax from media surfaces, any change of media colors from those indicated under "Uninoculated" in the REAGENTS section, and any indication of growth on media surfaces. The following precautions pertain to the individual compartments indicated: ? Glucose: The wax overlay in this compartment produces the degree of anaerobiosis necessary to

allow only the true fermentation reaction characteristic of all Enterobacteriaceae. If this medium remains red after inoculation and incubation, the strain does not belong to the Enterobacteriaceae. Examples of oxidase-negative non-Enterobacteriaceae that fail to ferment glucose under anaerobic conditions in BBL Enterotube II are glucose negative Acinetobacter species. Although these organisms can be differentiated from Enterobacteriaceae using BBL Enterotube II (in the Interpretation Guide, use the database for the oxidase negative nonfermenters), BBL Oxi/Ferm Tube II may be required for their further identification. ? Phenylalanine (PA): The medium may become a deeper shade of green after incubation. This should be considered negative.

Warning: When preparing and handling indole and VP reagents, observe appropriate handling instructions and Risk and Safety Phrases. Consult GENERAL INSTRUCTIONS FOR USE document for aseptic handling procedures, biohazards, and disposal of used product.

STORAGE AND SHELF LIFE

On receipt, store BBL Enterotube II in the dark at 2 to 8? C, in the original box until just prior to use. Avoid freezing and overheating. The tubes may be inoculated up to the expiration date and incubated for the recommended incubation times. Tubes from opened packages can be used up to the expiration date. Opened tubes must be used immediately.

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USER QUALITY CONTROL

Inoculate BBL Enterotube II with the strains mentioned below. For inoculation, incubation, and reading, proceed as described in Test Procedure.

Glucose Gas Lysine Ornithine H2 S Indole Adonitol Lactose Arabinose Sorbitol V P Dulcitol Phenylalanine Ure a Citrate

ORGANISMS

Acceptable biocodes

Proteus mirabilis

+ +/- - + + - - - - - - - + + +/- 66007 / 66006 /

ATCCTM 12453

46007

Proteus vulgaris

+/(+) +/- - - + + - - - - - - + + + 63007 / 43007

ATCC 6380

Providencia alcali-

+ +/- - - - + + - - - - - + - -/+ 61404 / 41405 /

faciens ATCC 9886

41404 / 61405

Serratia marcescens + +/- + + - - +/- - - + + - - - + 74461 / 54461 /

ATCC 13880

74061 / 54061

Escherichia coli

+ +/- + + - + - + + + - - - - - 75340 / 55340

ATCC 25922

Klebsiella

+ +/- + - - - + + + + + + - -/+ + 50771 / 70771 /

pneumoniae

70773 / 50773

ATCC 27736

Pseudomonas

- - - - - - - - -/+ - - - - -/(+) +

aeruginosa

*

ATCC 27853

* P. aeruginosa is oxidase positive and is, therefore, not included in the BBL Enterotube II databases. However, this strain is useful

as a control organism to detect negative reactions in the glucose and gas compartments.

+= positive; - = negative; (+) weakly positive; +/- = positive or (rarely) negative; +/(+) = positive or (rarely) weakly positive; -/+=

negative or (rarely) positive.

PROCEDURE

Materials Provided BBL Enterotube II. Microbiologically controlled. Results Pad and Color Reaction Chart for BBL Enterotube II.

Materials Not Provided BBL Enterotube II Interpretation Guide (codebook), document no. CM-273176.CE, which is divided into three databases: (1) Oxidase negative nonfermenters, (2) Enterobacteriaceae ? method without VP, and (3) Enterobacteriaceae ? method with VP. Kovacs` indole reagent (Indole Dropper Reagent, 50 ampoules, cat. no. 261185) and Voges-Proskauer reagents (Voges-Proskauer A Dropper Reagent, 50 ampoules, cat. no. 261192; Voges-Proskauer B Dropper Reagent, cat. no. 261193) reagents are available from BD. Materials and reagents to perform the supplemental tests as mentioned in the BBL Enterotube II Interpretation Guide. BBL Oxi/Ferm II identification system (cat. no. 212116) for the identification of non-Enterobacteriaceae. Ancillary culture media, reagents and laboratory equipment as described.

Kovacs` reagent may be prepared in the laboratory as follows:

Amyl or Isoamyl Alcohol

75 ml

p-Dimethylaminobenzaldehyde

5 g

Hydrochloric acid, concentrated

25 ml

Dissolve p-dimethylaminobenzaldehyde (the reagent should be light yellow in color) in alcohol and then slowly add the acid. The prepared reagent should have a straw yellow color and be stored in a brown bottle under refrigeration for maximum stability. Any reagent having a dark brown color should not be used.

Voges-Proskauer reagents may be prepared as follows:

5% Alpha-naphthol solution

alpha-Naphthol

5 g

Ethanol (ethyl alcohol), absolute

100 ml

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20% Potassium hydroxide solution Water Potassium hydroxide (pellets) Creatine

100 ml 20 g 300 mg

After preparation, Voges-Proskauer reagents are stable in tightly closed bottles for two weeks at 2 to 8? C in the dark.

Specimen Types BBL Enterotube II is a biochemical identification system and must not be used directly with clinical specimens. Use isolates from appropriate media (see Test Procedure). BBL Enterotube II may be used to identify aerobic, facultatively anaerobic Gram negative rods (Enterobacteriaceae) isolated from any specimen.

