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Microbact Biochemical Identification Kits

Sector: Industrial

STAPHYLOCOCCAL 12S IDENTIFICATION SYSTEM

CODE: MB1561

Introduction
Staphylococci remain an important component of the human commensal flora, with coagulase-negative staphylococci representing the major proportion 1. Despite their frequency as contaminants, coagulase-negative staphylococci have become important nosocomial pathogens, in part because of the increased use of indwelling medical devices. Because of their increased importance it has now become imperative for microbiology laboratories to identify these coagulase-negative staphylococci to the species level 2.

Intended use
Microbact™ Staph 12S system is based upon conventional identification systems 3,10. Using a combination of sugar utilisation and colorimetric enzyme detection substrates, the 12S is able to identify 22 of the most important clinical species of staphylococci 2. This includes both coagulase-negative and coagulase-positive staphylococci.
The Microbact™ Staph 12S system is intended for the identification of Staphylococcus species only. Staphylococci are Gram-positive cocci (0.5 to 1.5 µm in diameter) they are non-motile, non-spore forming, catalase-positive facultative anaerobes.
Micrococcus and related genera can be eliminated by their inability to grow anaerobically and resistance to Furazolidone (100µg disc) 5.

Principle of the Test
The Microbact™ Staph 12S system is a standardised micro-substrate system designed to emulate conventional biochemical substrates used for the identification of Staphylococcus species. Each identification strip consists of 12 tests. Reactions that occur during the incubation period are demonstrated by an easily interpreted colour change. The sugar utilisation tests rely on a pH indicator colour change, whilst the enzyme detection substrates produce a coloured end product or react with an added indicator.
Organism identification is based on pH change and substrate utilisation as established in published references 3,4,6,7,8,9. Refer to the table of substrates/reactions for the specific reaction principles and colour changes.
Each species of Staphylococcus produces a different pattern of reactions. When the results of the reactions are entered into the Microbact™ software, using a numeric code, a probable identification of the Staphylococcus species in question is produced. Oxoid is continually extending and improving its independent database of reactions for use with the Microbact™ Staph 12S.

TABLE OF SUBSTRATES AND REACTIONS:

Well No.
Designation
Reaction principle
Reaction
Comments
Negative
Positive
1
Maltose
Utilisation of specific sugars
resulting in the production of
acidic end products
Red
Yellow
Phenol red indicator
changes colour when
appropriate sugar is
utilised producing acid
2
2 Mannitol
Red
Yellow
3
Mannose
Red
Yellow
4
Sucrose
Red
Yellow
5
Trehalose
Red
Yellow
6
N-acetyl glucosamine
Red
Yellow
7
Arginine utilisation
Detection of arginine
dihydrolase at 24 hours
Yellow/
Olive green
Green/blue
Arginine dihydrolase converts
arginine into ornithine, ammonia and carbon dioxide. The pH rise is indicated by bromothymol blue.
Green reactions occurring at
48 hours should be interpreted as negative.
8
Urease test
Urea hydrolysis
straw/yellow
pink/red
Urease splits urea into carbon dioxide and ammonia. The pH rise is indicated by phenol red.
9
Beta-glucosidase
Hydrolysis of p-nitrophenyl- ß-D-glucopyranoside by action of ß-glucosidase
Pale Yellow/
Colourless
Bright
Yellow
Hydrolysis of colourless p-nitrophenyl- ß-d-glucopyranoside releases yellow p-nitrophenol
10
Alkaline phosphatase
Hydrolysis of p-nitrophenyl-phosphate by action of alkaline phosphatase
Pale Yellow/
Colourless
Bright
Yellow
Hydrolysis of colourless p-nitrophenyl-phosphate releases yellow p-nitrophenol
11
Beta-glucuronidase
Hydrolysis of p-nitrophenyl-ß-D-glucuronide by action of ß-glucuronidase
Pale Yellow/
Colourless
Bright
Yellow
Hydrolysis of colourless p-nitrophenyl- ß-d-glucuronide releases yellow p-nitrophenol
12
Beta-galactosidase

Hydrolysis of ß-naphthyl-ß-d-galactopyranoside by action of ß-Glucosidase
Pale Yellow/
Colourless
Plum purple

Hydrolysis of colourless ß-naphthyl- ß-d-galactopyranoside releases ß-naphthol which is detected by Fast blue BB salt which turns purple

THE FOLLOWING SPECIES CAN BE IDENTIFIED USING THE MICROBACT™ STAPH 12S SYSTEM.

