Technical Data

Hi25™ Enterobacteriaceae Identification Kit KB003

Intended use
KB003 is a standardized, colorimetric identification system, a combination of 25 tests for identification of
Enterobacteriaceae species from clinical specimen and non clinical samples using pure isolate.
Kit Contains :
Each kit contains sufficient material to perform 5/10/20 tests.
1. 5/10/20 kits of Part I. 7. TDA reagent (R036) for Phenylalanine Deaminase test.
2. 5/10/20 kits of Part II. 8. Baritt reagent A (R029) for Voges-Proskauer's test.
3. Oxidase reagent discs (DD018) 9. Baritt reagent B (R030) for Voges-Proskauer's test.
4. Technical product insert. 10.Methyl Red Indicator (I007) for Methyl Red test
5. Result Interpretation Chart and Result Entry 11.Kovac's reagent (R008) for Indole test
Datasheet. 12.Sulphanilic acid (R015) for Nitrate test
6. Identification Index. 13.N, N-Dimethyl-1-Napthylamine Reagent (R009) for Nitrate test.
Material Required but not supplied :
1.McFarland standard
2.Inoculation loops, pipettes
3.Enrichment medium / Isolation media
Direction
Preparation of inoculum
• KB003 cannot be used directly on clinical specimens. The organisms to be identified have to be first isolated and
purified. Only pure cultures should be used. Isolate the organism to be identified on a common medium like Nutrient
Agart (M001)/ Nutrient agar for oxidase (M1274) or a differential medium like MacConkey Agar (M082).
• Pick up a single isolated colony and inoculate in 5 ml BHI Broth (M210) and incubate at 35-37°C for 4-6 hours until the
inoculum turbidity is 0.1 OD at 620nm or 0.5 McFarland standard. Some fastidious organisms may require more than 6
hours of incubation. In this case incubate till the inoculum turbidity reaches 0.1 OD at 620nm. Alternatively,
prepare the inoculum by picking 1-3 well isolated colonies and make a homogenous suspension in 2-3ml sterile saline.
The density of the suspension should be 0.1 OD at 620nm.
• Perform Oxidase test on the organism to be tested. The test is performed using Oxidase disc (DD018) provided with the
kit.
• Pick up a well isolated colony and rub it on a single oxidase disc. Positive reaction is indicated by development of deep
purple colour within 10 seconds. Colour change in 10-60 seconds indicates a delayed positive reaction. Colour
development after 60 seconds or no change in colour indicates a negative reaction. Note the result in the Result Entry
Datasheet. Oxidase test must be performed as it is an integral part of the identification system. It must be performed to
differentiate Enterobacteriaceae from other Gram negative rods.
Inoculation of the kit
1.Open the kit aseptically. Peel off the sealing foil.
2.Inoculate each well with 50 µl of the above inoculum by surface inoculation method.
3.Alternatively, the kit can also be inoculated by stabbing each individual well with a loopful of inoculum

Incubation
Temperature of incubation: 35-37°C. Duration of incubation: 18-24 hours.
Interpretation of results :
Interpret results as per the standards given in the identification index. Addition of reagents in well no 5,6,9,10, and 11of strip
1 should be done at the end of incubation period that is after 18 - 24 hours.

Please refer disclaimer Overleaf.

 HiMedia Laboratories Technical Data

Principle
Each Hi25™ kit is a standardized colorimetric identification system utilizing thirteen conventional biochemical tests and
eleven carbohydrate utilization tests. The tests are based on the principle of pH change and substrate utilization. On
incubation organisms undergo metabolic changes which are indicated by a colour change in the media that is either visible
spontaneously or after addition of a reagent. Oxidase test is performed separately using oxidase reagent disc provided with
the kit.
Type of specimen
Pure isolate from clinical specimen and non clinical sample
Specimen collection and handling
Refer direction
Warning and Precautions :
In Vitro diagnostic Use. For professional use only. Read the label before opening the pack. Wear protective gloves/protective
clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture.
Standard precautions as per established guidelines should be followed while handling clinical specimens. Aseptic conditions
should be maintained during inoculation and handling of the kits. Reagents should not come in contact with skin, eyes or
clothing. Safety guidelines may be referred in individual safety data sheets.
Limitations:
1.Allow the reagents to come to room temperature after removal from the refrigerator .
2.In case of carbohydrate fermentation test some microorganisms show weak reaction. In this case record the reaction as ±
and incubate further upto 48 hours. Orange colour after 48 hours of incubation should be interpreted as a negative
reaction.
3.At times organisms give conflicting result because of mutation or the media used for isolation, cultivation and
maintenance.
4.The identification index has been compiled from standard references and results of tests carried out in the laboratory.
5.Erroneous false negative results may be obtained if the inoculum turbidity is less than 0.1 OD.
6.Results are more prominent if an enriched culture is used instead of a suspension.
7.It cannot be used directly for clinical specimens. The microorganisms to be identified have to be first isolated on
appropriate isolation media. Only pure cultures should be used.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when
stored at recommended temperature.
Quality Control
Appearance
Two Sterile white opaque strips with 12 wells each. Strip 1 containing sterile media for ONPG, Lysine utilization,
Ornithine utilization, Urease detection, Phenylalanine deamination (TDA), Nitrate reduction, H2S production, Citrate
utilization, Voges Proskauer’s, Methyl red, Indole, Malonate, Strip 2 containing Esculin hydrolysis tests and 11 different
carbohydrates utilization test - Arabinose, Xylose, Adonitol, Rhamnose, Cellobiose, Melibiose, Saccharose, Raffinose,
Trehalose, Glucose, Lactose. Oxidase disc are given seperately.
Quantity of medium
0.8 ml of medium in each well.
Sterility Check
Passes release criteria
Interpretation of results :
Interpret results as per the standards given in the identification index. Addition of reagents should be done at the end of
incubation period that is after 18 - 24 hours.
Part I :
ONPG Test : Well No. 1
• Colour change from colorless to yellow indicates positive reaction.
• No colour change indicates negative reaction

