12.6.6: The EnteroPluri-Test
- Page ID
- 123453
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)A number of techniques can be used for the identification of specific species and subspecies of Enterobacteriaceae. Speciation is important because it provides data regarding patterns of susceptibility to antimicrobial agents and changes that occur over a period of time. It is also essential for epidemiological studies such as determination of nosocomial infections and their spread.
In an effort to simplify the speciation of the Enterobacteriaceae and reduce the amount of prepared media and incubation space needed by the clinical lab, a number of self-contained multi-test systems have been commercially marketed. Some of these multi-test systems have been combined with a computer-prepared manual to provide identification based on the overall probability of occurrence for each of the biochemical reactions. In this way, a large number of biochemical tests can economically be performed in a short period of time, and the results can be accurately interpreted with relative ease and assurance.
The EnteroPluri-Test is a self-contained, compartmented plastic tube containing 12 different agars (enabling the performance of a total of 15 standard biochemical tests) and an enclosed inoculating wire. After inoculation and incubation, the resulting combination of reactions, together with a Computer Coding and Identification System (CCIS), allows for easy identification. The various biochemical reactions of the EnteroPluri-Test and their correct interpretation are discussed below. Although it is designed to identify members of the bacterial family Enterobacteriaceae, it will sometimes also identify common biotypes of Pseudomonas and other non-fermentative Gram-negative bacilli. It does not identify Pseudomonas aeruginosa.
IDENTIFYING MEMBERS OF THE ENTEROBACTERIACEAE WITH THE ENTEROPLURI-TEST
The EnteroPluri-Test contains 12 different agars that can be used to carry out 15 standard biochemical tests (see Fig. Fig. \(\PageIndex{1\)). Interpret the results of your EnteroPluri-Test using the instructions below and record them on the EnteroPluri-Test table on your Results page.
For more detail on the 15 biochemical tests in the EnteroPluri-Test. See Table below
Table \(\PageIndex{1\)): Interpretation of the Enterotube II.
| Compartment | Reaction | Negative | Positive |
|---|---|---|---|
| 1 | glucose fermentation | red/orange | yellow |
| 1 | gas production | wax not lifted | wax lifted |
Remarks: Glucose - Any degree of yellow is positive. Acid end products from glucose fermentation turn the pH indicator from red (alkaline) to yellow (acid).
Remarks: Gas - Positive is a definite and complete separation of the white wax overlay from the surface of the glucose medium. Detects gas from glucose fermentation.
| Compartment | Reaction | Negative | Positive |
|---|---|---|---|
| 2 | lysine decarboxylase activity | yellow | purple |
Remarks: Any degree of purple is positive. Alkaline end products from the decarboxylation of lysine changes the pH indicator from pale yellow (acid) to purple (alkaline).
| Compartment | Reaction | Negative | Positive |
|---|---|---|---|
| 3 | ornithine decarboxylase activity | yellow | purple |
Remarks: Any degree of purple is positive. Alkaline end products from the decarboxylation of ornithine changes the pH indicator from pale yellow (acid) to purple (alkaline).
| Compartment | Reaction | Negative | Positive |
|---|---|---|---|
| 4 | hydrogen sulfide production | beige | black |
| 4 | indole production (done last) | colorless | red |
Remarks: Hydrogen sulfide - Only a true black is positive. Reduction of thiosulfate produces hydrogen sulfide which reacts with iron salts to produce black ferric sulfide.
Remarks: Indole - This test is not interpreted until all other compartments have been read. Kovac's Reagent must be added before reading. Indole, produced from the breakdown of tryptophan, reacts with Kovac's reagent turning it red.
| Compartment | Reaction | Negative | Positive |
|---|---|---|---|
| 5 | adonitol fermentation | red | yellow |
Remarks: Any degree of yellow is positive. Acid end products from adonitol fermentation turn the pH indicator from red (alkaline) to yellow (acid).
| Compartment | Reaction | Negative | Positive |
|---|---|---|---|
| 6 | lactose fermentation | red | yellow |
Remarks: Any degree of yellow is positive. Acid end products from adonitol fermentation turn the pH indicator from red (alkaline) to yellow (acid).
| Compartment | Reaction | Negative | Positive |
|---|---|---|---|
| 7 | arabinose fermentation | red | yellow |
Remarks: Any degree of yellow is positive. Acid end products from adonitol fermentation turn the pH indicator from red (alkaline) to yellow (acid).
| Compartment | Reaction | Negative | Positive |
|---|---|---|---|
| 8 | sorbitol fermentation | red | yellow |
Remarks: Any degree of yellow is positive. Acid end products from adonitol fermentation turn the pH indicator from red (alkaline) to yellow (acid).
| Compartment | Reaction | Negative | Positive |
|---|---|---|---|
| 9 (not normally used) | Voges-Proskauer | colorless | red |
Remarks: This test is not used unless required later as a confirmatory test. Acetoin produced during the production of butylene glycol from glucose fermentation reacts with the added reagents KOH and alphanaphthol and turns red.
| Compartment | Reaction | Negative | Positive |
|---|---|---|---|
| 10 | dulcitol fermentation | not yellow | yellow |
| 10 | phenylalanine deaminase activity | not black/smoky gray | black/smoky gray |
Remarks: Dulcitol - Yellow or pale yellow is positive. Any other color is negative. Acid from dulcitol fermentation turns the pH indicator from green (alkaline) to yellow (acid).
