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Rosco Diagnostica Instructions for Use for Roscozym™ Roscozym™ Identification Kit for Enterobacteriaceae

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DBV0015D 20.04.2007 English

Manufacturer ROSCO Diagnostica A/S, Taastrupgaardsvej 30, DK-2630 Taastrup, Denmark, www.rosco.dk Intended use To be used in vitro for the identification of Enterobacteriaceae and oxidase negative non-fermentative Gram-negative rods. Principle The Roscozym kit combines a total of 11 enzymatic tests using 10 tablets with chromogenic and modified conventional substrates contained in a panel with 11 mini-tubes attached to each other (tube No. 11 contains no tablet, but can be used for a supplementary test). Preformed enzymes contained in the bacterial suspension react with the substrates of the tablets and after 4 hours' incubation at 35-37 °C the results of the tests are read, appearing as colour reactions (without or after addition of reagents). Reading may also be performed after overnight incubation, without risk of erroneous results. Storage instructions On receipt store the outer container in a refrigerator at 2-8° C. The expiry date on the panel applies only to Roscozym panels with lids stored at the correct temperature. Reagents The Roscozym kit contains 10 different tablets with accurately determined amounts of substrate in a Roscozym panel with 11 tests for the detection of enzymatic activity.1 Precautions Follow the directions for use. Roscozym performance depends on use of proper inoculum, incubation time and correct interpretation of colour after incubation. Only properly trained technicians should use the products. Precautions should be taken against microbiological hazards by properly sterilizing specimens, containers, media, and panel after use. Kits and agar plates with potential pathogens are disposed of as microbiological waste. Specimen Only oxidase negative, Gram-negative rods should be tested with the Roscozym system. The specimen should be collected and handled following recommended guidelines. The specimen should be a pure culture fully typical of the species to be identified.

Procedure Materials provided: 20 Roscozym panels, Roscozym record slip, Roscozym 4-Hour ENT Index. Materials required but not provided: Culture media, Aminopeptidase reagent (Rosco #92231), Kovacs' reagent (Rosco #92031), quality control organisms, and laboratory equipment necessary to perform identification e.g. inoculating loops, swabs, pipettes and collection containers.

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Rosco Diagnostica I. Direction for use I.1. Inoculum preparation • Select morphologically identical well isolated colonies from the primary plate (McConkey agar, Drigalski agar, TSA or similar) and make a suspension in 3 ml physiological saline (0.9% NaCl) to achieve a turbidity equal to at least McFarland No. 2 standard. McConkey and Drigalski agar are preferable. • Use only fresh cultures (18-24 hours). • Perform a test for purity by streaking on a non-selective agar plate. I.2. Inoculation and incubation • Open the panel and dispense 0.25 ml portions of the bacterial suspension into each of the 10 tubes. • Add 3 drops of sterile paraffin oil to tube No. 7 and 8 (LDC and ODC) and close the panel by pressing the lid firmly over all tubes (the two handles pointing in opposite directions). • Incubate at 35-37 °C for 4 hours (or overnight), e.g. using a kit tray. • Sugar fermentation tests (ADON, SORB, XYL) showing doubtful results after 4 hours should be reincubated and read again after overnight incubation. I.3. Reading Interpretation of the reactions (positive/negative) is made by colour comparison with the "Roscozym Record Slip" included in the box. • • • •

First read the spontaneous urease (UR) reaction (10) in tube No. 10 (double test tablet UR/IN). After 18-24 hours' incubation only strong red and purple are positive. Open one half of the flexible lid and add 3 drops of Aminopeptidase reagent (Rosco #92231) to tube No. 9 (PYR) and add 3 drops of Kovacs' reagent (Rosco #92031) to tube No. 10 (UR/IN) and close the panel. Shake gently for 2-3 seconds. Note all the remaining reactions: No. 1-9 and reaction No. 11 (indole). After 18-24 hours' incubation only strong blue/violet is positive for LDC and ODC. If reagents have been added after 4 hours, these reactions (No. 9 and 11) cannot be read again after overnight incubation. Use the 4-hour results (No. 9, 10 and 11) for the overnight numerical profile.

II. Results • • •

The 11 tests are divided into 3 groups of 3 tests and 1 group of 2 tests. Negative results are registered as 0 and positive results as 1 for the first test (ONPG, ADON, LDC and UR), 2 for the second test (PGUA, SORB, ODC and IN) and 4 for the third test (ß-XYL, XYL and PYR) of each group (see the interpretation table). A 4-digit number is obtained by adding the values of the 3 tests (or 2) in each of the four groups. This 4-digit number is the numerical profile of the unknown microorganism. Record and calculate the numerical profile of the unknown microorganism using the "Roscozym Record Slip". ROSCOZYM RECORD SLIP

Example: Date: Test Values of positive Results SUM of positive Code number:

ONPG

PGUA

1

2

4

+

+

-

3 3632

ß-XYL ADON

Specimen LDC ODC

SORB

XYL

1

2

4

1

-

+

+

+

6

PYR

UR

IN

2

4

1

2

+

-

-

+

3

2

Identification:

Compare the obtained numerical profile with the profiles given in the Roscozym Index included in the box. Code number/Numerical profile: 3632 Identification: E. coli. Unexpected or non-conclusive results: Repeated testing and testing of QC-strains are recommended. The use of additional test and a new specimen can in some cases be useful. Alternatively the strain may be sent to a reference laboratory for further identification.

