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Microbact Biochemical Identification Kits

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GRAM-NEGATIVE IDENTIFICATION SYSTEM

MICROBACT™ 12A (12E) AND 24E (12A (12E) + 12B)
GRAM-NEGATIVE IDENTIFICATION SYSTEM

CODES: MB1073, MB1074, MB1076, MB1077, MB1130, MB1131, MB1132, MB1133.

Intended use
The Microbact™ Gram-negative system is to be used for the identification of aerobic and facultatively anaerobic Gram-negative bacteria (Enterobacteriaceae and miscellaneous Gram- negative bacteria) 2,3,4,5,6,7.

Principle of the test
The Microbact™ Gram-negative system is a standardised micro-substrate system designed to simulate conventional biochemical substrates used for the identification of Enterobacteriaceae and common miscellaneous Gram-negative bacilli (MGNB). Organism identification is based on pH change and substrate utilisations as established by published reference methodologies 4,6,8,9. Refer to the Table of Reactions below for the substrates contained in each well, the specific reaction principle, and colour changes.
The Microbact™ Gram-negative product consists of two separate substrate strips, 12A and 12B. Each strip consists of 12 different biochemical substrates. The 12A strip may be used alone for identification of oxidase-negative, nitrate-positive glucose fermenters comprising 15 genera and may be useful for screening pathogenic Enterobacteriaceae from enteric and urine specimens or identification of other common isolates. The 12B strip can be used in conjunction with the 12A strip for the identification of oxidase-positive, nitrate-negative, and glucose-nonfermenters (MGNB) as well as the Enterobacteriaceae.
Note: The 12 substrates contained in the 12A strips are available in a solid microplate format, referred to as 12E. The 12B strips can be used alongside the 12E, but in a separate tray. The 24E solid microplate format contains the 24 substrates contained in the combination of both the 12A and 12B strips.

THE FOLLOWING SPECIES OF OXIDASE-NEGATIVE, GRAM-NEGATIVE BACILLI CAN BE IDENTIFIED USING THE 12A (12E) ALONE:
Enterobacteriacea

Acinetobacter spp. Shigella spp.

P. stuartii

A. baumannii

Shigella serogp. AB&C

P. alcalifaciens
A.lwoffii S. sonnei Salmonella spp.
A. haemolyticus Hafnia sp. Salmonella spp.
Citrobacter spp. H. alvei S. typhi
C. freundii Klebsiella spp. S. cholerae-suis

C. diversus

K. pneumoniae S. paratyphi A
Enterobacter spp.

K. oxytoca

S. arizonae
E. aerogenes K. ozaenae

Serratia spp.

E. cloacae K. rhinoscleromatis S. marcescens
E. agglomerans Morganella sp. S. liquefaciens
E. gergoviae M. morganii S. rubidaea

E. sakazakii

Proteus spp. Tatumella sp.
Escherichia spp. P. mirabilis T. ptyseos
E. coli P. vulgaris Yersinia spp.
E. coli-inactive Providencia spp. Y. enterocolitica
E. vulneris P. rettgeri Y. pseudotuberculosis

ADDITIONAL OXOIDASE-NEGATIVE, GRAM-NEGATIVE BACILLI CAN BE IDENTIFIED USING THE 12A (12E) + 12B COMBINED OR BY USING 24E:

Acinetobacter spp.

Ewingella sp.

Salmonella subsp.3B
A. baumannii E. americana Salmonella subsp.4
A. lwoffii Hafnia sp. Salmonella subsp.5
A. haemolyticus H. alvei Salmonella subsp.6
Budvicia sp. H. alvei biogp 1 Serratia spp.
B. aquatica Klebsiella spp. S. marcescens

Buttiauxella sp.

