MICROGEN IDENTICATION SYSTEM

Effective Date: 
Thu, 08/01/2013
Reviewed: 
Tue, 01/10/2017
Revised: 
Fri, 08/25/2017
Policy: 

The Microgen GN-ID system comprises two separate microwell test strips (GN A and GN B). Each microwell test strip contains 12 standardised biochemical substrates which have been selected on the basis of extensive computer analysis  of published databases for the identification of the family Enterobacteriaceae and commonly encountered non-fastidious oxidase positive and negative gram negative bacilli. The dehydrated substrates in each well are reconstituted with a saline suspension of the organism to be identified. If the individual substrates are metabolised by the organism, a colour change occurs during incubation or after addition of specific reagents (see Substrate Reference Table). The permutation of metabolised substrates can be interpreted using the Microgen Identification System Software (MID-60) to identify the test organism.
The GN A microwell test strip is intended for the identification of oxidase negative, nitrate positive glucose fermenters comprising the most commonly occurring genera of the family Enterobacteriacea.
The GN A and GN B microwell test strips are used together to produce a 24 substrate system to identify non-fastidious gram negative bacilli (oxidase negative and positive) in addition to all currently recognised species of the family Enterobacteriacea (28 genera) - see data tables.
The GN B microwell test strip is designed to be used in conjunction with the GN A microwell test strip and not on its own.
 

Procedure: 

INOCULATION AND INCUBATIONS

1. Carry out an oxidase test on the isolate. Oxidase positive organisms can only be identified by inoculating both GN A and GN B microwell test strips.

2. Emulsify a single colony from an 18-24 hour culture in 3mL sterile 0.85% saline for the GN A microwell test strip. If both GN A and GN B microwell test strips are to be inoculated, the colony should be emulsified in 3-5mL sterile 0.85% saline. Mix thoroughly.

3. Carefully peel back the adhesive tape sealing the microwell test strip(s). Do NOT discard the sealing strip(s) as they will be required later.

4. Using a sterile pasteur pipette, add 3-4 drops (approximately 100μL) of the bacterial suspension to each well of the microwell test strip(s).

5. As a purity check, transfer 1 drop of the bacterial suspension on to a purity plate using a nonselective differential medium. Incubate the plate aerobically at 35-37°C for 18-24 hours.

6. After inoculation, overlay wells 1,2 3 and 9 (GN A microwell test strip counting from the tabbed end) and wells 20 and 24 (GN B microwell test strip - well 13 is at the tabbed end) with 3-4 drops of mineral oil. (Do NOT overlay well 20 if isolate is oxidase positive). These wells are highlighted with a black circle around the well to assist in adding oil to the correct wells.

7. Seal the top of the microwell test strip(s) with the adhesive tape removed earlier and incubate at 35-37°C. Ensure that the punctures in the adhesive tape are over wells 7, 11 and 12 in the GN A microwell test strip and over well 14 in the GN B microwell test strip.

8. The GN A and GN B microwell test strips are read after 18-24 hours incubation for Enterobacteriaceae, and after 48 hours for oxidase positive isolates.

READING AND ADDITION OF REAGENTS

GN A Strip

1. Remove the adhesive tape and record all positive reactions with the aid of the colour chart(included in this booklet). Record the results on the forms provided.

2. Add the appropriate reagents to the following microwells:

a) Add 2 drops of Kovac's reagent to well 8. Read and record the results after 60 seconds. Formation of a red colour indicates a positive result

b) Add 1 drop of VP I reagent and 1 drop of VP II reagent to well 10 and read after 15-30minutes.  Formation of a deep pink/red colour indicates a positive result.

c) Add 1 drop of TDA reagent to well 12 and read after 60 seconds. Formation of a cherry red colour indicates a positive result.

3. For Oxidase Positive organisms, Perform the nitrate reduction test on well 7 after reading and recording the ONPG result. Add 1 drop of Nitrate A reagent and 1 drop of Nitrate B reagent to the well and read after 60 seconds. The development of a red colour indicates that nitrate has been reduced to nitrite. If well 7 remains yellow or colourless after addition of nitrate reagents, add a small amount of zinc powder. This will indicate whether nitrate has been completely reduced to nitrogen gas.

After addition of Nitrate A + B:

Red = Positive
Colourless/Yellow = Negative

After addition of zinc powder:

Colourless/Yellow = Positive
Red = Negative

Record these additional results on the forms provided.