Please refer disclaimer Overleaf.

 HiMedia Laboratories Technical Data

Lysine utilization: Well No. 2

• Colour change to Purple / Dark Purple indicates positive reaction
• No colour change or yellow colour indicates negative reaction.
Ornithine utilization: Well No. 3
• Colour change to Purple / Dark Purple indicates positive reaction
• No colour change or yellow colour indicates negative reaction.
Urease Test: Well No. 4
• Colour changes to pink indicates positive reaction.
• No colour change indicates negative reaction.
Phenylalanine Deamination Test : Well No. 5
• Add 2-3 drops of TDA reagent (R036).
• Development of dark green colour within one minute indicates a positive reaction.
• No change in colour denotes a negative reaction.
Nitrate Reduction Test : Well No. 6
• Add 1-2 drops of Sulphanilic acid (R015) and 1-2 drops of N,N-Dimethyl-1-Napthylamine Reagent (R009).
• Immediate development of pinkish red colour on addition of reagent indicates positive reaction.
• No change in colour indicates a negative reaction.
H2S production: Well No. 7
• Orangish yellow colour to black indicates a positive reaction
• No color change or slight yellowish brown indicates a negative reaction.
Citrate utilization: Well No.8
• Positive test is indicated by a colour change to blue colour.
• Green or no colour change indicates a negative reaction.
Voges-Proskauer's Test: Well No.9
• Add 2-3 drops of Baritt reagent A (R029) and 1 drop of Baritt reagent B (R030).
• Positive test is indicated by a development of pinkish red colour in 5 - 10 minutes.
• No colour change or a copper colour (due to reaction of Reagent A and Reagent B) indicates a negative reaction.
Methyl red Test : Well No. 10
• Add 2-3 drops of Methyl red indicator (I007).
• Positive test is indicated by a development of red colour.
• Yellowish orange colour indicates a negative reaction.
Indole Test : Well No. 11
• Add 2-3 drops of Kovac's reagent (R008)
• Positive test is indicated by a development of pinkish red ring.
• No red ring indicates a negative reaction.
Malonate utilization: Well No. 12
• Positive test is indicated by a colour change to blue colour.
• Light green or no colour change indicates a negative reaction.

Part II
Esculin hydrolysis: Well No. 1
• Cream colour changes to black colour indicates a positive reaction
• No colour change or brownish yellow indicates a negative reaction
Carbohydrate fermentation Test : Well No. 2-12
• Positive test is indicated by a colour change to yellow colour.
• Red or no colour change indicates a negative reaction.
Oxidase test
• Change in colour from colourless to deep purple colour within 10 seconds indicates positive reaction.
• Colourless after 60 seconds indicates negative reaction

Please refer disclaimer Overleaf.

