13.Analyses Microbiologiques
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Chapitre III
Diagnostic et évaluation
4. établissement des procédures de vérification : La vérification de la méthode HACCP est une activité mise en œuvre pour s’assurer que ce système fonctionne efficacement. 4.1.
Les analyses microbiologiques :
Les produits alimentaires peuvent contenir une flore microbienne plus au moins abondante qui peut être nuisible pour leur qualité, et pour la santé du consommateur. Des micro-organismes sont utilisés comme des agents technologiques et leur présence est parfois indispensable pour certaines industries alimentaires. L’analyse bactériologique des produits alimentaires est indispensable pour : pour :
Assurer au produit une bonne qualité et une longue conservation.
Garantir la qualité hygiénique du produit. 4.1.1. La matière première :
a) L’eau de reconstitution : 1) Technique de prélèvement et d’échantillonnage : Le prélèvement s’effectue directement par le robinet de tank de lancement, ceci après avoir flambé l’orifice du robinet probablement désinfecté, on laisse couler un moment et à l’aide d’un flacon stérile, on prélève 250 ml. 2) Dilution : On travail avec la solution mère qui représente le prélèvement de 250 ml de l’eau de reconstitution. 3) Recherche microbiologique : L’eau constitue un élément essentiel dans l’industrie laitière les micro-organismes micro-organismes rencontré dans l’eau sont très variés, leur nature dépend de celle de l’eau à anal ysée. Les germes recherchés dans l’eau de reconstitution sont :
Recherche et dénombrement des Germes Totaux.
Recherche et dénombrement des germes de contamination fécale (Coliforme Totaux et Fécaux)
30
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Chapitre III
Diagnostic et évaluation
Recherche et dénombrement des Entérocoques (Streptocoques Fécaux).
Recherche et dénombrement des Clostridies Sulfito-Reducteurs.
3.1) Recherche et dénombrement des Germes Totaux : schéma n° 01 Leur dénombrement nous renseigne sur la charge microbienne du produit, et i l chiffre le degré de contamination. La technique utilisée utilisée est celle de numération en milieu solide (P.C.A ou T.G.E.A ) en boites de petries avec ensemencement dans la masse. Méthodologie :
La technique consiste à introduire aseptiquement 1 ml de la solution mère dans chaque boites de petries (02 boites), couler par la l a suite environ 20 ml de la l a gélose P.C.A. Faire des mouvements circulaires pour bien mélanger et laisser les boites solidifier sur la paillasse. Incubation :
-
La 1ère boite sera incubée à 37°C pendant 24 heures. heures.
-
La 2eme boite sera incubée à 22°C pendant 72 heures.
Lecture :
Les colonies de germes totaux se présentent sous forme lenticulaire en masse. Dénombrement :
Il s’agit de compter toutes les colonies ayant poussé sur les boites en tenant compte du facteur suivant : -
Ne dénombrer que les boites contenant entre 30 et 300 colonies.
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Chapitre III
Diagnostic et évaluation
Eau à analysée
1 ml
1 ml
- Ajouter 20 ml de gélose PCA ou TGEA - Laisser solidifier sur paillasse - Incuber pendant :
22°C (72 heures)
37°C (24 heures) - Dénombrer les colonies lenticulaires en masse.
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Chapitre III
Diagnostic et évaluation
c ontaminations fécales (Coliformes 3.2) Recherche et dénombrement des contaminations Totaux et Fécaux) : schéma n° 02 La méthode adapter pour cette analyse est la technique du dénombrement en tube multiple (NPP). (Annexe Le milieu utilise est le BCPL (Bouillon Lactose au Pourpre Bromocréol). La technique en milieu liquide fait appel à deux tests consécutifs à savoir :
Le test de présomption : réservé à la recherche des Coliformes.
Le test de confirmation : (test Mac Kenzie) réservé à la recherche des Coliformes Fécaux (à partir des tubes positifs du test de présomption).
Test de présomption :
Porter aseptiquement de l’échantillon d’eau à analysée : -
50 ml dans un flacon contenant 50 ml du milieu BCPL (D/C) muni d’une cloche de durham.