S. aureus subsp. aureus S. saprophyticus
S. epidermidis S. cohnii subsp. cohnii
S. capitis subsp. capitis S. cohnii subsp. urealyticum
S. capitis subsp. ureolyticus S. xylosis
S. caprae S. simulans
S. warneri S. carnosus
S. haemolyticus S. intermedius
S. hominisS. hyicus
S. lugdunensis S. chromogenes
S. schleiferi S. sciuri
S. auricularis S. lentus

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 12S strips in a CO2 incubator as substrates and/or enzyme reactions could be adversely affected, giving false reactions.

Storage Instructions
Store at 2–8°C. The test strips are stable until the expiry date specified if kept unopened in the foil envelopes at this temperature.

Kit Presentation
Each kit contains the following:
20 Microbact™ Staph 12S test strips consisting of 12 wells each containing a different dehydrated substrate as described in the table of substrates.
21 x 3 mL vials of Microbact™ Staph 12S suspending media consisting of buffering agents and peptones for preparing the inoculum.
1 Holding Tray.
Technical Product Insert.
Organism ID Report Form including Colour Interpretation Chart.
Report Forms.
Materials not provided
The following material may be required but are not provided:
MicrobactTM Staph Fast Blue Reagent 2 x 10mL (code MB1588)
Microbact™ Computer Aided Identification Package (code MB1244)
Sterile Mineral Oil (code MB1093)
Inoculating Loops
Sterile Pipettes
Staphylococcal latex (codes DR0850, DR0850)/Coagulase reagent
DNase plates and 1N HCL
Incubator (35 +/ 2°C)

Proceedure
Isolation
The basic procedure for culture and isolation of bacteria from either Staphylococcus spp. or staphylococci clinical specimens has been well documented. When isolating Staphylococcus, every specimen should be plated onto blood agar and other media appropriate for the specimen.
On blood agar, abundant growth of Staphylococcus spp. or staphylococci occurs within 18-24 hours. Since most species cannot be distinguished from one another during this time period, colonies should be picked at this time only for preliminary identification testing. Colonies should be allowed to grow for an additional 2-3 days before the primary isolation plate is confirmed for species composition.
Failure to hold the plates for this length of time can lead to mixed cultures being used in the identification process, producing false results. Specimens from heavily contaminated sources should also be streaked onto media selective for Staphylococcus spp. or staphylococci, such as Mannitol-Salt Agar or Columbia Colistin-Nalidixic Acid Agar. These media inhibit the growth of Gram-negative organisms but allow Staphylococcus spp., staphylococci and certain other Gram-positive cocci to grow.
Additional Tests
Before using the Microbact™ Staph 12S system some additional testing will need to be performed. These results will be recorded on the report form and entered into the computer software to aid in identification.

Test results required:
1. Coagulase/Staphylococcal Latex results
There are many systems available for detecting coagulase, but the slide agglutination test using human or rabbit plasma is one of the most common. Staphylococcal latex is another fast and convenient way of detecting coagulase. Oxoid Staphytect Plus is an accurate and highly specific test which can be used in conjunction with the 12S system for efficient Staphylococcal identification.
2. DNase Detection
DNase detection is an easy and useful test that can help greatly in the identification of various Staphylococcus species.
Staphylococcal species other than Staphylococcus aureus that can produce DNase include:Staphylococcus caprae, Staphylococcus hyicus, Staphylococcus chromogens, Staphylococcus intermedius.
3. Pigment
Pigment production can be a useful adjunct to the Staphylococcal identification process. Positive pigment is defined as the visual detection of carotenoid pigments (e.g., yellow, yellow–orange or orange) during colony development at normal incubation or room temperatures.
Pigmented colonies are often hard to visualise. To aid visualisation a white swab can be used to remove one or more colonies from the agar plate. The swab can then be checked for pigment colour, using the white background for contrast. Pigment production on blood containing media is often poor. Pigments may be enhanced by the addition of milk, fat, glycerol monoacetate or soap to media such as Tryptone Soya Agar (TSA).
Colony colour on this agar is more easily visualised than on blood containing agar.
Staphylococcus species that can produce pigment include: Staphylococcus aureus, Staphylococcus chromogens, Staphylococcus scuiri, Staphylococcus lugdunensis.