HiMedia Laboratories Technical Data

Strip I Result Interpretation chart

No. Test Reagents to be added Principle Original colour Positive Negative

after incubation of the medium reaction reaction
1 ONPG — Detects galactosidase activity Colourless Yellow Colourless

2 Lysine Olive green to Purple / Dark

utilization — Detects Lysine decarboxylation Light Purple Purple Yellow

3 Ornithine Olive green to Purple / Dark

utilization — Detects Ornithine decarboxylation Light Purple Purple Yellow

4 Urease — Detects Urease activity Orangish yellow Pink Orangish yellow

5 Phenylalanine 2-3 drops of TDA reagent Detects Phenylalanine Colourless Green Colourless
Deamination deamination activity

6 Nitrate 1-2 drops of sulphanilic acid Detects Nitrate reduction Colourless Pinkish Red Colourless
reduction and 1-2 drops of N, N-
Dimethyl-1-Napthylamine

7 H2S production — Detects H2 S production Orangish yellow Black Orangish yellow

8 Citrate utilization — Detects capability of organism to Green Blue Green

utilize citrate as a sole carbon source

9 Voges 1-2 drops of Baritt reagent A and Detects acetoin production Colourless / Light Pinkish red Colourless/
Proskauer's 1-2 drops of Baritt reagent B Yellow slight copper

10 Methyl red 1-2 drops of Methyl red indicator Detects acid production Colourless Red Yellowish- orange

11 Indole 1-2 drops of Kovac's reagent Detects deamination of tryptophan Colourless Pinkish Red Colourless

12 Malonate — Detects capability of organism to Light green Blue Light green

utilization utilize sodium malonate as a sole
carbon source

Strip II Result Interpretation chart

No. Test Principle Original colour Positive Negative

of the medium reaction reaction
13 Esculin hydrolysis Esculin hydrolysis Cream Black Cream

14 Arabinose Arabinose utilization Pinkish Red / Red Yellow Red / Pink

15 Xylose Xylose utilization Pinkish Red / Red Yellow Red / Pink

16 Adonitol Adonitol utilization Pinkish Red / Red Yellow Red / Pink

17 Rhamnose Rhamnose utilization Pinkish Red / Red Yellow Red / Pink

18 Cellobiose Cellobiose utilization Pinkish Red / Red Yellow Red / Pink

19 Melibiose Melibiose utilization Pinkish Red / Red Yellow Red / Pink

20 Saccharose Saccharose utilization Pinkish Red / Red Yellow Red / Pink

21 Raffinose Raffinose utilization Pinkish Red / Red Yellow Red / Pink

22 Trehalose Trehalose utilization Pinkish Red / Red Yellow Red / Pink

23 Glucose Glucose utilization Pinkish Red / Red Yellow Red / Pink

24 Lactose Lactose utilization Pinkish Red / Red Yellow Red / Pink

25 Oxidase Done on Oxidase disc separately. Colourless Deep purple White/

Detects cytochrome oxidase production. within 10 seconds Purple after 60 seconds

Please refer disclaimer Overleaf.

HiMedia Laboratories Technical Data

KB003 : Hi25TM Enterobacteriaceae Identification Kit

Identification Index of various Enterobacteriaceae species

Tests ONPG Lysine Ornithine Urease TDA Nitrate H2S Citrate Utilization Voges Proskauer's Methyl Red Indole Malonate

Budvicia aquatica
Buttiauxella agrestis
Cedecea davisae
Cedecea lapagei
Cedecea neteri
Citrobacter amalonaticus
Citrobacter diversus
Citrobacter freundii
Enterobacter aerogenes
Enterobacter amnigenus (Biogroup I)
Enterobacter amnigenus (Biogroup II)
Enterobacter taylorae (E. cancerogenus)
Enterobacter cloacae

Enterobacter gergoviae
Enterobacter sakazakii
Escherichia coli
Escherichia coli, inactive
Escherichia blattae
Escherichia fergusonii
Escherichia hermannii
Escherichia vulneris
Ewingella americana
Hafnia alvei
Klebsiella oxytoca
Klebsiella pneumoniae
subspecies ozaenae

Klebsiella pneumoniae subspecies pneumoniae

Klebsiella pneumoniae subspecies
rhinoscleromatis
Klebsiella terrigena
Kluyvera ascorbata
Leclercia adecarboxylata
(Escherichia adecarboxylata)
Morganella morganii subspecies morganii
Morganella morganii subspecies sibonii
Pantoea agglomerans
Pantoea dispersa
Proteus mirabilis
Proteus myxofaciens
Proteus penneri
Proteus vulgaris
Providencia alcalifaciens
Providencia rettgeri
Providencia rustigianii
Rahnella aquatilis
Salmonella Bongori
Salmonella Choleraesuis subspecies Arizonae
Salmonella Choleraesuis
subspecies Choleraesuis
Salmonella Choleraesuis subspecies Darizonae
Salmonella Choleraesuis subspecies Houtenae
SalmonellaCholeraesuis subspecies Indica
Salmonella Choleraesuis subspecies Salamae
Salmonella Enteritidis
Salmonella Typhi
Salmonella Typhimurium
Serratia entomophila
Serratia ficaria
Serratia fonticola
Serratia marcescens
Serratia odorifera (Biogroup I)
Serratia odorifera (Biogroup II)
Serratia plymuthica
Serratia proteamaculans
Serratia rubidaea
Shigella boydii, Shigella
flexneri, Shigella dysenteriae
Shigella sonnei

Yersinia enterocolitica
Please
Yersinia refer disclaimer
frederiksenii Overleaf.
Yersinia intermedia
Yersinia pestis
Yersinia pseudotuberculosis

Please refer disclaimer Overleaf.

