Foodborne

Microorganisms1.

Spoilage microorganism2.

Indicator microorganism3.

Pathogenic microorganism4.

Desirable microorganism

แบคทีเรียท่ีกอใหเกิดโรคอาหาร

เปนพิษ

จําแนกเปน 2 กลุม

แบคทีเรียท่ีกอใหเกิดโรคอาหาร

เปนพิษจําแนกเปน 2 กลุม

1.

Food-Borne Intoxications

Clostridium botulinum

Staphylococcus aureus

Bacillus cereus

2.

Food-Borne Infections

- Salmonellae Shigellae- Enteropathogenic

E.coli- Listeria monocytogenes- Vibrio cholerae- Campylobacter jejuni / coli

- V. parahaemolyticus- Yersenia enterolitica- Clostridium perfringens- และอ่ืนๆ

Enumeration of Mesophilic

Aerobes in Food (APHA Compendium 2001)

Homogenize and sample(50 g sample + 450 mL

Phosphate buffer1:10, 1:102, 1:103)

1 mL

Plate (duplicate)

Standard plate count agar (SPC)35 o

C *

48 h.

Count (25-250 colonies)

Report(bacteria / g.)

* 32 o

C For daily product

25 o

C 5-7 days

Enumeration of Yeast and Mold in Food

(APHA Compendium 2001)

Count (15-150 colonies)

Homogenize and sample( 50 g sample + 450 mL

Phosphate buffer 1:10, 1:102, 1:103)

1 mL

Plate (duplicate)

Standard plate count agar (SPC)*+

Chloramphenicol

Report(Yeasts and Molds / g.)

* Aw > 0.95

ใช DRBC * Aw ≤

0.95

ใช DG18

Enumeration of Lactic acid bacteria in Food (APHA Compendium 2001)

Homogenize and sample(50 g sample + 450 mL

Phosphate buffer1:10, 1:102, 1:103)

1 mL

Plate (duplicate)

De Man, Rogosa

and sharpe

agar (MRS)Anaerobe 30 oC

72

h.

Count (25-250 colonies)

Report(bacteria / g.)

confirm

1.Gram +

2.Catalase -

5-10 colonies

Calculated

Examination of Coliforms, Fecal coliformsand Escherichia coli in Food(APHA Compendium 2001)

Homogenize and dilute sample(1:10, 1:102, 1:103)

3 x 1 mL

3 dilution

Positive tubes

35 o

C 24-48 h.2% BGLB

Lauryl

sulphate

tryptose

(LST) broth

Coliforms E.coli

EC broth45.5 o

*

C 24-48 h.Positive tubes Positive tubes

Refer to MPN L-EMB3 typical colonies

TSI and IMViC

Refer to MPN

1.

In liquid medium (MPN method)

Presumptive

Negative tubes

Coliformsabsent

Confirm

35 o

C 24-48 h.

* water and shellfish used

44.5oC

slantTSI

Indole Positive (+)

Citrate

Negative (-)

butt. acid (A)gas Positive (+)H2

S Negative (-)

acid/alkaline (A/K)

Methyl Red (MR) Positive (+)

Voges Proskauer

Negative (-)

+ - + + -TSI Citrate Indole

-

+ + -

+ + -

+ + Gas

Blackish and/or metallic sheen colonies

LST 2% BGLB EC

E.coli on EMB agar

Biochemical Test

Homogenize and dilute sample(1:10,

1:102, 1:103…..)1 mL

2% BGLB

Violet-Red Bile Agar (VRBA)or Tryptic Soy Agar (TSA*)

(Lactose positive)

Report

2.

