BD Micro, GUIDE ENTEROTUBE II INTERPRETATION GUIDE, Enterotube, Each. BD Micro, GUIDE ENTEROTUBE II INTERPRETATION GUIDE, Enterotube. Remarks: Glucose – Any degree of yellow is positive. Acid end products from glucose fermentation turn the pH indicator from red (alkaline) to yellow (acid). of a Multitest System (Enterotube) for Enterotube, a multiple-test system which combines nine biochemical tests useful in the identification . (ii) Modified lysine-lactose me- dium: a .. Bergey’s manual of determinative bacteriology.

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Biol Lab Manual: Interpretation of Enterotube II

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 ihterpretation because it contains the enterobacterial common antigen.

The identification of Enterobacteriaceae is based on biochemical reactions as described by Ewing and by Farmer. For details, consult the references 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.

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 codebookallow identification of interpfetation significant Enterobacteriaceae. 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 medium is covered with wax to provide anaerobic conditions and to allow detection of gas formation.

The medium is covered with wax to provide anaerobic conditions. Beige to light amber. 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.

The following precautions pertain to the individual compartments indicated: 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.

The medium may become a deeper shade of green after incubation. This should be considered negative. When preparing and handling indole and VP reagents, observe appropriate handling instructions and Risk and Safety Phrases. 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. For inoculation, incubation, and reading, proceed as described in Test Procedure.

However, this strain is useful as a control organism to detect negative reactions in the glucose and gas compartments. CM CE, which is divided into three databases: Kovacs indole reagent Indole Dropper Reagent, 50 ampoules, cat. 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: 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. The culture used for the inoculation should be at least 18 hours old, but should generally not be older than 48 hours.

Prepare one sheet from the coding pad for the isolate by entering patient name, specimen number, and date. 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.

The tip of the inoculating wire is under the white cap. Do not flame interretation. A visible amount of inoculum should be seen at the tip and the side of the wire. Avoid touching agar enterotuge wire. Inoculate BBL Enterotube II by first twisting wire, then withdrawing wire enterotbue all twelve compartments applying a turning motion Figure 2. Reinsert wire without sterilizing into BBL Enterotube II, using a turning motion through all 12 compartments, until the notch interpretatoon 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. Allow for air circulation between incubated tubes. Interpret and record all reactions with exception of indole and Voges-Proskauer.

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 Unterpretation Addition: To perform the indole test: Add drops of Kovacs reagent cat.

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.

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. A interpretarion test is indicated by the development of a red color within 20 min. Do not wait longer than 20 min before reading the results! 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 inyerpretation below to identify the isolate. Information on the appearance of positive and negative reactions is provided in Table 1.

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. 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 interpretatjon the colors of the media in the tube after incubation interprteation 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.

Indicate each positive test result by circling the number appearing below the appropriate compartment on the Results Pad Figure 6. Finally, perform the indole and VP tests. If positive, circle the appropriate numbers on the prepared sheet.


The VP test is inetrpretation only if the database with VP is used. Add circled numbers in the bracketed section and enter the sum in the space provided below the arrow Figure 6. Locate the five digit number in the Interpretation Guide codebook and find the best answer s in the column entitled Enterotubr 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 enterotue Klebsiella pneumoniae is obtained. The end products of bacterial fermentation of glucose are either acid, or acid and gas. The shift in ph due to the production of acid is indicated by change in the color of the indicator in the medium from red alkaline to yellow acidic.

Any degree of yellow should enterotubw interpreted as Gas production wax not lifted wax lifted a positive reaction; orange should be considered negative. This is evidenced by definite and guive separation of the wax overlay from entreotube surface of the glucose medium, but not by bubbles in the medium.

Since the amount of gas produced by different bacteria varies, the amount of separation between medium and overlay will also vary with the strain being tested.

Bacterial decarboxylation of entetotube, which results in the formation of the alkaline end product cadaverine, is indicated by a change in the color of entfrotube indicator in the medium from pale yellow acidic to purple alkaline.

The medium remains yellow if decarboxylation of lysine does not occur. Decarboxylation of ornithine, which results in the formation of the alkaline end product putrescine, is indicated by a change in the color of the indicator in the medium from pale yellow acidic to purple alkaline.

The medium remains yellow if decarboxylation of ornithine does not inteerpretation. Hydrogen sulfide is produced by light amber bacteria capable of reducing sulfur-containing compounds, such as peptones and sodium thiosulfate present in the medium. The hydrogen sulfide reacts with the iron salts also present in the medium guuide form a black precipitate of ferric sulfide usually along the line of inoculation.

Some Providencia strains may produce a diffuse brown coloration in this medium, however, this should not be confused with true H 2S production as indicated by the presence of black color.

The production of indole from the metabolism of tryptophan by the bacterial enzyme tryptophanase is detected by the development of a pink to red color after the addition of Kovacs indole reagent, which is added into the compartment after 18 to 24 h incubation of the tube see Test Procedure section. Bacterial fermentation of adonitol, lactose, arabinose, and sorbitol result in the formation of acidic end products, is indicated by a change interpretaation color of the indicator present in the medium from red alkaline to yellow acidic.

Any sign of yellow should be interpreted as a entrrotube reaction; orange should be considered negative. Acetylmethylcarbinol acetoin is an intermediate enteeotube the production of butylene glycol from glucose fermentation.

The guidf of acetoin is detected after the addition of the VP reagents after incubation see Test Procedure section. The presence of acetoin is indicated by the development of a red color within 5 to 20 min. Bacterial fermentation of dulcitol, which results in the formation of acidic end products, is indicated by a change in color of the indicator present in the medium from green alkaline to yellow or pale yellow acidic.