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, Bogor
Agricultural University
Southeast Asian Food and
Agriculture Science &
Technology Center, Bogor
Agricultural University
OXOID Seminar, J akarta 21
th
May 2009
Harsi D. Kusumaningrum, OXOID Seminar Jakarta 21th may 2009
Brief history of Microbiology
McKaneand Kandel, 1996
McKaneand Kandel, 1996
Detecting New/Emerging Foodborne Pathogens
< 1900 V. cholerae, T. spiralis, C. botulinum, Salmonella, Shigella
1900-10 B. melitensis
1910-20 S. aureus, foodbornepolio
1920-30
1950-60 L. monocytogenes, C. perfringens, V. parahaemolyticus, Anisakidae
1960-70 B. cereus, V. parahaemolyticus, V. vulnificus, aflatoxinand other
mycotoxin
1980-90 L. monocytogenes, E.coli O157:H7, E. sakazakii
1990-00 Cyclospora, Cryptosporodium, nvCJd
1970-80 C. jejuni, Y. enterocolitica, Norwalk virus, Giardia, vomitoxin
1930-40 S.aureus, hepatitis A
1940-50 B. cereus, C. perfringens, V. parahaemolyticus
2000-10
Tomkin, 2007
WHY ?
BACTERIA
Salmonella spp.
Clostridium botulinum
Staphylococcus aureus
Campylobacter jejuni
Yersinia enterocolitica
Listeria monocytogenes
Vibrio cholerae O1
Vibrio cholerae non-O1
Vibrio parahaemolyticus
Vibrio vulnificus
Clostridium perfringens
Bacillus cereus
Aeromonas hydrophila
Shigella 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 PROTOZOA
Giardia lamblia
Entamoeba histolytica
Cryptosporidium parvum
Cyclospora cayetanensis
Diphyllobothrium spp.
Nanophyetus spp.
Eustrongylides sp.
Acanthamoeba
VIRUSES PRION
ALGA MOLD
FOODBORNE PATHOGEN
Microorganisms Hospital
(%)
PUSKESMAS
(%)
Public
(%)
Rotavirus 40-45 23.9 16.2
E. coli (enterotoxigenik) 4-14 6.8 12.2
Salmonella spp. 3-20 0.8 4
Vibrio cholerae 1.5-73 - 0.6
Vibrio NAG 0.3-0.6 2.6 0.8
V. Parahaemolyticus 1.7 - 2.1
Campylobacter spp. 0.2-8.2 - 1.6
Yersinia enterocolitica 0.2 5.1 1.3
Cryptosporidium 1.3 0.8 -
Etiology of Diarrhea in Indonesia (1980-1990)
Source: Punjabi, 2004
Pathogen Percentages
Vibrio 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 Habitat
enterica (I) 1.454 Hewanberdarahpanas
salamae (II) 489
arizonae (IIIa) 94
diarizonae (IIIb) 324
houtenae (IV) 70
indica (VI) 12
Salmonella bongori (V) 20
Total 2.463
Hewanberdarahdingin
danlingkungan
Salmonella enterica
Salmonella species and subspecies
Source: Brenner et al, 2000
nomenclature system
Old System New System
. 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 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. enterica
ser. Enteritidis (Salmonella Enteritidis)
. Salmonella enterica subsp. enterica
ser. Paratyphi (Salmonella Paratyphi)
Salmonella nomenclature according CDC since 2000
Source: Brenner et al, 2000
nomenclature system
Iversen, 2008
Iversen et al. IJ SEM J une 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 M
Areobiccolony counts 3.5 5.0 ISO 4833 2.1.1. Carcasses of
cattle, sheep,
goats and horses
Enterobacteriaceae 1.5 2.5 ISO 21528-2
Areobiccolony counts 4.0 5.0 ISO 4833 2.1.2. Carcasses of
pigs
2.5
Analytical
Reference
Method
Enterobacteriaceae 3.0 ISO 21528-2
Food Category Microorganisms
regulation
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 matrix
2. Other microorganisms (background flora)
3. Cell attachments
4. Inhibitory effects of food
5. Method limitation
6. Physiological state of microorganisms
7. Non-uniform distribution
Types of Microbiological Methods
Quantitative Qualitative Traditional Rapid
1 Culture
2 Microscopic
3 Chemical
4 Physical
5 Biochemical
6 Molecular
7 Immunological
8 Bioassay
Quantitative Analysis
U.S. Food & Drug Administration, Bacteriological Analytical Manual Online, J anuary 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 * n
1
) + (0.1 * n
2
) ] * (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 01-3751-2006
Tepung terigu sebagai bahan makanan
Spiral Plater
Accelerated Bacterial Colony Enumeration
AOAC Official Methods of Analysis
sec. 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 0.00256
3b 0.00644
3a 0.01208
4c 0.02036
4b 0.03232
4a 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. Sampling
2. Amplification
Enrichment cultures, PCR, etc
3. Detection
Selective medium, electrophoresis, Elisa, etc
4. Confirmation
Serotyping, API strips, DNA finger printing, etc
microbiological analysis
Introduction
Several changes are being introduced in this edition of BAM (8thEdition). 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 selenitecystine(SC) broth for the analysis of all foods, except guar gum. In addition,
RV medium replaces lauryl tryptosebroth 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 positve
Salmonella 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
Restaino, 2005
Taiwin, 2005
Restaino, 2005
Restaino, 2005
The conventional media for the detection of Salmonella
by H
2
S 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 Chromogenic
Medium 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
Restaino, 2005
Rambach agar
RAMBACHAgar 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 coliforms
from Salmonellae, the medium contains a chromogene
indicating the presence of -galactosidasesplitting, a
characteristic for coliforms. Coliformmicroorganisms
grow as blue-green or blue-violet colonies. Other
Enterobacteriaceaeand 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

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Harsi D.

Kusumaningrum, OXOID Seminar Jakarta 21th may 2009

ENTEROVIRULENT Escherichia coli GROUP (EEC Group)

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