K. pneumoniae S. marcescens biogp 1
B. agrestis K. oxytoca S. liquefaciens
Cedecea spp. K. ornithinolytica S. rubidaea
C. davisae K. planticola S. odorifera biogp 1
C. lapagei K. ozaenae S. odorifera biogp 2
C. neteri K. rhinoscleromatis S. plymuthica

Cedecea sp 3

K. terrigena S. ficaria
Cedecea sp 5 Klebsiella gp 47 S. entomophila
Citrobacter spp. K. ascorbata S. fonticola
C. freundii

K. cryocrescens

Shigella spp.
C. diversus Leclercia sp.
Shigella serogp.AB&C
C. amalonaticus L. adecarboxylata S. sonnei
C. amalonaticus biogp 1 Leminorella spp.
Trabulsiella sp.
C. farmeri L. grimontii T.guamensis

C. youngae

L. richardii Xenorhabdus spp.
C. braakii Moellerella sp.
X. luminescens(25C)
C.werkmanii M. wisconsensis X. luminescens gp 5
C. sedlakii Morganella sp.
X. nematophilis (25C)
Citrobacter sp 9 M. morganii Xanthomonas sp.
Citrobacter sp 10 M. morganii ssp. morganii X. (S. )maltophilia
Citrobacter sp 11 M. morganii biogp 1 Yersinia spp.

Edwardsiella spp.

M. morganii ssp. Siboni 1 Y. enterocolitica
E. tarda Obesumbacterium sp.
Y. frederiksenii
E. tarda biogp 1 O. proteus biogp 2 Y. intermedia
E. hoshinae Pragia sp.
Y. kristensenii
E .ictaluri P.fontium Y. rohdei
Enterobacter spp. Proteus spp.
Y. aldovae
E. aerogenes P. mirabilis Y. bercovieri

E. cloacae

P. vulgaris Y. mollaretii
E. agglomerans P. penneri Y. pestis
E. gergoviae P. myxofaciens Y. pseudotuberculosis
E. sakazakii Providencia spp. Y. ruckeri
E. taylorae P. rettgeri

Yokenella sp.

E. amnigenus biogp 1 P. stuartii Y. regensburgei
E. amnigenus biogp 2 P. alcalifaciens Enteric Gp17
E. asburiae P. rustigianii Enteric Gp41
E. hormaechei P. heimbachae Enteric Gp45

E. intermedium

Rahnella sp. Enteric Gp58
E. cancerogenus R. aquatilis Enteric Gp59
E. dissolvens Salmonella spp. Enteric Gp60
E. nimipressuralis Salmonella subsp.1 Enteric Gp63
Escherichia spp. S. typhi Enteric Gp64
E. coli S. cholerae-suis Enteric Gp68
E. coli-inactive S. paratyphi A

Enteric Gp69

E. fergusonii S. gallinarum  
E. hermannii S. pullorum  
E. vulneris Salmonella subsp.2  

E. blattae

S. arizonae subsp.3A  

For further information refer to the Help File in the Microbact™ Computer Aided Identification Package.

AND THE FOLLOWING OXIDASE-POSITIVE BACTERIA CAN BE IDENTIFIED USING THE 12A (12E) + 12B COMBINED OR BY USING 24E:

Pseudomonas spp. Flavobacterium spp.

Moraxella spp.

Ps. aeruginosa

F. meningosepticum
(Chryseobacterium meningosepticum)

Moraxella spp.
Ps. fluorescens-25 F. odoratum (Myroides odoratus) Plesiomonas sp.
Ps. fluorescens-35 F. breve  (Empedobacter brevis) P. shigelloides
Ps. putida F. indologenes
(Chryseobacterium indologenes)
Aeromonas spp.
Ps. stutzeri Vibrio spp. A. hydrophila
Ps. diminuta

V. fluvialis

A. veronii bio sobria
Burkholderia spp. V. furnissii A. veronii bio veronii
B. cepacia V. mimicus A. caviae
B. pseudomallei V. vulnificus Weeksella spp.
Shewanella sp. V. hollisae W .virosa

S. putrefaciens

V. cholerae W. zoohelcum
Alcaligenes spp. V. parahaemolyticus Pasteurella spp.
A. faecalis type 11 V. alginolyticus P. multocida
A. faecalis   P. haemolytica

A. xylosoxidans spp. xylos
(Achromobacter xylosoxidans}

  Actinobacillus spp.
    Actinobacillus sp.

