401606-Aloa Confirmation Eng

Biolife Italiana S.r.l. Technical Sheet N°401606.DOC B E-0 03/2010 Pagina 1 di 3

Biolife Italiana Viale Monza 272, 20128 Milano. Tel. n° 02-25209.1 Fax n° 02-2576428 E mail: [email protected]; Internet: www.biolifeitaliana.it

ALOA® CONFIRMATION BASE

ALOA® ENRICHMENT- SELECTIVE SUPPLEMENTS

ALOA® CONFIRMATION AGAR

Dehydrated medium base, enrichment and selective supplements, ready to use plates for the rapid confirmation of L.monocytogenes colonies isolated onto ALOA Agar plates.

TYPICAL FORMULAS ALOA® Confirmation Base (g/l) Peptones 15,00 Organic and inorganic salts 38,40 X-Glucoside 0,05 Rhamnose 5,50 Phenol red 0,25 Agar 15,00

ALOA® SELECTIVE SUPPLEMENT (vial contents) Nalidixic Acid 0,010 g Ceftazidime 0,010 g Cicloheximide 0,025 g Polymyxin B sulphate 38.350 IU

ALOA® ENRICHMENT SUPPLEMENT (vial contents L-�-fosphatidylinositol 1,0 g

ALOA® Confirmation Agar (piastre pronte (g/l) Peptones 15,00 Organic and inorganic salts 38,40 X-glucoside 0,05 Ramnose 5,50 Phenol red 0,25 Agar 15,00 Nalidixic Acid 0,020 Ceftazidime 0,020 Cicloheximide 0,050 Polymyxin B sulphate 76.700 IU L-�-fosphatidylinositol 2,0

DIRECTIONS FOR PREPARATION FROM DEHYDRATED MEDIUM Suspend 37.1 g in 500 ml of cold distilled water, heat to boiling with frequent agitation and sterilise by autoclaving at 121°C for 15 minutes. Cool to 48-50°C add the contents of one vial of ALOA Enrichment Supplement pre-warmed to 48-50°C, and the contents of one vial of ALOA Selective Supplement, reconstituted with 5 ml of ethanol/sterile distilled water (1:1). Mix well and distribute in sterile Petri dishes. Final pH 7.3 ± 0.2

INTENDED USE ALOA Confirmation Agar is a selective, chromogenic and differential medium to be used only for the confirmation test of suspected L.monocytogenes colonies cultivated onto Agar Listeria Ottaviani & Agosti (ALOA) during the procedures for detection or enumeration of L.monocytogenes in foods, animal feeling stuffs and environmental samples. PRINCIPLE OF THE METHOD ALOA Confirmationagar is based on 3 biochemical characteristics of Listeria monocytogenes: - Phospholipase activity. This appears as an opaque halo around Listeria monocytogenes colonies. - ß-glucosidase activity. The hydrolysis of the chromogenic substrate produces a blue-green coloration

of Listeria monocytogenes colonies. - Rhamnose fermentation, associated with a pH indicator, gives a yellow background to colonies. Phospholipase activity and ß-glucosidase activity are the same detected with ALOA plates.



1

2

3 5

4

6

Rhamnose fermentation allows to differentiate between L. monocytogenes and other bacteria that can grow with similar characteristics on ALOA, such as L. ivanovii and some Bacillus cereus. TECHNIQUE READING AND RESULTS INTERPRETATION Confirm the streaked colonies as follow:

PROFILES GROWTH COLOUR

OPAQUE HALO

COLOUR CHANGE OF INDICATOR

INTERPRETATION

1 Blue-green + + L.monocytogenes 2 Blue + - A 3 Blue or other

colours - +/- B

Legenda A : Discordant result B : Non typical colony, a second sampling of colony must be carried out. In the event of a discordant result (profiles 2 and 3: positive with ALOA Agar plates and not characteristic on ALOA® Confirmation Agar, the sufficient means have to be carried out by the Laboratory as to ensure the validity of the result.

LIMITATIONS AND PRECAUTIONS

• Confirmation test can be done on a single colony; however if ALOA plate shows confluent growths with no well defined colonies, a further subculture step will be required on another ALOA plate prior to streak the ALOA Confirmation Agar plate.

• For a good observation of the opaque halo surrounding the streak, it is better to examine the plate using a light source or compare it to an un-inoculated ALOA Confirmation agar plate.

• If more than one colony is to be confirmed on an ALOA Confirmation plate, enough space must be left between streaks to avoid crossed contamination or confluent growth between samples.

• ALOA Confirmation plates can be read up to 48 hours after incubation if kept refrigerated as the low temperature prevents any further development of the reactions. However, after this time, the plates must be discarded.

• ALOA Confirmation plates may contain a slight precipitate before inoculation that disappears during incubation. This is a characteristic of the agar and will not affect results.

USER QUALITY CONTROL

Pick a single typical colony (blue to blue green colony, round, regular, surrounded by an opaque halo) from an ALOA plate and streak it on a ALOA Confirmation Agar plate. 6 typical colonies can be streaked on a the same ALOA Confirmation Agar plate (see the scheme) Incubate the inoculated plates at 37°C +/- 1°C for 24h +/- 2h.

Streaks 1, 2, 4, 6: L.monocytogenes Streaks 3, 5:negative test



STRAIN INCUBATION EXPECTED RESULTS L.monocytogenes ATCC 13932 37°C - 24 h Blue-green growth surrounded by an opaque halo

with a colour change of the indicator from red to yellow

L.ivanovii ATCC 19119 37°C – 24h Blue-green growth surrounded by an opaque halo with any change of the colour indicator

E.faecalis ATCC 19433 37°C - 24 h Growth inhibited STORAGE AND PRECAUTIONS Dehydrated medium: store at a +2 +8°C°; keep the bottle tightly closed away from the bright light; do not ingest, do not inhale the product. Selective/Enrichment Supplement: store at a +2 +8°C°; consult the material Safety Data Sheet before the use Ready to use plates: store at a +2 +8°C° The products here described are for Laboratory use only. ALOATM is a trademark of Biolife Italiana Srl PACKAGING DEHYDRATED MEDIUM AND SUPPLEMENT 4016062 ALOA Confirmation Base 500 g (6.7 L) 423501 ALOA Enrichment Selective Supplements, 4+4 vials, each for 500 ml of medium,

READY TO USE PLATES 541606 ALOA CONFIRMATION AGAR 20 plates, Ø 90 mm

401606-Aloa Confirmation Eng

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