 HiMedia Laboratories Technical Data

KB003 : Hi25TM Enterobacteriaceae Identification Kit

Identification Index of various Enterobacteriaceae species

Tests Esculin
hydrolysis
Arabinose Xylose Adonitol Rhamnose Cellobiose Melibiose Saccharose Raffinose Trehalose Glucose Lactose

Budvicia aquatica
Buttiauxella agrestis
Cedecea davisae
Cedecea lapagei
Cedecea neteri
Citrobacter amalonaticus
Citrobacter diversus
Citrobacter freundii
Enterobacter aerogenes
Enterobacter amnigenus (Biogroup I)
Enterobacter amnigenus (Biogroup II)
Enterobacter taylorae (E. cancerogenus)
Enterobacter cloacae

Enterobacter gergoviae
Enterobacter sakazakii
Escherichia coli
Escherichia coli, inactive
Escherichia blattae
Escherichia fergusonii
Escherichia hermannii
Escherichia vulneris
Ewingella americana
Hafnia alvei
Klebsiella oxytoca
Klebsiella pneumoniae
subspecies ozaenae

Klebsiella pneumoniae subspecies pneumoniae

Klebsiella pneumoniae subspecies rhinoscleromatis
Klebsiella terrigena
Kluyvera ascorbata
Leclercia adecarboxylata
(Escherichia adecarboxylata)
Morganella morganii subspecies morganii
Morganella morganii subspecies sibonii
Pantoeaagglomerans
Pantoea dispersa
Proteus mirabilis
Proteus myxofaciens
Proteus penneri

Proteus vulgaris
Providencia alcalifaciens
Providencia rettgeri
Providencia rustigianii
Rahnella aquatilis
Salmonella Bongori
Salmonella Choleraesuis subspecies Arizonae
Salmonella Choleraesuis subspecies Choleraesuis
Salmonella Choleraesuis subspecies Diarizonae
Salmonella Choleraesuis subspecies Houtenae
Salmonella Choleraesuis subspecies Indica
Salmonella Choleraesuis subspecies Salamae
Salmonella Enteritidis

Salmonella Typhi
Salmonella Typhimurium
Serratia entomophila
Serratia ficaria
Serratia fonticola
Serratia marcescens
Serratia odorifera (Biogroup I)
Serratia odorifera (Biogroup II)
Serratia plymuthica
Serratia proteamaculans
Serratia rubidaea
Shigella boydii, Shigella flexneri,
Shigella dysenteriae
Shigella sonnei
Yersinia enterocolitica
Yersinia frederiksenii
Yersinia intermedia
Yersinia pestis
Yersinia pseudotuberculosis

Please refer disclaimer Overleaf.

HiMedia Laboratories Technical Data

Storage and Shelf Life

On receipt store between 2-8°C. Use before expiry date on the label. Product performance is best if used within
stated expiry period.

Disposal
After use, kits and the instruments used for isolation and inoculation (pipettes, loops etc.) must be disinfected using a
suitable disinfectant and then discarded by incineration or autoclaving in a disposal bag. Follow established laboratory
procedures in disposing of infectious materials and material that comes into contact with clinical sample must be
decontaminated and disposed of in accordance with current laboratory techniques (2,3).

Reference
1.Bergey's Manual of Systematics of Archaea and Bacteria (BMSAB), 2015.
2.Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
3.Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of
Clinical Microbiology, 11th Edition. Vol. 1.

Revision:01/2023

HiMedia Laboratories Pvt. Limited, IVD In vitro diagnostic

Plot No.C-40, Road No.21Y, 8°C Storage temperature
medical device
MIDC,WagleIndustrial Estate,
Thane (W) -400604, MS, India 2°C

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CE Marking
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Do not re-use

Disclaimer :
User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in this and
other related HiMedia™ publications. The information contained in this publication is based on our research and development work and is to the best
of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes to specifications and information related
to the products at any time. Products are not intended for human or animal or therapeutic use but for laboratory,diagnostic, research or further
manufacturing use only, unless otherwise specified. Statements contained herein should not be considered as a warranty of any kind, expressed or
implied, and no liability is accepted for infringement of any patents.

HiMedia Laboratories Pvt. Ltd. Corporate Office : Plot No.C-40, Road No.21Y, MIDC, Wagle Industrial Estate, Thane (W) - 400604,
India. Customer care No.: 022-6147 1919 Email: [email protected] Website: www.himedialabs.com