Remarks: PA - Pyruvic acid produced from deamination of phenylalanine reacts with ferric salts in the medium turning it black.
| Compartment | Reaction | Negative | Positive |
|---|---|---|---|
| 11 | urea hydrolysis | beige | red/purple |
Remarks: Hydrolysis of urea forms ammonia which causes the pH indicator to turn from yellow (acid) to pink, red, or purple (alkaline).
| Compartment | Reaction | Negative | Positive |
|---|---|---|---|
| 12 | citrate utilization | green | blue |
Remarks: Any degree of blue is positive. Utilization of citrate produces alkaline products turning the pH indicator from green (acid) to blue (alkaline).
1. Interpret the results of glucose fermentation in compartment 1.
- Any yellow = +; red = -
- If positive, circle the number 4 under glucose on your Results page.
2. Interpret the results of gas production also in compartment 1.
- White wax lifted from the yellow agar = +; wax not lifted from agar = -
- If positive, circle the number 2 under gas on your Results page.
3. Interpret the results of lysine decarboxylase in compartment 2.
- Any violet = +; yellow = -
- If positive, circle the number 1 under lysine on your Results page.
4. Interpret the results of ornithine decarboxylase in compartment 3.
- Any violet = +; yellow = -
- If positive, circle the number 4 under ornithine on your Results page.
5. Interpret the results of H2S production in compartment 4.
- Black/brown = +; beige = - (The black may fade or revert back to negative if the EnteroPluri-Test is read after 24 hours of incubation.)
- If positive, circle the number 2 under H2S on your Results page.
6. Indole production also in compartment 4. Do not interpret the indole test at this time. Add Kovac's reagent only after all other tests have been read (see step 16 below).
7. Interpret the results of adonitol fermentation in compartment 5.
- Any yellow = +; red = -
- If positive, circle the number 4 under adonitol on your Results page.
8. Interpret the results of lactose fermentation in compartment 6.
- Any yellow = +; red = -
- If positive, circle the number 2 under lactose on your Results page.
9. Interpret the results of arabinose fermentation in compartment 7.
- Any yellow = +; red = -
- If positive, circle the number 1 under arabinose on your Results page.
10. Interpret the results of sorbitol fermentation in compartment 8.
- Any yellow = +; red = -
- If positive, circle the number 4 under sorbitol on your Results page.
11. Voges-Praskauer (VP) test in compartment 9. Do not interpret the VP test at this time. Add the reagents alpha-naphtol and potassium hydroxide (KOH) only after all other tests have been read (see step 17 below).
12. Interpret the results of dulcitol fermentation in compartment 10.
- Yellow = +; green or dark brown = -
- If positive, circle the number 1 under dulcitol on your Results page.
13. Interpret the results of PA deaminase also in compartment 10.
- Dark brown= +; green or yellow= -
- If positive, circle the number 4 under PA on your Results page.
14. Interpret the results of urea hydrolysis in compartment 11.
- Pink, red or purple = +; beige = -
- If positive, circle the number 2 under urea on your Results page.
15. Interpret the results of citrate utilization in compartment 12.
- Any blue = +; green = -
- If positive, circle the number 1 under citrate on your Results page.
16. Your instructor will add 2-3 drops of Kovac's reagent to the indole test compartment.
- Pink/red = +; yellow = -
- If positive, circle the number 1 under indole on your Results page.
17. Your instructor will add 3 drops of alpha-naphthol reagent and 2 drops of potassium hydroxide (KOH) to the VP test compartment.
- Red = +; colorless = -
- If positive, circle the number 2 under VP on your Results page.
18. Add all the positive test number values in each bracketed section and enter each sum in its code box on the EnteroPluri-Test chart on your Results page.
19. The 5 digit number is the CODICE number. Look that number up in the Codebook beginning on page 2 and identify your unknown. (Should more than one organism be listed, the confirmatory tests indicated in the CCIS would normally then have to be performed. In addition, an identification of Salmonella or Shigella would usually be confirmed by direct serologic testing as will be described in Lab 17.)
Contributors and Attributions
Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)