Oxoid Presentation Print Version 2009 Final

Harsi D. Kusumaningrum, OXOID Seminar Jakarta 21th may 2009

MICROBIOLOGICAL ANALYSIS ON SUPPORTING QUALITY AND SAFETY

OF FOOD AND BEVERAGES

Harsi D. Kusumaningrum

Dept. Food Science & Technology, BogorAgricultural University

Southeast Asian Food and Agriculture Science & Technology Center, BogorAgricultural University

OXOID Seminar, Jakarta 21th May 2009


Brief history of Microbiology

McKane and Kandel, 1996

Harsi D. Kusumaningrum, OXOID Seminar Jakarta 21th may 2009McKane and Kandel, 1996


Detecting New/Emerging Foodborne Pathogens

< 1900 V. cholerae, T. spiralis, C. botulinum, Salmonella, Shigella1900-10 B. melitensis1910-20 S. aureus, foodborne polio1920-30

1950-60 L. monocytogenes, C. perfringens, V. parahaemolyticus, Anisakidae1960-70 B. cereus, V. parahaemolyticus, V. vulnificus, aflatoxin and other

mycotoxin

1980-90 L. monocytogenes, E.coli O157:H7, E. sakazakii1990-00 Cyclospora, Cryptosporodium, nvCJd

1970-80 C. jejuni, Y. enterocolitica, Norwalk virus, Giardia, vomitoxin

1930-40 S.aureus, hepatitis A1940-50 B. cereus, C. perfringens, V. parahaemolyticus

2000-10 Tomkin, 2007


WHY ?


BACTERIASalmonella spp.Clostridium botulinumStaphylococcus aureusCampylobacter jejuniYersinia enterocoliticaListeria monocytogenesVibrio cholerae O1Vibrio cholerae non-O1 Vibrio parahaemolyticusVibrio vulnificusClostridium perfringensBacillus cereus Aeromonas hydrophilaShigella spp.Streptococcus

ENTEROVIRULENT Escherichia coli GROUP (EEC Group)Escherichia coli - enterotoxigenic (ETEC) Escherichia coli - enteropathogenic (EPEC) Escherichia coli O157:H7 enterohemorrhagic (EHEC) Escherichia coli - enteroinvasive (EIEC)

PARASITIC PROTOZOAGiardia lambliaEntamoeba histolyticaCryptosporidium parvumCyclospora cayetanensisDiphyllobothrium spp. Nanophyetus spp.Eustrongylides sp.Acanthamoeba VIRUSES PRION

ALGA MOLD

FOODBORNE PATHOGEN


Microorganisms Hospital (%)

PUSKESMAS (%)

Public (%)

Rotavirus 40-45 23.9 16.2E. coli (enterotoxigenik) 4-14 6.8 12.2Salmonella spp. 3-20 0.8 4Vibrio cholerae 1.5-73 - 0.6Vibrio NAG 0.3-0.6 2.6 0.8V. Parahaemolyticus 1.7 - 2.1Campylobacter spp. 0.2-8.2 - 1.6Yersinia enterocolitica 0.2 5.1 1.3Cryptosporidium 1.3 0.8 -

Etiology of Diarrhea in Indonesia (1980-1990)

Source: Punjabi, 2004


Pathogen PercentagesVibrio cholerae O1 37.1%Shigella spp 27.3%Salmonella spp 17.7%V. parahaemolyticus 7.3%Salmonella typhi 3.9%Campylobacter jejuni 3.6%V. cholerae non-O1 2.4%Salmonella paratyphi A 0.7%

Source: Tjaniadi P, et al., 2003 (Am. J. Trop. Med. Hyg.),

Characteristic of 2,812 strains of pathogen isolated from stool samples of patients presenting with diarrhea from 1995 to 2001, Indonesia

Gastroenteritis in Indonesia


Old system. Salmonella bongori. . Salmonella choleraesuis. . Salmonella choleraesuis subsp. arizonae. . Salmonella choleraesuis subsp. choleraesuis. Salmonella choleraesuis subsp. diarizonae. . Salmonella choleraesuis subsp. houtenae. . Salmonella choleraesuis subsp. indica. . Salmonella choleraesuis subsp. salamae. . Salmonella enteritidis. . Salmonella paratyphi. - Salmonella typhi. Salmonella typhimurium

Salmonella Nomenclature

Continuous updating of nomenclature system


Species Subspecies Serovar Habitatenterica (I) 1.454 Hewan berdarah panassalamae (II) 489arizonae (IIIa) 94diarizonae (IIIb) 324houtenae (IV) 70indica (VI) 12

Salmonella bongori (V) 20Total 2.463

Hewan berdarah dingindan lingkungan

Salmonella enterica

Salmonella species and subspecies

Source: Brenner et al, 2000 Continuous updating of nomenclature system


Old System New System . Salmonella bongori. . Salmonella choleraesuis. . Salmonella choleraesuis subsp. arizonae. . Salmonella choleraesuis subsp. choleraesuis. Salmonella choleraesuis subsp. diarizonae. . Salmonella choleraesuis subsp. houtenae. . Salmonella choleraesuis subsp. indica. . Salmonella choleraesuis subsp. salamae. . Salmonella enteritidis.