Identification
On non-selective agars, isolated colonies of Staphylococcus are 1-3 mm in diameter after 24 hours and 3-8 mm in diameter by 3 days of incubation in air at 35 ± 2°C. Some species such as Staphylococcus auricularis can require 24–36 hours of incubation before detectable colony development.
The Microbact™ Staph 12S system is to be used for the identification of Staphylococcus spp. only. Before testing, isolates should be checked to ensure that they belong to the Staphylococcus genus.
Staphylococci are Gram-positive cocci (0.5 to 1.5 µm in diameter) that occur singly and in pairs, tetrads, short chains (three or four cells) and irregular grape-like structures. They are non-motile, non-spore forming, catalase- positive facultative anaerobes.
Micrococcus and related genera can be eliminated by their inability to grow anaerobically and resistance to Furazolidone (100µg disc) 5.

Preparation of inoculum
Pick 2-5 isolated colonies (depending on colony size) from an 18–24 hour pure culture and emulsify in 3 ml of staphylococcal suspending medium. Mix thoroughly to prepare a homogeneous suspension.
Inoculation
1. Remove a test strip from its foil pouch and place in the holding tray. Label appropriately.
2. Remove the lid from the test strip.
3. Using a sterile Pasteur pipette add 4 drops (100µl) of the bacterial suspension to each well.
4. Overlay well no.7, arginine (indicated by a black circle on the test strip) with 2 drops of Mineral Oil (code MB1093). Replace the lid.
5. Place 1 drop of the inoculum onto an appropriate non-selective medium (e.g. TSA or Columbia Agar) for a purity check and incubate at 35 ± 2°C for 24 hours. If growth on the plate indicates that the suspension was not pure, then the test must be considered invalid and repeated.

Incubation
Incubate the inoculated test strips at 35 ± 2°C aerobically for 24 hours. If the arginine result cannot be interpreted confidently after 24 hours the strips can be replaced in the incubator and reread after further incubation.

Reading the test strip
1. If necessary, reconstitute the Fast Blue reagent (code MB1588) by adding the entire contents of the diluent bottle to the reagent bottle. Mix thoroughly and write the date of reconstitution on the bottle. Once reconstituted Fast Blue reagent has a shelf life of 8 weeks.
2. Remove test strip from the incubator.
3. Remove the lid.
4. Add one drop of Fast Blue reagent to well no. 12, indicated by a green circle on the test strip (Beta-galactosidase). A colour change within 5-10 seconds to plum purple will occur if the test is positive.
5. Record all test results onto the Microbact™ Organism ID Report Forms provided in the kit. Use the colour chart on the inside flap of the pad to aid in the interpretation of the colour changes in each well of the 12S strip. Page 2 of this booklet contains a table of substrates/reactions that can be used as a guide for interpreting reactions. Positive reactions are indicated by a + and negative reactions by a -.

Interpretation
Once each reaction is recorded on the report form, convert each block of three reactions into a numeric value. If the reaction in the well is considered positive the numeric value below the result (reaction index number) will be included. Add the three numbers together to obtain each digit of the Microbact™ code which is either compared to the profile register or entered into the computer package.

Example:

 
Result
Reaction Index
Sum of Positive
reactions
Maltose
+
4
4
Mannitol
-
2
Mannose
-
1
Sucrose
+
4
4
Trehalose
-
2
N-Acetyl glucosamine
-
1
Arginine
+
4
6
Urease
+
2
Beta-Glucosidase
-
1
Alkaline Phosphatase
+
4
4
Beta-Glucuronidase
-
2
Beta-Galactosidase
-
1
Latex/Coagulase
-
4
0
Dnase
-
2
Pigment
-
?