-
5 fois 10 ml dans 05 tubes contenant 10 ml de milieu BCPL (D/C) muni d’une cloche de durham.
-
5 fois 1 ml dans 05 tubes contenant 10 ml de milieu BCPL (S/C) muni d’une cloche de durham.
Incubation :
L’incubation se fait à 37°C pendant pendant 24 heures. Lecture :
Les tubes et flacon positifs présentent à la fois :
Un dégagement gazeux (supérieur au 1/10 de la hauteur de la cloche.
Un trouble microbien accompagné d’un virage de couleur du milieu au jaune (ce qui constitue le témoin de la fermentation du lactose présent dans le milieu).
Les résultats sont négatifs si les tubes et flacon ne présentent aucun trouble et aucun dégagement gazeux.
La lecture se fait selon la prescription de la table de Mac Grady (Annexe
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Chapitre III
Diagnostic et évaluation
Test de confirmation au test de Mac Kenzie :
Les tubes de BCPL trouvés positifs lors du dénombrement des Coliformes Totaux, ferrant l’objet d’un repiquage dans le milieu de BCPL muni d’une cloche de durham et EPEI (Eau Peptone exemple d’Indol). Incubation :
L’incubation se fait à 44°C pendant 24 2 4 heures. Lecture :
Les tubes et flacon positifs présentent à la fois :
Un dégagement gazeux (supérieur au 1/10 de la hauteur de la cloche).
Un anneau rouge en surface témoin de la production d’indol par l’Echérichiacoli après adjonction de 02 à 03 gouttes de réactif de Kowacs.
La lecture finale se fait selon la prescription de la table de (NPP) en tenant compte du fait qu’Echérichiacoli est à la fois producteur de gaz et d’indol à 44°C.
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Chapitre III
Diagnostic et évaluation
250ml
Eau a analysée
Test de présomption 10 ml
50 ml
1 ml
10
10
10
10
10
10
10
10
10
10
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
BCPL (D/C) + Cloche durham
BCPL (D/C)
BCPL (S/C)
Incubation à 37°C pendant 24 H
1O
Trouble microbien +
ml
Gaz dans la cloche Réaction positive
Réaction négative Test de confirmation
BCPL
+2 gouttes du kowacs
EPEI Incubation à 44°C pendant 24 H
Anneau rouge
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Chapitre III
Diagnostic et évaluation
3.3) Recherche et dénombrement des Clostridium Sulfito-Réducteurs : schéma n°03 Les Clostridium sulfito-Réducteurs sont des germes anaérobies qui ont le pouvoir de réduire le sulfite en sulfure, et capables de former des spores dans les conditions défavorables. Méthodologie :
Prendre 05 tubes vides et stériles, mettre 5ml d’eau à analysée dans chacun des 04 tubes et 1ml d’eau à analysée dans le 5 ème tube. Puis, porter les tubes au bain marie à 80°C pendant 10mn, et après refroidir immédiatement ces tubes sous jet d’eau froide. Ensuite, ajouter environ 20ml de gélose VF (viande foie) (après avoir ajouté à cette dernière une ampoule de 5ml de sulfite de sodium et une ampoule de 1ml de l’al un de fer) dans chaque tubes. Incubation :
Incuber les 05 tubes à 46°C pendant 48 heures. Lecture :
La 1ere lecture se fait impérativement après 16 heures d’incubation. Dans le cas où il n’y pas de colonies noires, réincuber les tubes et effectuer une 2ème lecture après 24 heures voir 48 heures. Dénombrement :
On compte les colonies noires directement dans les tubes positifs.
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Chapitre III
Diagnostic et évaluation
Eau à analysée
Chauffage à 80°C, 10 minutes Refroidissement brutal sous l’eau de robinet 5 ml
5 ml
5 ml
5ml
Ajouter environ 20 ml de gélose VF (+02 ampoules contenant : 5 ml de sulfite de sodium, 1 ml de l’alun de fer) Puis incuber à 46°C pendant 16-24 ou au plus tard 48heures.