. Salmonella paratyphi.

. Salmonella bongori.

. Salmonella enterica.

. Salmonella enterica subsp. arizonae.

. Salmonella enterica subsp. enterica.

. Salmonella enterica subsp. diarizonae

. Salmonella enterica subsp. houtenae.

. Salmonella enterica subsp. indica.

. Salmonella enterica subsp. salamae.

. Salmonella enterica subsp. entericaser. Enteritidis (Salmonella Enteritidis)

. Salmonella enterica subsp. entericaser. Paratyphi (Salmonella Paratyphi)

Salmonella nomenclature according CDC since 2000

Source: Brenner et al, 2000 Continuous updating of nomenclature system


Iversen, 2008


Iversen et al. IJSEM June 2008


Commission Regulation (EC) No. 2073/2005 of 15 November 2005 on microbiological criteria of foodstuffs

Sampling plan

Limits( log cfu/cm2; daily mean log)

n c m MAreobic colony counts 3.5 5.0 ISO 48332.1.1. Carcasses of

cattle, sheep, goats and horses

Enterobacteriaceae 1.5 2.5 ISO 21528-2

Areobic colony counts 4.0 5.0 ISO 48332.1.2. Carcasses of pigs 2.5

Analytical Reference Method

Enterobacteriaceae 3.0 ISO 21528-2

Food Category Microorganisms

Continuous updating of regulation


Continuous updating of regulation


Reason to detect microorganisms in food

Food Quality Shelf life Marketing strategy

Food Safety HACCP Consumer protection

Legal


Challenges in the Microbiological Analysis of Foods

1. Complex matrix2. Other microorganisms (background flora)3. Cell attachments4. Inhibitory effects of food5. Method limitation6. Physiological state of microorganisms7. Non-uniform distribution


Types of Microbiological Methods

Quantitative Qualitative Traditional Rapid1 Culture2 Microscopic3 Chemical4 Physical5 Biochemical6 Molecular7 Immunological8 Bioassay


Quantitative Analysis


U.S. Food & Drug Administration, Bacteriological Analytical Manual Online, January 2001

Short review

Aerobic Plate Count

Conventional Plate Count Method

Spiral Plate Method


Conventional Plate Count Method

Normal plates (25-250), select spreader-free plate(s).

Count all colony forming units (CFU), including those of pinpoint size, on selected plate(s).

Record dilution(s) used and total number of colonies counted.


Short review

Aerobic Plate Count


Plates with 25-250 CFU

Formula: N = C / [ (1 * n1) + (0.1 * n2) ] * (d)

where N = Number of colonies per ml or g of product C = Sum of all colonies on all plates counted n1 = Number of plates in first dilution counted n2 = Number of plates in second dilution counted d = Dilution from which the first counts were obtained



SNI 01-3751-2006Tepung terigu sebagai bahan makanan


Spiral Plater

Accelerated Bacterial Colony Enumeration

AOAC Official Methods of Analysissec. 977.27

The Spiral Plate Count (SPLC)

Official method for microorganisms in milk, foods, and cosmetics of - the Association of Official Analytical Chemists (AOAC)

- the American Public Health Association (APHA)


http://www.interscience.fr/html/content/sp_met_e.htm

Loading Syringe

Disposing Syringe

Spiral Spreading

Spiral Plate Method

A mechanical plater inoculates a rotating agar plate with liquid sample.

The sample volume dispensed decreases as the dispensing stylus moves from the center to the edge of the rotating plate.


Spiral Plate Method

One inoculation determines microbial densities between 500 and 500,000 microorganisms/ml.

Additional dilutions may be made for suspected high microbial concentrations.


Area Volume Sample (ml)3c 0.002563b 0.006443a 0.012084c 0.020364b 0.032324a 0.05

A mask/grid is supplied for use with 100 mm dishes; counting grid is divided into 8 equal wedges

Each wedge is divided by 4 arcs labeled l, 2, 3, and 4 from outside grid edge.