Microbact™ code = 44640

Results of the test reactions, via the five digit code number, are entered into the Microbact™ Computer Aided Identification Software producing a probable species ID of the Staphylococcus in question. Results can also be entered individually into the software if preferred. Please consult the Microbact™ software help for full details.
The software also allows Individual reactions to be changed once entered. In cases where reaction results are hard to interpret or not known a question mark (?) can be used. Of the 12 reactions a limit of 2 can be entered as question marks. For full details please consult the Microbact™ software help.
The Microbact™ Computer Aided Identification Software 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 part of the total probabilities for all choices.

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

Staphylococcus aureus ATCC®25923

Oxoid Cultiloops® C7010L

Staphylococcus epidermidis ATCC®12228
Oxoid Cultiloops® C6500L
Staphylococcus saprophyticus ATCC®15305

Oxoid Cultiloops® C7014L

The following chart gives the expected results on the Microbact™ 12S system after 18 – 24 hours of incubation.

 
S. aureus
ATCC ®25923
S. epidermidis
ATCC®12228
S. saprophyticus
ATCC®15305
Maltose
+
+
+
Mannitol
+
-
+
Mannose
+
-
-
Sucrose
+
+
+
Trehalose
+
-
+
N-Acetyl glucosamine
+
-
-
Arginine
+
+
-
Urease
+
+
+
Beta-Glucosidase
+
-
-
Alkaline Phosphatase
+
+
-
Beta-Glucuronidase
-
-
-
Beta-Galactosidase
-
-
+

Limitations
1. Some Staphylococcal strains may have atypical biochemical reactions due to unusual nutritional requirements and may be difficult to identify.
2. Prolonged incubation, insufficient incubation, improper filling of wells or inadequate inoculum may lead to false results.
3. Reactions obtained using the Microbact™ Staph 12S System may differ from published results using other substrate formulations.
4. Species with a low frequency of occurrence may require additional testing.
5. The interpretation of mathematically calculated identification results requires trained personnel who should use judgement and knowledge in conjunction with the following information before accepting the identification of an organism: Gram-stain, colonial morphology, source of isolate, percentage probability, tests against, additional tests, frequency of ID choice and the antibiogram.

References
1. Rhoden, DL, Miller, JM Four-year prospective study of STAPH-IDENT system and conventional method for reference identification of Staphylococcus, Stomatococcus, and Micrococcus spp. J. Clin. Microbiol. 1995 33: 96-98
2. Kloos, WE, Bannerman, TL Update on clinical significance of coagulase-negative staphylococci Clin. Microbiol. Rev. 1994 7: 117-140
3. Kloos, WE, Bannerman, TL 1999. Staphylococcus and Micrococcus, p. 264 – 282. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, R.H. Yolken (ed) Manual of Clinical Microbiology, 7th ed. American Society of Microbiology, Washington D.C.
4. Geary, C, Stevens, M, Sneath, PHA, Mitchell, CJ Construction of a database to identify Staphylococcus J. Clin. Pathol. 1989;42:289 - 294
5. Hebert, GA, Crowder, GC, Hancock, GA, Jarvis, WR, Thornsberry, C Characteristics of Coagulase-Negative Staphylococci That Help Differentiate These Species and Other Members of the Family Micrococcaceae J. Clin. Microbiol. 1988 26:1939-1949
6. Ieven, M, Verhoeven, J, Pattyn, SR, Goossens, H Rapid and Economical Method for Species Identification of Clinically Significant Coagulase-Negative Staphylococci J. Clin. Microbiol. 1995 33:1060-1063
7. Bascomb, S Enzyme Tests in Bacterial Identification Methods in Microbiology 1987 Volume 19:Chapter 3, 105-160
8. Bascomb, S, Manafi, M Use of Enzyme Tests in Characterisation and Identification of Aerobic and Facultatively Anaerobic Gram-positive Cocci
Clin. Microbiol. Rev. 1998 11: 318-340
9. McTaggart, L, Elliot, TSJ Is resistance to novobiocin a reliable test for confirmation of the identification of Staphylococcus saprophyticus ?
J. Med. Microbiol. 1989 30: 253-266
10. McFaddin, JF Biochemical Tests for Identification of Medical Bacteria Lippincott Williams & Wilkins 3rd Edition 2000

 
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