For further information refer to the Help File in the Microbact™ Computer Aided Identification Package.

Warning and precautions
1. These strips are intended for in vitro use only; for use by qualified laboratory personnel using aseptic techniques and established precautions against microbiological hazards.
2. Used materials should be autoclaved, incinerated, or immersed in germicide before disposal.
3. DO NOT incubate strips in a CO2 incubator as substrates and/or enzyme reactions could be adversely affected, giving false reactions.

Storage instructions
The test strips are stable if kept unopened in the foil envelopes until the expiry date specified when stored at 2-8°C. Once the foil pouch has been opened, unused strips must be placed back in the foil pouch, and the foil pouch taped closed. Strips stored in this manner must be used within 7 days.

Kit presentation
Each kit contains the following:

1 Holding Tray.
Technical Product Insert.
Organism ID Report Forms including Colour Interpretation Chart.

Small Kits
MB1130A Microbact 12E (10 pouches, 80 identifications)
MB1131A Microbact 24E (10 pouches, 40 identifications)
MB1132A Microbact 12A (10 pouches, 60 identifications)
MB1133A Microbact 12B (10 pouches, 60 identifications)
Large Kits
MB1073A Microbact 12E (20 pouches, 160 identifications)
MB1074A Microbact 24E (20 pouches, 80 identifications)
MB1076A Microbact 12A (20 pouches, 120 identifications)

MB1077A Microbact 12B (20 pouches, 120 identifications)


Materials Required but not provided

The following materials may be required but are not provided:

Order Code Product Name
MB0209 Indole
MB0181 VP I
MB0184 VP II
MB0180 TDA
MB0186 NIT A
MB0187 NIT B
MB1093 Mineral Oil

MB0266

Oxidase Strips

These can be purchased individually or as a set by ordering product code MB1082:
Reagent Set D (Indole, VPI, VPII, TDA, NIT A/B)
MB1244 Microbact™ Computer Aided Identification Package
MB0266A Oxidase reagent
Motility medium
2.5/5.0ml suitable saline solution, 0.85%
5.0ml peptone water (for use with selective media)
SR0035 Sterile serum
Zinc powder
Inoculating loop
Sterile pipettes
Fanless Incubator (or use container in Incubator with fan) (35° ± 2°C), Non - CO2

Set up proceedure
Isolation

An 18-24 hour pure culture of the organism to be identified must be obtained. Appropriate agar media, for example MacConkey (CM0007), Eosin Methylene Blue (CM0069), Blood or Chocolate (CM0331 and SR0050), may be used to grow organisms.
Qualified Personnel should collect specimens according to standards routinely required for specimen handling 10.
Before use, perform an oxidase test on the organism to be identified.
Note: Oxidase positive organisms cannot be identified using the Microbact™ 12A (12E) alone and should be examined with the Microbact™ 24E (12A (12E) + 12B).
Refer to the procedure chart for a condensed version of the following procedures.

Procedure chart:

 
12A (or 12E)
Enterobacteriaceae
24E (12A (or 12E) + 12B)
Enterobacteriaceae
24E (12A (or 12E) + 12B)
Miscellaneous Gram-Negative Bacilli
Oxidase
Negative
Negative
Positive
Preparation of Inoculum

1-2 colonies
2.5 ml saline

2-3 colonies
5.0 ml saline

2-3 colonies
5.0ml saline

When Actinobacillus or Pasteurella spp. are suspected, add 1 drop sterile serum per ml of saline suspension
Strip Inoculation
4 drops to each well
4 drops to each well
4 drops to each well
Oil Overlay
Well 1(lysine)
Well 2 (ornithine)
Well 3 (H2S)
12B well 8 (arabinose)
24E well 20(arabinose)
12B well 12 (arginine)
24E well 24 (arginine)
12B well 12 (arginine)
24E well 24 (arginine)
Incubation Temperature
35° ± 2°C
35° ± 2°C
35° ± 2°C
(25°C for Ps. fluorescens)
Test Reading:
Read and record all positive test results
Reagent Addition:
Well 8 – Indole
= 2 drops Kovacs, read within 2 minutes
Well 10 – VP
= 1 drop of each, VPI and VPII,
read at 15 – 30 minutes
Well 12 – TDA
= 1 drop TDA, read immediately