Other lines within these arcs are added for ease of counting.


ProtoCOL SR is a comprehensive and fully automatic colony counting and zone sizing system specifically designed for microbiology and virology applications.

This colony counter and zone sizer system is suitable for reading a wide range of types of microbiology and virology media.

The ProtoCOL SR colony counter


Colonies can be automatically counted on standard pour plates, surface inoculated plates, spiral plates, 3M Petrifilm , filters, culture flasks and numerous other media.

Zones can be automatically measured for potency testing, antibacterial susceptibility and virology applications

The ProtoCOL SR colony counter


Qualitative Analysis


Microbiological Analysis Steps

1. Sampling2. Amplification

Enrichment cultures, PCR, etc3. Detection

Selective medium, electrophoresis, Elisa, etc4. Confirmation

Serotyping, API strips, DNA finger printing, etc


Continuous updating of microbiological analysis


Introduction

Several changes are being introduced in this edition of BAM (8th Edition). The first change involves the expanded use of Rappaport-Vassiliadis (RV) medium for foods with both high and low levels of competitive microflora. In the previous edition, RV medium was recommended only for the analysis of shrimp. Based on the completion of AOAC precollaborative (5, 6) and collaborative (7, 8) studies, RV medium is now being recommended for the analysis of high microbial and low microbial load foods. RV medium replaces selenite cystine (SC) broth for the analysis of all foods, except guar gum. In addition, RV medium replaces lauryl tryptose broth for use with dry active yeast. Tetrathionate (TT) broth continues to be used as the second selective enrichment broth. However, TT broth is to be incubated at 43C for the analysis of high microbial load foods and at 35C for the analysis of low microbial load foods, including guar gum.


The recent revision of ISO 6579 for Salmonella testing is a result of the growing incidence of Lactose positveSalmonella spp. isolated from cases of food poisoning.

Traditionally Salmonella are considered to be non-lactose fermenting organisms - however a small but important number of this highly diverse group are capable of lactose fermentation and may be incorrectly identified or missed altogether by conventional Salmonella selective media.

Note:


Innovative Microbiological Testing with Chromogenic Media

Harsi D. Kusumaningrum, OXOID Seminar Jakarta 21th may 2009Restaino, 2005


Taiwin, 2005


The conventional media for the detection of Salmonellaby H2S character has a very poor specificity creating an abondance of false positives (Citrobacter, Proteus, etc. as suspect colonies) among the rare real positive Salmonella.

In order to distinguish the real positive, the conventional method requires the tedious examination of 5 colonies per suspected sample.

The use of chromogenic media can eliminate most false positives and allow the technicians to focus all attention on rare suspected samples

Salmonella detection


OSCM II (Oxoid)

The Oxoid Salmonella Rapid Culture Method combines the features of two Oxoid products - ONE Broth-Salmonella and Oxoid Salmonella ChromogenicMedium Mark II (OSCM II). Chromogens within the medium enable differentiation of Salmonella colonies (bright purple) from any remaining organisms that are able to grow, such as Klebsiella and Enterobacter, thus reducing the number of false-positives requiring confirmation


Rambach agar

RAMBACH Agar enables species of Salmonella to be differentiated unambiguously from other bacteria by adding propylene glycol to the culture medium. Salmonellae form acid with propylene glycol, so that, in combination with a pH indicator, the colonies have a characteristic red color. In order to differentiate coliformsfrom Salmonellae, the medium contains a chromogeneindicating the presence of -galactosidase splitting, a characteristic for coliforms. Coliform microorganisms grow as blue-green or blue-violet colonies. Other Enterobacteriaceae and Gram-negative bacteria, such as Proteus, Pseudomonas, Shigella, S. typhi and S. parathyphi A grow as colorless-yellow colonies.


Source: Oxoid


Adavantages of rapid testing methods

Same (or greater sensitivity, accuracy and specificity as conventional methods

Saving of space and materials Reduction in human errors and labor cost

(when automated methods are used)

The microbiologist can judge data, organize activities and repeats tests if necessary


Thank you

Thank you

Thank you

Thank you

MICROBIOLOGICAL ANALYSIS ON SUPPORTING QUALITY AND SAFETY OF FOOD AND BEVERAGES Reason to detect microorganisms in foodChallenges in the Microbiological Analysis of Foods Types of Microbiological MethodsQuantitative AnalysisQualitative AnalysisMicrobiological Analysis StepsInnovative Microbiological Testing with Chromogenic Media Adavantages of rapid testing methods

Documents

Oxoid Presentation Print Version 2009 Final