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Rosco Diagnostica INTERNAL QUALITY CONTROL A quality control procedure using ATCC strains or known positive or negative strains should be used to monitor the performance of Roscozym. Quality control should be performed in accordance with established laboratory quality control procedures. Negative/positive test results are mentioned below for ATCC strains. A correct numeric code for the respective ATCC strains indicate correct performance of the entire procedure. Quality control table Values of positive ATCC strain E. coli ATCC 25922 Enterobacter cloacae ATCC 13047 Proteus vulgaris ATCC 13315

1

2

4

1

2

4

1

2

4

1

2

ONPG

PGUA

ß-XYL

ADON

SORB

XYL

LDC

ODC

PYR

UR

IN

+

+

0

0

+

+

+

+

0

0

+

3632

+

0

+

0

+

0

0

+

+

0

0

5260

0

0

0

0

0

+

0

0

0

+

+

0403

Code

If aberrant quality control results are noted, patient results should not be reported. LIMITATIONS OF METHOD • Roscozym should only be used to differentiate members of the family Enterobacteriaceae. The database also includes the oxidase negative members of the non-fermenters group. • Pure cultures of microorganisms should be used, since a mixed microbial population will result in aberrant results. • The biochemical identification of Salmonella and Shigella must be considered as presumptive and should be confirmed by serology. • In a few cases, extra tests are necessary for complete identification (see Roscozym 4-Hour ENT Index). • Use of Roscozym for the identification of microorganisms and interpretation of results require a technician trained in general microbiological methods, who should judiciously make use of knowledge, experience, specimen information, and other pertinent procedures before reporting identity of the isolate. • Results from additional tests may affect the final identification result. REFERENCES 1.

User’s Guide Diatabs. www.rosco.dk

2.

Brison et al. Interet taxonomique de la recherche de la beta-xylosidase chez les Enterobacteriaceae. Ann. Inst. Pasteur, 123, 341-347, 1972.

3.

Casals J.B., Pringler N. Rapid identification of Enterobacteriaceae using the ROSCOZYM 4-HOUR ENT kit", 2. Congress of the Spanish Society of Clinical Microbiology and Infectious Diseases/European Society of Clinical Microbiology Symposium , May 1986, Mallorca (Spain).

4.

Dibb W.L., Bottolfsen K.L. Evaluation of Rosco Diagnostic beta-glucuronidase Tablets in the identification of urinary isolates of E. coli. Acta Pathol. Microbiol. Scand. Sect.B, 92, 261-264, 1984.

5.

Dominguez A et al. Use of a commercial double-test tablet (Rosco PGUA/Indole) for screening of E. coli. Diagn. Microbiol. infect. 15, 291-294, 1992.

6.

Godsey J.H. et al. Rapid identification of Enterobacteriaceae with microbial enzyme activity profiles. J. Clin. Microbiol., 13, 483-490, 1980.

7.

Hansen W., Yourassowsky E. Detection of beta-glucuronidase in lactose fermenting members of the family Enterobacteriaceae and its presence in bacterial urine cultures. J. Clin. Microbiol., 20, 1177-1179, 1984.

8.

Kilian M., Bülow P. Rapid diagnosis of Enterobacteriaceae. I Detection of bacterial glycosidases. Acta Pathol. Microbiol. Scand. Sect. B, 84, 245-251, 1976.

9.

Mulczyk M., Szewczuk A. Pyrrolidonyl aminopeptidase in bacteria: a new colorimetric test for differentiation of Enterobacteriaceae. J. Gen. Microbiol. 61, 9-13, 1970.

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Rosco Diagnostica Roscozym interpretation table Test No. 1 2 3 4 5 6 7 8 9 10 * 11 *

Reactive Ingredient (Active ingredients per tablet) Beta-Galactosidase (o-Nitrophenyl-β-D-Galactopyranoside 0.4 mg) Beta-Glucuronidase (p-Nitrophenyl-ß-D glucuronic acid 0.3 mg) Beta-Xylosidase (p-Nitrophenyl ß-D-xylopyranoside 0.3 mg) Adonitol (Adonitol 3 mg) Sorbitol (D-Sorbitol 2 mg) d-Xylose (d-Xylose 2 mg) Lysine Decarboxylase (LDC) (L-Lysine 3 mg) Ornithine Decarboxylase (ODC) (L-Ornithine HCl 3 mg) Pyrrolidonyl Aminopeptidase (L-Pyrrolidonyl-ß-Naphthylamide 0.2 mg) Urease (Urea 4 mg) Indole (L-Tryptophane 1 mg)

Code marked Reagent on panel

Value

Positive Reaction

Negative Reaction

ONPG

-

1

Yellow

Colourless

PGUA

-

2

Yellow

Colourless

ß-XYL

-

4

Yellow

Colourless

ADON

-

1

SORB

-

2

XYL

-

4

LDC

Oil

1

ODC

Oil

2

PYR

I

4

Red

Yellow/orange

UR

-

1

Red/purple

Yellow/orange

IN

II

2

Yellow, Red, orange red yellow-orange Yellow, Red, orange red yellow-orange Yellow, Red, orange red yellow-orange Blue, violet Yellow, green, Strong violet grey, light blue Blue, violet Yellow, green, Strong violet grey, light blue

Red Yellow/orange (surface layer)

*) Tests Nos. 10 and 11, Urease/Indole: This is a double test diagnostic tablet permitting the performance of two tests using one tablet in the same mini-tube. Reagents to be added before incubation: Oil: Add 3 drops of paraffin oil before incubation. Reagents to be added after incubation: I. Add 3 drops of Aminopeptidase reagent (Rosco #92231) and read colour reaction within 5 minutes. II. Add 3 drops of Kovacs' reagent (Rosco #92031), shake and read colour of the surface layer after 3 minutes.

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http://www.rosco.dk/pdf/IFU-Roscozym-uk-200407_RIV_7183