1ml
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Chapitre III
Diagnostic et évaluation
b) La poudre de lait écrémé et entier : 1) Technique de prélèvement et d’échantillonnage : Le lieu de prélèvement est le laboratoire des analyses microbiologiques, 03 échantillons sont prélevés à partir de 03 sacs de 25kg de poudre de lait à 0%, et à 26% de matière grasse, cette poudre est conditionnée dans des sacs en polyéthylènes et doublés en papier hermétiquement fermé. La surface des sacs a été nettoyée et désinfectée par l’alcool et la couche supérieure a été écartée écartée à l’aide d’une spatule stérile. On procède à un échantillonnage à l’aide d’une sonde métallique stérile à proximité de la flamme. 2) Dilutions : Introduire aseptiquement 25 g de poudre dans un flacon stérile contenant 225ml de TSE puis homogénéisé l’ensemble a fin d’obtenir la solution mère. A partir de cette dernière, on prélève 1ml à l’aide d’une pipette stérile et on le porte dans un tube contenant 9ml d’eau physiologique, c’est la dilution 10 -1. On prend 1ml de la dilution 10 -1 à l’aide d’une autre pipette et on le porte dans un autre tube contenant 9 ml d’eau physiologique ce qui nous donne la dilution 10 -2. La même technique est répète pour obtenir la dilution 10 -3. 3) Recherche microbiologique : Les germes recherchés dans la poudre de lait sont : -
La Flore Mésophile Aérobie Total (F.M.A.T)
-
Les Coliformes Totaux.
-
Les Clostridiums Sulfito-Réducteurs.
-
Les levures et moisissures.
-
Les Salmonella.
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Chapitre III
Diagnostic et évaluation
3.1) Recherche et dénombrement de la Flore Aérobie Mésophile Total (F.M.A.T) : (schéma n°04) Méthodologie :
A partir des dilutions décimales allant de 10 -1, 10-2, 10-3, porter aseptiquement 1ml de l’échantillon dans 03 boites de petries vides et stériles préparées à cet usage. Compléter ensuite avec environ 20 ml de gélose P.C.A fondue, puis faire des mouvements circulaires pour permettre à l’inoculum de ce mélangé à la gélose utilisée. Laisser les boites solidifier sur paillasse quelques minutes. Incubation :
Les boites seront incubées couvercles en bas à 30°C pendant 72 heures. Lecture :
Les colonies de F.M.A.T se présentent sous forme de lentic ulaire en masse. Dénombrement :
Il s’agit de compter toutes les colonies ayant poussé ayant poussé sur les boites en tenant compte des facteurs suivants :
Ne dénombrer que les boites contenant entre 30 et 300 colonies.
Multiplier le nombre trouvé par l’inverse de sa dilution.
Faire ensuite la moyenne arithmétique des colonies entre les différentes dilutions.
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Chapitre III
Diagnostic et évaluation
A partir des dilutions décimales
10-1
10
1 ml
-2
10
1 ml
Ajouter 20ml de gélose PCA
-3
1 ml
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Chapitre III
Diagnostic et évaluation
3.2) Recherche et dénombrement des coliformes totaux : schéma n°05 Méthodologie :
Préparer 03 dilutions 10 -1, 10 -2, 10 -3, prendre 03 boites de petries vides et stériles et mettre dans chacune 1 ml de l’échantillon. Couler ensuite, environ 20 ml de la gélose Désoxycholate fondue dans un chaque boites puis faire des mouvements circulaires pour obtenir un mélange homogène. Laisser les boites solidifier sur paillasse quelques minutes. Incubation :
Les boites seront incubées couvercles en bas à 37°C pendant 24 heures. Lecture :
Les Coliformes Totaux se présentent sous forme de colonies rouge foncées). - Le dénombrement des boites se fait par le comptage des colonies (rouges foncés). Le nombre trouvé sera multiplié par l’inverse de la dilution de chaque chaque boite de petries, puis faire la moyenne arithmétique des 03 boites.
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Chapitre III
Diagnostic et évaluation
A partir des dilutions décimales
-1
10
-2
1 ml
C.Totaux
-3
1 ml
C.Totaux
- Ajouter 20 ml de la gélose Désoxycholate.