Reagent Addition:
See 12A/12E
Well 1 (12B) Well 13 (24E)
Interpret Gelatin at
24-48 hours
Well 12 (12B) Well 24 (24E)
Arginine
Yellow - Negative
Green-blue - Positive

Reagent Addition:
See 12A/12E
Well 1 (12B) Well 13 (24E)
Interpret Gelatin at 48 hours
Well 12 (12B) Well 24 (24E)
Arginine
Yellow-green - Negative
Blue - Positive

Preparation of inoculum
Pick 1-3 isolated colonies from an 18-24 hour culture and emulsify in 2.5ml of sterile saline solution if 12A/E alone is being used or 5.0 ml of sterile saline if 24E (12A/E and 12B) are being used. Mix thoroughly to prepare a homogeneous suspension.
If the organism has been grown on a selective medium and the colony is small or inhibited, it may be necessary to emulsify the colony in 5.0 ml of peptone water and incubate at 35° ± 2°C for four hours. Using a sterile Pasteur pipette, transfer one drop of the peptone water culture into the appropriate volume (see Procedure Chart) of sterile saline solution (0.85%).

Inoculation
The wells of individual substrate sets can be exposed by cutting the end tag of the sealing strip and slowly peeling it back.
Place the strip or plate in the holding tray and using a sterile Pasteur pipette add 4 drops (approximately 100 µl) of the bacterial suspension, or half fill each well in the set. When Actinobacillus or Pasteurella sp. are suspected (no growth on media containing bile salts or on media deficient in blood or serum) add one drop of sterile serum (SR0035) per ml of saline suspension.
Using a sterile pipette or dropper bottle, overlay the substrates underlined on the holding tray with sterile mineral oil, i.e. wells 1, 2 and 3 for 12A (12E) or 24E and wells 8 and 12 for 12B or wells 20 and 24 for 24E. (Well 8 for 12B and 20 for 24E is not overlayed with oil for oxidase- positive, miscellaneous Gram-negative bacilli.)

Incubation
Reseal the inoculated rows with the adhesive seal and write the specimen identification number on the end tag with a marker pen. Incubate at 35° ± 2°C for 18-24 hours. When Ps. fluorescens appears as the organism of choice, repeat the test at an incubation temperature of 25° ± 2°C.To determine the purity of the inoculum, it is advisable to inoculate a solid non-selective medium with the test suspension to act as a culture purity check.

Reading the test strip
1. The 12A (12E) strip should be read at 18-24 hours. The 12B/24E strip is read at 24 hours when identifying Enterobacteriaceae. All systems should be read after 48 hours for the identification of Miscellaneous Gram-negative bacilli.
2. Remove the strips or tray from the incubator, peel back the sealing tape. Record all positive results. The reactions are evaluated as positive or negative by comparing them with the colour chart. Record the results under the appropriate heading on the report form. For aid in interpreting reactions, refer to the Table of Reactions.

1. 12A (12E) or 24E
Add the following reagents: Well 8 (Indole production) - add 2 drops of Indole (Kovacs) reagent. Evaluate within 2 minutes of the addition of the reagent.
Well 10 (Voges-Proskaüer reaction) - add 1 drop each of VPI reagent and VPII reagent. Evaluate 15 to 30 minutes after the addition of reagents.
Well 12 (Tryptophan Deaminase) - add 1 drop of TDA reagent. Test can be evaluated immediately after the addition of the reagent.