On solid medium (Enumeration or pour plate method)

(Gram negative short rod)

Plate (duplicate)

Mix and solidily

Count purple-red

colonies (15-150 colonies)

Select 5-10 colonies

confirm

Gram stain

Calculation

VRBA35 oC **

24 h

*

Expected to be Stressed or Damaged

** 32 o

C For daily product

Homogenize and dilute sample(1:10,

1:102, 1:103…..)1 mL

5-8 mL Violet-Red Bile Agar (VRBGA*)

Report

(Enumeration of enterobacteriaceae)(APHA 2001)

Count purple-red

colonies (15-150 colonies)

Calculation

35 oC**

20-24 h

10 mL Violet-Red Bile Agar (VRBGA*)Mix and solidify

35 oC 24 h

* VRBA +1% Glucose

** 32 o

C

For daily product

Homogenize and dilute sample(1:10,

1:102, 1:103…..)1 mL

Report

(Enumeration of Enterococci or feacal Streptococci) (APHA 2001)

Count pink

colonies (15-150 colonies)

Calculation

35 oC 48 ±2 h

10 mL KF agarMix and solidify

* VRBA +1% Glucose

** 32 o

C

For daily product

5-10 colonies

confirm

CONFIRMATION

Typical colonies

morphology Catalase test (-)

Microaerobic

41.5

0C 24 - 48 h

Gram-positive cocci, elongated, in pairs and

occasionally short chains

Growth *1. at 10 0 C2. 6.5 % NaCl3. pH 9.6

Enterococci (+)

Feacal streptococci

(+/-)

Examination of Staphylococcus aureus in Food(BAM online 2001)

Baird-Parker Egg Yolk-Tellurite Medium

Homogenize and dilute sample(1:10, 1:102, 1:103)

3 x 1mL3 dilution

coagulase test

35 o

C 45 -48 h.

Calculation and report

10% NaCl TSB +

1% Sodium Pyruvate

coagulase test (3-5 colonies)

Typical colony

Refer to MPN

Baird-Parker Egg Yolk-

Tellurite MediumEnumerate grey-black coloniessurrounded with opaque zone an

outer clear zone

MPN methodSurface plating method

0.3 , 0.3 , 0.4 mL

35 o

C 48± 2 h.

Enumeration Method

(20-200 colonies)

coagulase test

Negative Positive

Coagulase Test

Typical colonies of

S. aureus

BPGrey-black colonies surrounded with

opaque zone and outer clear zone

Detection of Clostridium perfringens in Food(BAM online 2001)

Homogenize and dilute sample(1:10, 1:102, 1:103)

Biochemical test1.

Lactose-gelatin medium2.

Motility-nitrate medium

anaerobe 35 o

C 24 h.

Yellow colonies Surrounded by opaque zone

Nagler testor

Lecithinase Inhibition test

Modified Brain Heart Infusion Egg yolk Agar

Cooked meat medium (duplicate)1mL

35 o

C 24 h.

positive

C.perfringens

C.perfringens Lactose +Gelatin +Nitrate +Motility -Salicin -Raffinose +

additional

35 o

C 24 h.

Enumeration of Clostridium perfringens in Food(BAM online 2001)

Homogenize and dilute sample(1:10, 1:102, 1:103)

Biochemical test1. Lactose-gelatin medium2. Motility-nitrate medium

anaerobe 35 o

C 24 h.

20-200 black colonies

Nagler testor Lecithinase Inhibition test

TSC agar without egg yolk (6-7 ml)1mL

35 o

C

24 h.

positive

C.perfringens

C.perfringens Lactose +Gelatin +Nitrate +Motility -Salicin -Raffinose + additional

TSC agar without egg yolk (15 ml)mixed

Lactose Yellow +Gelatin liquid +Nitrate +Motility -( Salicin -

, Raffinose + )

Nagler test or Lecithinase Inhibition test

Black colonies

+

--

+

Nagler Test

Biochemical Test

C.perfringens

Tryptose sulfite cycloserine Agar

Lactose gelatin medium Nitrate motility medium

Modified Brain Heart Infusion Egg yolk Agar

Detection of Salmonellae

in Food(ISO 6579 : 2002)

25 g sample + 225 mL BPW 37 o

C

24 h.

10 mL MKTTn37 o

C 24 h.

1. XLD agar2. HE agar

37 o

C

24 h.Typical colonies of Salmonellae

1. TSI2. LIM

TSI LIMslant alkalinebutt acidgas + H2

S +

Lysine decarboxylase +Indole -Motility +

5. Serological test Agglutination with Salmonella-antisera(OMA-

OMG,( Vi ),A , B , C , D , E,...)