1 ml
C.Totaux
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Chapitre III
Diagnostic et évaluation
3.3) Recherche et dénombrement des Clostridiums Sulfito-Réducteurs : schéma n°06 Méthodologie :
Faire subir aux tubes des dilutions 10 -1, 10-2, un chauffage de 80°C pendant 1O mn et un refroidissement immédiat sous jet sous jet d’eau. Puis a partir de ces tubes portes aseptiquement 1 ml d’échantillon dans 02 tubes à essai contenant environ 20 ml du milieu VF (+ 1ampoule de l’alun l’ alun de fer et une ampoule de sulfite de sodium) et bien mélanger. Incubation :
Ces tubes seront ainsi incubés à 46°C pendant 16, 24, ou au plus tard 48 heures. Lecture :
La première lecture doit se faire impérativement à 16 heures d’incubation, car : car :
D’une part, les colonies de Clostridium sont envahissantes dans ce cas on ce trouverait en face d’un tube complètement noir rendant alors l’interprétation des résultats impossible.
D’autre part, il faut absolument repérer toute colonie noir ayant poussé en masse et d’un diamètre supérieur à 0,5 mm.
Dans les cas d’absence de colonies caractéristiques, ré incuber incuber les tubes une deuxième fois et
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Chapitre III
Diagnostic et évaluation
10-1
-
10-2
Chauffage à 80°C pendant 10mn. Refroidissement immédiat sous et d’eau
1 ml
1 ml
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Chapitre III
Diagnostic et évaluation
3.4) Recherche et dénombrement des Levures et Moisissures : schéma n°07 Les levures et moisissures sont des germes aérobies, leur dénombrement se fait sur milieu de Sabouraud. Méthodologie :
On porte aseptiquement 1 ml à partir des dilutions allant de 10 -1, 10-2, 10-3, dans 03 boites de petries vides et stériles. Verser ensuite, environ 20 ml de gélose de Sabouraud fondue, faire des mouvements circulaires afin d’homogénéiser le mélange, puis laisser les boites solidifier sur paillasse. Incubation :
Elle se fait à 20 – 20 – 25°C 25°C pendant 3 à 5 jours avec couvercles en haut. Lecture :
Après incubation, on effectue le comptage des Levures à part (grandes colonies rondes) et des Moisissures (des colonies ramifiées) d’autre part. Multiplier ensuite le nombre trouvé par l’inverse de sa dilution, puis faire la somme des 03 bo ites
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Chapitre III
Diagnostic et évaluation
10-1
10
1 ml
-2
10-3
1 ml
1 ml
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Chapitre III
Diagnostic et évaluation
3.5) Recherche et dénombrement des Salmonella : schéma n°08 La recherche du genre Salmonella nécessite une prise d’essai a part : Jour 1 : pré-enrichissement :
Prélever 25g de la poudre de lait et la porter dans un flacon de 225 ml de T.S.E, incuber à 37°C pendant 12 – 12 – 18 18 heures. Jour 2 : Enrichissement :
L’enrichissement doit s’effectuer sur un milieu sélectif : sélectif : S.F.B (bouillon au sélénite cystéine) (S/C) repartie à raison de 100ml par flacon auquel on ajoute 2 ml d’additif « sélénite acide de sodium ». Prendre 10 ml à partir du milieu de pré-enrichissement et les mettre dans le flacon de S.F.B puis
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Chapitre III
Diagnostic et évaluation
Pré-enrichissement
T.S.E
25 g du produit à analyser
225ml - Homogénéisation et incubation 37°C pendant 12 – 12 – 18 18 heures 10ml
Enrichissement
Additif : sélénite acide de sodium (2ml) SFB
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Chapitre III
Diagnostic et évaluation
4.1.2. Lait après pasteurisation 1) Technique de prélèvement et d’échantillonnage : La prise d’échantillon est réalisée au niveau de robinet du tank de repos. Après stérilisation du robinet par flambage, on laisse couler le lait quelques secondes (30 secondes) puis à l’aide d’un tube à essai, on prélève le liquide jusqu’à ju squ’à remplissage du tube. 2) Dilution : On transfère à l’aide d’une pipette pasteur stérile, 1 ml de la suspension mère dans un tube qui contient 9 ml d’eau physiologique et on agite bien, pour obtenir la dilution 10 -1. De ce dernier on va porter 1 ml par une autre pipette graduée dans un autre tube stérile contenant 9 ml d’eau physiologique ou on on obtient la dilution 10 -2. De la même façon, on obtient la dilution 10 -3. 3) Recherche microbiologique :
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Chapitre III -
Deuxième lecture à 48 heures.