2. 12B/24E
The gelatin well (well 1 for 12B and well 13 for 24E) must be read at 24-48 hours for Enterobacteriaceae and at 48 hours for miscellaneous Gram-negative bacilli (MGNB). Hydrolysis of gelatin is indicated by dispersal of the black particles throughout the well.
The arginine reaction (well 12 for 12B and well 24 of 24E) is interpreted differently at 24 hours and 48 hours incubation.
24 Hours Incubation (Enterobacteriaceae):
Yellow - Negative
Green-blue - Positive
48 Hours Incubation (MGNB):
Yellow-green - Negative
Blue - Positive

Additional Test

Nitrate Reduction Test (o-nitrophenyl-ß-d-galactopyranoside (ONPG))
This test is performed in well 7 (ONPG) AFTER reading the ONPG reaction. One drop of Nitrate reagent A and 1 drop of Nitrate reagent B is added to the well. Production of a red colour within a few minutes of the addition of the reagent indicates that nitrate reduction to nitrite (NO2) has occurred. A small amount of zinc powder should be added to those wells which exhibit a yellow colour after the addition of the nitrate reagents. This will determine whether nitrate has been reduced completely to nitrogen gas (N2). The results should be interpreted as follows:

After the addition of Nitrate reagents A and B:
Red colour
Positive
NO2 Positive
Yellow colour
Negative
NO2 Negative
On the addition of zinc powder:
Yellow colour
Positive
(N2+)
Red colour
Negative
(N2-)

All organisms belonging to the family Enterobacteriaceae reduce nitrates to nitrites and give a positive reaction.
Note: Gram-negative bacilli which do not reduce nitrates can only be identified by utilising the 24E (12A (12E) + 12B) system.

TABLE OF SUBSTRATES AND REACTIONS (12A/12E/24E):

Well No.
Designation
Reaction Principle
Reaction colours
Comments
Negative
Positive
1
Lysine
Lysine decarboxylase
Yellow
Blue-green
Green or blue is positive reaction. Bromothymol blue indicates formation of the specific amine cadaverine.
2
Ornithine
Ornithine
decarboxylase
Yellow-green
Blue
Green should be regarded as a negative reaction. The pH shift indicated by bromothymol blue caused by formation of the specific amine putrescine is greater than that caused by lysine decarboxylation.
3
H2S
H2S production
Straw colour
Black
H2S is produced from thiosulphate. H2S reacts with ferric salts in the medium to form a black precipitate.
4
Glucose
Glucose
fermentation
Blue-green
Yellow

Bromothymol blue indicator changes from blue to yellow when the carbohydrate is utilised to form acid.

5
Mannitol
Mannitol
fermentation
Blue-green
Yellow
6
Xylose
Xylose
fermentation
Blue-green
Yellow
7
ONPG
Hydrolysis of o-nitrophenyl-ß-d-galactopyranoside
(ONPG)by action
of ß-galactosidase
Colourless
Yellow
ß-galactosidase hydrolysis of the colourless ONPG releases yellow orth-onitrophenol.
8
Indole
Indole production
from tryptophan
Colourless
Pink-red

Indole is formed from metabolism of tryptophan.. Indole Kovacs reagent forms a pink-red complex with indole.

9
Urease
Urea hydrolysis
Straw colour
Pink-red
Ammonium released from splitting of urea causes the pH to rise - indicated by phenol red changing from yellow to pink-red
10
VP
Acetoin production
(Voges-Proskaüer
reaction)
Straw colour
Pink-red
Acetoin is produced from glucose indicated by the formation of a pink-red complex after the addition of alpha-naphthol and creatine.
11
Citrate
Citrate utilization
(citrate is the only
source of carbon)
Green
Blue
Citrate is the sole carbon source, which if utilized results in a pH rise, indicated by bromothymol blue, with a colour change from green to blue.
12
TDA
Production of
indolepyruvate by
deamination of
tryptophan
Straw colour
Cherry red

Tryptophan deaminase forms indolepyruvic acid from tryptophan which produces a brown colour in the presence of ferric ions. Indole positive organisms may produce a brown colour. This is a negative reaction.

