Part of Thermo Fisher Scientific
STAPHLOCOCCAL 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. hominis | S. 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 | - |
? |
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