-
Troisième lecture à 72 heures.
Diagnostic et évaluation
Dénombrement :
Il s’agit de compter toutes les colonies ayant poussé sur les boites en tenant des facteurs suivants :
Ne dénombrement que les boites contenant entre 30 et 300 300 colonies.
Multiplier le nombre trouvé par l’inverse de sa dilution.
Faire ensuite la moyenne arithmétique des colonies entre les différentes dilutions.
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Chapitre III
Diagnostic et évaluation
Schéma n°09 : Recherche des Germes Totaux dans le lait pasteurisé.
A partir des dilutions décimales
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Chapitre III
Diagnostic et évaluation
3.2) Recherche et dénombrement des germes de contamination fécal (Coliformes Totaux et Fécaux) : schéma n°10 Les Coliformes sont dénombrés sur le milieu solide Désoxycholate. Méthodologie :
Après la préparation des dilutions décimales 10 -1, 10-2, 10-3, on porte aseptiquement 1
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Chapitre III
Diagnostic et évaluation A partir des dilutions décimales
10
-1
10-2
-3
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Chapitre III
Diagnostic et évaluation
4.1.3. Le produit fini (lait UHT) : 1) Technique de prélèvement et d’échantillonnage : Le lait UHT est prélevé dés sa sortie de la conditionneuse chaque 02 heures, 1 fardeau contenant 4 briques est retiré :
1 brique analysée le jour même. 1 brique incubée à 55°C pendant 7 jours. 1 brique incubée à 30°C pendant 15 jours.
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Chapitre III
Diagnostic et évaluation
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Chapitre III 3.2) Le test d’alcool
Diagnostic et évaluation
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Chapitre III 4.2.
Les analyses physico-chimiques :
Diagnostic et évaluation
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Chapitre III a.2) Titre alcalimétrique complet (TAC) :
Diagnostic et évaluation
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Chapitre III Solution tampon pH = 10.
Diagnostic et évaluation
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Chapitre III Mode opératoire :
Diagnostic et évaluation
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Trusted by over 1 million members
Try Scribd FREE for 30 days to access over 125 million titles without ads or interruptions! Start Free Trial Cancel Anytime.
Trusted by over 1 million members
Try Scribd FREE for 30 days to access over 125 million titles without ads or interruptions! Start Free Trial Cancel Anytime.
Trusted by over 1 million members
Try Scribd FREE for 30 days to access over 125 million titles without ads or interruptions! Start Free Trial Cancel Anytime.
Trusted by over 1 million members
Try Scribd FREE for 30 days to access over 125 million titles without ads or interruptions! Start Free Trial Cancel Anytime.
Trusted by over 1 million members
Try Scribd FREE for 30 days to access over 125 million titles without ads or interruptions! Start Free Trial Cancel Anytime.
Trusted by over 1 million members
Try Scribd FREE for 30 days to access over 125 million titles without ads or interruptions! Start Free Trial Cancel Anytime.
Trusted by over 1 million members
Try Scribd FREE for 30 days to access over 125 million titles without ads or interruptions! Start Free Trial Cancel Anytime.
Trusted by over 1 million members
Try Scribd FREE for 30 days to access over 125 million titles without ads or interruptions! Start Free Trial Cancel Anytime.
Trusted by over 1 million members
Try Scribd FREE for 30 days to access over 125 million titles without ads or interruptions! Start Free Trial Cancel Anytime.
Trusted by over 1 million members
Try Scribd FREE for 30 days to access over 125 million titles without ads or interruptions! Start Free Trial Cancel Anytime.
Trusted by over 1 million members
Try Scribd FREE for 30 days to access over 125 million titles without ads or interruptions! Start Free Trial Cancel Anytime.
Trusted by over 1 million members
Try Scribd FREE for 30 days to access over 125 million titles without ads or interruptions! Start Free Trial Cancel Anytime.
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