TABLE OF SUBSTRATES AND REACTIONS (12B/24E):

Well No.
12B/24E.
Designation
Reaction Principle
Reaction colours
Comments
Negative
Positive
1/13
Gelatin
Gelatin liquefaction
Colourless
Black
Liquefaction of gelatin by proteolytic enzymes diffuses the black pigment. Solid gelatin particles which may drift across the well after rehydration should be considered as a negative reaction.
2/14
Malonate
Malonate inhibition
Green
Blue
Sodium malonate is the sole carbon source and this inhibits the conversion of succinic acid to fumaric acid. An organism unable to utilize this substrate results in the accumulation of succinic acid and the organism cannot grow. Bromothymol blue is the indicator. Yellow-green is indicative of a negative result. Utilisation of Na malonate at the same time that ammonium sulphate is utilised as the nitrogen source produces sodium hydroxide resulting in increased alkalinity and a blue colouration.
3/15
Inositol
Inositol
fermentation
Blue-green
Yellow

Bromothymol blue indicator changes from blue to yellow when the carbohydrate is fermented.

4/16
Sorbitol
Sorbitol
fermentation
Blue-green
Yellow
5/17
Rhamnose
Rhamnose
fermentation
Blue-green
Yellow
6/18
Sucrose
Sucrose
fermentation
Blue-green
Yellow
7/19
Lactose
Lactose
fermentation
Blue-green
Yellow
8/20
Arabinose
Arabinose
fermentation
Blue-green
Yellow
9/21
Adonitol
Adonitol
fermentation
Blue-green
Yellow
10/22
Raffinose
Raffinose
fermentation
Blue-green
Yellow
11/23
Salicin
Salicin
fermentation
Blue-green
Yellow
12/24
Arginine
Arginine
dihydrolase

Argine dihydrolase converts arginine into ornithine, ammonia and carbon dioxide. This causes a pH rise as indicated by bromothymol blue. Green reactions occurring at 48 hours should be interpreted as negative

24 hours
Yellow
Green-blue
48 hours
Yellow-green
Blue

Interpretation
An octal coding system has been adopted for Microbact™ 1. Each group of three reactions produces a single digit of the code. Using the results obtained, the indices of the positive reactions are circled. The sum of these indices in each group of three reactions forms the code number. This code is entered into the computer package.

Computer aided identification Package
The Microbact™ Computer Aided Identification Package should be consulted for the identification choices. The percentage figure shown against the organism name is the percentage share of the probability for that organism as a part of the total probabilities for all choices.
Note: Miscellaneous Gram-negative bacilli - Weakly positive reactions are recorded as negative results. The results of tests for oxidase, nitrate reduction and motility are included as part of the reaction pattern. Using the results obtained, from each group of three reactions a 9 (nine) digit code number is produced.

Quality control
The overall performance of the system should be monitored by testing appropriate control strains. The following organisms are recommended for independent laboratory assessment.

Proteus mirabilis ATCC® 12453 Escherichia coli ATCC® 25922
Klebsiella pneumoniae ATCC® 13883

Acinetobacter baumannii ATCC®19606

The following chart gives the expected results on the Microbact System after an 18-24 hour incubation:

 
Escherichia coli
ATCC® 25922
Klebsiella pneumoniae
ATCC® 13883
Proteus mirabilis ATCC® 12453
Acinetobacter baumannii ATCC®19606
1
12E/A
24E
LYS
+
+
-
-
2
ORN
+
-
+
-
3
H2S
-
-
+
-
4
GLU
+
+
+
+
5
MAN
+
+
-
-
6
XYL
+
+
-
+
7
ONP
+
+
-
-
8
IDN
+
-
-
-
9
URE
-
+
+
-
10
VP
-
+
-
-
11
CIT
-
+
+
+
12
TDA
-
-
+
-
7
NIT
+
+
-
-
1
24E/
12B
GEL
-
-
-
-
2
MAL
-
+
-
+
3
INO
-
+
-
-
4
SOR
+
+
-
-
5
RHA
+
+
-
-
6
SUC
-
+
-
-
7
LAC
+
+
-
-
8
ARA
+
+
-
+
9
ADO
-
+
-
-
10
RAF
-
+
-
-
11
SAL
-
+
-
-
12
ARG
-
-
-
-

Note 1: Serratia marcescens (ATCC® 43861) can be run as supplemental QC to test the sensitivity of the GEL reaction. The expected results is positive.
Note 2: Flavobacterium multivorum (ATCC® 35656) may be used to show a negative glucose result.