GN B Strip

1. Remove the adhesive tape and record all positive reactions with the aid of the colour chart.
2. Record the results on the forms provided.
3. Read specific well as follows:

a) the gelatin well (13) must be read after 18-24 hours for Enterobacteriaceae and after 48 hours for oxidase positive isolates. A positive gelatin liquefaction result is indicated by black particles visible throughout the well.

b) The arginine well is interpreted differently after 24 hours and 48 hours incubations:

24 hours (Enterobacteriaceae)
Yellow = Negative
Green/Blue = Positive

48 hours (Oxidase positive organisms)
Yellow/Green = Negative
Blue = Positive

IDENTIFICATION

On the Microgen GN-ID A+B Report Form, the substrates have been organised into triplets (sets of 3 reactions) with each substrate assigned a numerical value (1, 2 or 4). The sum of the positive reactions for each triplet forms a single digit of the Octal Code that is used to determine the identity of the isolate. The Octal Code is entered into the Microgen Identification System Software (MID-60), which generates a report of the five most likely organisms in the selected database.
The software provides an identification based on on probability, % probability and likelihood with an analysis of the quality of differentiation. Full definition of these terms and an explanation of their usefulness in interpretation is provided with the software Help manual.

Note: For oxidase positive organisms (miscellaneous gram negative bacilli):
• Record weak reactions as negative. The results for oxidase, nitrate reduction and motility must be included to form a 9 digit Octal Code

Limitations

Results should be interpreted in the context of all available laboratory information.
The Microgen ID system is intended for identification of those organisms included in the database. It should not be used to identify any other bacteria.

Test only pure, single colonies since mixed colonies may give erroneous results.

Reactions obtained using Microgen GN-ID may differ from published data obtained using alternative substrate formulations or reagents.

Some bacterial strains may have atypical biochemical reactions and may be difficult to identify.

Computer generated identification results should be interpreted by suitably trained personnel.

When determining the final identification of an isolate, the source of the isolate, gram staining, colonial morphology, additional tests and tests against the suggested identification should be considered.

Motility and nitrate tests must be performed on oxidase positive, gram negative bacilli. A 9 digit Octal Code is required to interpret the results using the Microgen Identification System Software.

The GN-ID A microwell test strip may not be able to differentiate accurately between Klebsiella spp, Enterobacter spp and Serratia spp. Species within these three genera may be differentiated by using GN-ID A+B. Alternatively, additional tests such as motility and DNAse tests can be used.

 

Key Points: 

Reagents and Supplies:
MID-641 160 Test GN-ID A Panel
20 x A microwell plates each composed of 8 x microwell test strips containing 12 biochemical
substrates for identification of GN A organisms
(see data tables)

MID-65 24 Test GN-ID B Panel
24 x B Microwell test strips containing 12 biochemical substrates to be used with GN A microwell test strips for identification of GN B organisms
(see data tables)

Microgen Identification System Software (MID-60)
Oxidase Strips (6)
Mineral Oil
VP I and VP II Reagents (7)
Nitrate A&B Reagents (8)
TDA Reagent (9)
Kovac's Reagent (10)
Sterile 0.85% saline
Sterile pipettes and bacteriological loops
Incubator (35-37°C)

Reagent Storage:
• GN A and GN B microwell test strips are stable in unopened foil pouches at 2-8°C until the expiry date on the label.
• Opened and partially used pouches of microwell test strips can be stored for up to 14 days at 2-8°C provided that the pouch is resealed and contains the desiccant sachets.

Quality Control:
The performance of the Microgen GN-ID system should be monitored using appropriate control strains. The following cultures are recommended for independent laboratory assessment:

Klebsiella pneumoniae NCTC 9528
Acinetobacter baumannii ATCC 19606
Proteus mirabilis ATCC 14153
Escherichia coli ATCC 25922

For each new box of reagent all four of the above strains will be tested for quality control. Thereafter, one different strain will be tested each week (rotating through all four strains).  Acceptability limits for identification using the online Microgen ID system are 90% +/- 10% from a historical perspective. 

REFERENCES
Lapage S.P, Bascombe S, Willcox W.R and Curtis M.A. (1973) Identification of Bacteria byComputer: General Aspects and Perspectives J.Gen. Microbiol. 77: 273 -290

Murray, Baron, Pfaller, Tenover, Yolken Manual of Clinical Microbiology, 6th Edition

Ewing W.H. (1972) Identification of Enterobacteriaceae, 3rd Edition, Minneapolis: Burgess Printing Company

Ewing W.H. (1986) Edwards and Ewing’s Identification of Enterobacteriaceae, 4th Edition. Elsevier Science Publishing Co., New York, N.Y.

Murray P.R. (Ed) (1999) Manual of Clinical Microbiology 7th Edition. American Society forMicrobiology, Washington, DC

Cruickshank R, Duguid J.P, Marmion B.P, Swain R.H.A. The Practice of Medical Microbiology,Medical Microbiology, 12th Edition, pp180-181

Barritt M.M, (1936) The intensification of the Voges Proskauer reaction by the addition of alphanaphthol. J. Pathol. Bacteriol 42: 441

Conn H.J, (1936) On the detection of nitrate reduction. J. Bacteriol. 21: 225

Singer J. and Volcani B.E. (1955) An improved ferric citrate test for differentiating Proteus-Providencia group from other Enterobacteriaceae. J. Bacteriol. 69: 255

Gadebusch H.H and Gabriel S. (1956) Modified stable Kovacs reagent for the detection of indol Am. J. Clin. Pathol. 26: 1373