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KB003

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TM

KB003 Hi25 Enterobacteriaceae Identification Kit


Introduction
KB003 is a comprehensive test system that can be used for identification of gram-negative Enterobacteriaceae species. Organisms belonging to Enterobacteriaceae are gram negative,
oxidase negative, nitrate positive rods and are the most frequently isolated bacteria from clinical specimens. Hi25™ identification kit can be used for screening pathogenic organisms
from urine, enteric specimens and other relevant clinical samples. It can also be used for validating known laboratory strains. The complete list of organisms that can be identified with
this system is given in the identification index provided with the kit.

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.

Kit contents
1. Each kit contains sufficient material to perform 10 tests. 8. TDA reagent (R036) for Phenylalanine Deaminase test.
2. 10 kits of Part I. 9. Baritt reagent A (R029) for Voges-Proskauer's test.
3. 10 kits of Part II. 10. Baritt reagent B (R030) for Voges-Proskauer's test.
4. Oxidase reagent discs (DD018) 11. Methyl Red reagent (I007) for Methyl Red test
5. Technical product insert. 12. Kovac's reagent (R008) for Indole test
6. Result Interpretation Chart and Result Entry Datasheet. 13. Sulphanilic acid (R015)
7. Identification Index. 14. N,N-Dimethyl-1-Napthylamine Reagent (R009).

Instructions for use


1. 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/ M1274) or a differential medium like MacConkey Agar (M082)..
Pick up a single isolated colony and inoculate in 5 ml Brain Heart Infusion Broth and incubate at 35-37°C for 4-6 hours until the inoculum turbidity is ³ 0.1OD 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 0.1OD 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 0.1OD 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.
Note Erroneous false negative results may be obtained if the inoculum turbidity is less than 0.1 OD.
Results are more prominent if an enriched culture is used instead of suspension.

2. Inoculation of the kit


Open the kit aseptically. Peel off the sealing foil. Inoculate each well with 50 µl of the above inoculum by surface inoculation method.
Alternatively, the kit can also be inoculated by stabbing each individual well with a loopful of inoculum.
3. Incubation : Temperature of incubation: 35 ± 2°C. Duration of incubation: 18 - 24 hours.

Interpretation of results :
Interpret results as per the standards given in the identification index. Addition of reagents wherever required should be done at the end of incubation period that is after
18 - 24 hours.
Part I : 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.
Voges Proskauer's Test : Well No. 9 Add 2-3 drops of Baritt reagent A (R029) and 1 drop of Baritt reagent B(R030).
Pinkish red colour development within 5-10 minutes indicates a positive test.
No change in colour or a slight change in colour (due to reaction of Baritt reagent A with Baritt reagent B) denotes a negative reaction.
Methyl Red Test : Well No. 10 Add 1-2 drops of Methyl Red reagent (I007).
Reagent remains red in colour if the test is positive.
Reagent decolourises and becomes yellow if the test is negative.
Indole Test : Well No. 11 Add 1-2 drops of Kovac's reagent (R008).
Development of pinkish red colour within 10 seconds indicates positive reaction.
Reagent remains pale coloured if the test is negative.

Please refer disclaimer Overleaf.


HiMedia Laboratories Technical Data

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 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

Note : Based on % strains showing reactions following symbols have been assigned from laboratory results and standard references.
+ = Positive (more than 90%) = Negative (more than 90%) V = 11-89% positive.

Please refer disclaimer Overleaf.


HiMedia Laboratories Technical Data

KB003 : Hi25TM Enterobacteriaceae Identification Kit


Identification Index of various Enterobacteriaceae species

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

Note : Based on % strains showing reactions following symbols have been assigned from laboratory results and standard references.
+ = Positive (more than 90%) = Negative (more than 90%) V = 11-89% positive.

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

7 H2S production — Detects H2S 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 Detects acetoin production Colourless / Light Pinkish red Colourless/
Proskauer's and 1-2 drops of Baritt reagent B Yellow slight copper

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

11 Indole 1-2 drops of Kovac's red 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

Important points to be taken into consideration while interpreting the result :

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 for 48 hours.
Orange colour after 48 hours of incubation should be interpreted as a negative reaction.
3. In case of Lysine and Ornithine decarboxylation, incubation up to 48 hours may be required.
4. At times organisms give conflicting result because of mutation or the media used for isolation, cultivation and maintenance.
5. The identification index has been compiled from standard references and results of tests carried out in the laboratory.

Precautions :
Clinical samples and microbial cultures should be considered potentially pathogenic and handled accordingly.
Aseptic conditions should be maintained during inoculation and handling of the kits.
Reagents should not come in contact with skin, eyes or clothing.

Disposal of used material :

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.

Storage & Shelf-life


Store at 2-8°C. Shelf-life is 12 months.

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 diagnostic or therapeutic use but for
laboratory, 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. A-516,Swastik Disha Business Park,Via Vadhani Ind. Est., LBS Marg, Mumbai-400086, India. Customer care No.: 022-6147
1919 Email: techhelp@himedialabs.com

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