Limitations
1. Some bacterial strains may have atypical biochemical reactions due to unusual nutritional requirements or mutations and may be difficult to identify.
2. Reactions obtained using the Microbact System may differ from published results using other substrate formulations. Prolonged incubation, insufficient incubation, improper filling of wells, or inadequate inoculum may lead to false results.
3. Species with low frequency of occurrence require additional testing.
4. Acinetobacter calcoaceticus var. anitratus will include those strains that have been designated as Acinetobacter calcoaceticus, Acinetobacter baumannii, and unnamed genospecies 3; most clinical isolates that are glucose-positive and nonhemolytic are Acinetobacter baumannii.
5. The interpretation of mathematically calculated identification results requires trained clinical personnel who should use judgement and knowledge in conjunction with the following information before accepting the ID of an organism: Gram-stain, colonial morphology, source of isolate, percent probability (degree of separation), tests against, additional test indications and results, frequency of ID choice and antibioGram.
6. A Gram-stain and oxidase test should be performed prior to set-up of tests. In addition, motility and nitrate test should be performed for miscellaneous Gram-negative bacilli.
7. When using the 12E/A strip alone, Klebsiella spp., Enterobacter spp. and Serratia spp. should be reported as Klebsiella/Enterobacter/Serratia group. Twelve substrates provide insufficient data to speciate within this group as a single aberrant reaction may result in an incorrect identification. The lysine and ornithine decarboxylase reactions should be carefully interpreted. Motility and DNase tests are recommended for further speciation of this group. The inclusion of a 12B strip is strongly advised.
8. If further speciation is required for Yersinia spp. (i.e. other than Yersinia enterocolitica and Yersinia pseudotuberculosis), additional testing is required.

References
1. Identification of Bacteria by Computer: General Aspects and Perspectives. Lapage, S.P., et al (1973) J. Gen. Microbiology. 77, 273.
2. Comparison of Microbact 12E, API 20E and Conventional Media Systems for the Identification of Enterobacteriaceae. Mugg, P.A., (1979) The Australian Journal of Med. Tech. 10, 37-41.
3. Comparison of Microbact 12E and 24E systems and the API 20E systems for the Identification of Enterobactiaceae. Mugg, P.A. and Hill, A., (1981) J. Hyg. Camb. 87, 287.
4. Biochemical Identification of New Species and Biogroups of Enterobacteriaceae Isolated from Clinical Specimens. Farmer, J.J., et al, (Jan. 1985) J. Clin. Micro., 21 No. 1, 46-76.
5. Evaluation of the API20E and Microbact 24E Systems for the Evaluation of Pseudomonas pseudomallei. A.D. Thomas, (1983) Veterinary Microbiology. 8, 611-615.
6. Biochemical Characterisics of Enterotoxigenic Aeromonas sp. V. Burke, J. Robinson, H.M. Atkinson, and M. Gracey, (Jan. 1982) Journal of Clinical Microbiology. 48-52.
7. Comparison of five commercial methods for the Identification of Non-fermentative and Oxidase Positive Fermentative Gram-Negative Bacilli. Bilkey, Mary K., et al., (1988) N.Z.J. Med. Lab Technol., 8-12,
8. S.T. Cowen, K.J. Steel (1977) Manual for the Identification of Medical Bacteria, 2nd Edition Cambridge University Press.
9. A.Balows, W.J. Hausler, K.L. Herrmann, J.D. Isengerg, H. Jean Shadomy (eds). (1991) Manual of Clinical Microbiology, 5th Edition, American Society of Microbiology, Washington, D.C.

 
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