Test Procedure For the inoculation of BBL Enterotube II, growth from MacConkey (MAC), Eosin Methylene Blue (EMB), Salmonella Shigella (SS) or Hektoen Enteric (HE) agars, or from nonselective blood agar media can be used. The culture used for the inoculation should be at least 18 hours old, but should generally not be older than 48 hours. Make sure that the isolate that shall be identified with BBL Enterotube II is a pure culture of a Gram negative rod. BBL Enterotube II should be used only with oxidase-negative isolates since oxidase-positive organisms (non-Enterobacteriaceae) require the use of BBL Oxi/Ferm Tube II for their identification.

1. Prepare one sheet from the coding pad for the isolate by entering patient name, specimen number, and date. Note: It may be decided by the user if the databases with or without VP are used. Depending on this decision, use the frontside or the backside of the coding pad. If the database without VP is chosen, it may be necessary to perform the VP reaction as a supplemental test for selected organisms.

2. Take one BBL Enterotube II tube and enter patient name, specimen number, and date on the label. 3. Remove both caps. The tip of the inoculating wire is under the white cap. Do not flame wire. 4. Pick a well isolated colony directly with the tip of the BBL Enterotube II inoculating wire (Figure 1). A

visible amount of inoculum should be seen at the tip and the side of the wire. Avoid touching agar with wire. Utilize one or more BBL Enterotube II to pick additional colonies as experience dictates. 5. Inoculate BBL Enterotube II by first twisting wire, then withdrawing wire through all twelve compartments applying a turning motion (Figure 2). 6. Reinsert wire (without sterilizing) into BBL Enterotube II, using a turning motion through all 12 compartments, until the notch on the wire is aligned with the opening of the tube (Figure 3). The tip of the wire should be seen in the citrate compartment. Break wire at notch by bending. The portion of the wire remaining in the tube maintains anaerobic conditions necessary for true fermentation of glucose, production of as and decarboxylation of lysine and ornithine. 7. With the broken off part of the wire, punch holes through the foil covering the air inlets of the last eight compartments (adonitol, lactose, arabinose, sorbitol, Voges-Proskauer, dulcitol/PA, urea and citrate) in order to support aerobic growth in these compartments (Figure 4). Replace both caps. 8. Incubate at 35 to 37? C for 18 to 24 hours with BBL Enterotube II lying on its flat surface or in an upright position. Allow for air circulation between incubated tubes. 9. Interpret and record all reactions with exception of indole and Voges-Proskauer. (For complete instructions on how to read results of BBL Enterotube II, see Results section.) All other tests must be read before the indole and Voges-Proskauer tests are performed as the reagents added for these tests may alter the remainder of the BBL Enterotube II reactions.

Indole and Voges-Proskauer Reagent Addition: 1. Place BBL Enterotube II horizontally on the bench or vertically in a rack with glucose compartment

pointing downward. By using a needle or the broken-off end of a BBL Enterotube II inoculating wire or by melting with a hot inoculating wire, punch or melt a hole of about 3-4 mm in diameter in the plastic film above the Indole/H2S and VP compartments.

2. To perform the indole test: Add 3 - 4 drops of Kovacs` reagent (cat. no. 261185 or laboratory prepared; see section Materials Not provided) to the H2S/indole compartment. Allow reagent to contact the surface of the medium. A positive test is indicated by development of a red color in the added reagent within 10 sec.

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3. To perform the Voges-Proskauer test: This test is mandatory if the database with VP is chosen. It may also be necessary to perform this test if needed as a confirmatory test when using the database without VP. When using the laboratory prepared reagents (see section Materials Not Provided), add two drops of 20% potassium hydroxide solution containing 0.3% creatine and three drops of 5% alpha-naphthol in absolute ethyl alcohol. A positive test is indicated by the development of a red color within 20 min. Do not wait longer than 20 min before reading the results! When using Dropper Reagents, add 3 to 5 drops of Voges-Proskauer A reagent (cat. no. 261192) and 1 to 2 drops of Voges-Proskauer B reagent (cat. no. 261193). A positive reaction is indicated by the development of a cherry-red color within 5 to 15 min. A copper to brownish discoloration is read as negative. Do not wait longer than 15 min before reading the results!

Results Follow the instructions given below to identify the isolate. Information on the appearance of positive and negative reactions is provided in Table 1.

Note: If no color change, or an orange color, is observed in the glucose compartment and growth is evident by color changes in other compartments, the organism is not a member of the family Enterobacteriaceae. (See PRECAUTIONS section).

1. After 18 to 24 hours of incubation, interpret all reactions. With the exception of indole and VP, read the reactions in a sequential fashion by comparing the colors of the media in the tube after incubation with those given in the color scheme on the cover of the coding pad and (eventually) with an uninoculated BBL Enterotube II which must be brought to room temperature first (Figure 5). All reactions which should be positive may be of equal, greater or lesser intensity than the colors indicated in the Color Reaction Chart.

2. Indicate each positive test result by circling the number appearing below the appropriate compartment on the Results Pad (Figure 6).

3. Finally, perform the indole and VP tests. If positive, circle the appropriate numbers on the prepared sheet. The VP test is mandatory only if the database with VP is used.

4. Add circled numbers in the bracketed section and enter the sum in the space provided below the arrow (Figure 6).

5. Locate the five digit number in the Interpretation Guide (codebook) and find the best answer(s) in the column entitled "ID Value". Make sure to use the right database [(1) Oxidase negative nonfermenters, (2) Enterobacteriaceae ? method without VP, and (3) Entero-bacteriaceae ? method with VP]. In the example given in Figure 6, the identification as Klebsiella pneumoniae is obtained.

Figure 1

Figure 2

Figure 3

Figure 4 IA-273176.01

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