1. Pre-enrichment

2. Enrichment

3. Selective plating

4. Biochemical screening

10 mL RV medium41.5 ±

1

o

C

24 h.

1 mL 0.1 mL

Typical colonies of SalmonellaeHEagar XLD agar

TSI LIMslant alkalinebutt acidgas + H2

S +

Lysine +Indole -Motility +

Agglutination with Salmonella-antisera(OMA-

OMG,( Vi ),

A , B , C , D ,E,...)

Salmonella + Salmonella -

Biochemical Test

Aggutination Test

Examination of Bacillus cereus in Food(BAM online 2001)

Homogenize and dilute sample(1:10, 1:102, 1:103)

1 mL

30 o

C 24 h.

Calculation and report

TSB-

polymyxin or kanamycin

Report

MYP or MYK agar

MYP or MYK agar

Enumerated pink colony with opaque zone

Surface plating method

0.3 , 0.3 , 0.4 mL

35 o

C 48 h.

Enumeration Method

(20-200 colonies)

Confirm 5-10 colonies

Detection Method

30 o

C 24 h.

Typical colony

Confirm

long hair / root-like colony( B. mycoides)

incubate 30 ๐C or 35 ๐C 24 hrs.

ConfirmStreak onto NA plate

Incubate 30 ๐C 24 hrs.

Inoculate isolated colony into 1 mL TSB

incubate 35 ๐C 48 +

2 hrs.

VP mediumNitrate broth

Incubate 30 ๐C 24 hrs.

Phenol redglucose broth(AnO2

)

Sheep blood agarHemolysis

NA (dry plate 1-2 d before use)

+ red color(add reagents: 5%Alpha -

naphthol and 40% KOH)

red color +(add sulfanilic acid and alpha-naphthelamineif not become red,add zinc dust)

incubate 35 ๐C 24 hrs.

+ yellow color

incubate 30 ๐C24

hrs.

Beta-hemolysis (complete)-

strong for B. cereus

(2-4 mm.) -

weak for B. thuringiensis

and B. mycoides

incubate 30๐C 48-72 hrs.

*หมายเหตุ NO3

reduction ทั้ง 3

speciecs ใหผลบวก แต NO3 บางสวนลบ

Streak isolated colony onto NA

slantincubate 30 oC 24 hrs.

Bc motility medium

Motile for Bc

and Bt

Continue incubate

strain toxin crystal (B.thuringiensis)

at Troom

for 1-2 d

Incubate 35 ๐C 24 hrs.

inoculate

Pink colonies surroundedby an opaque zone

Haemolysis test

Typical colonies of B.cereus

1. B thuringensis

ATCC 10792 weak haemolyse2. B.cereus

G1 or G11 strong haemolyse 3. B.subtilis weak haemolyse4. Sample strong haemolyse 5. Sample weak

haemolyse 6. B.cereus var mycoides

ATCC 11778 weak haemolyse

3

1

45

6

2

MYP or MYK

Detection of Listeria monocytogenes in Food(BAM online 2003)

25 g sample + 225 mL BLEB (with 0.1% sodium pyruvate)

Add selective agents(Acriflavin, Nalidixic acid, Cycloheximide)

Oxford agar

Brown green colonies with halo zone

TSA-EY

Esculin Catalase Motile Mannitol -

Haemolysis+ + + Rhamnose + +

Xylose -

35 o

C 24 -48 h

35 o

C 24 -48 h

30 o

C 4 h

30 o

C 20 -44 h

Enumeration of Listeria monocytogenes in Food(ISO 11290-2:1998,2004)

25 g sample + 225 mL. half-fraser

Spread on Ottavini and Agosti (ALOA)

Enumerate the green-blue colonies surrounded by an opaque halo

Confirmation of Listeria spp. genus -

Inoculation on TSYEA Incubate 37o

C 24 h-

Catalase reaction-

Gram staining-

Motility test

Confirmation of Listeria monocytogenes-

Haemolysis test-

Carbohydrate Utilization- CAMP test

37 o

C 24 -48 h

20 o

C 1h0.1 ml.

Brown green colonies with halo Zone

Mannitol -Xylose -Rhamnose +

M X R

Typical colonies of

Listeria monocytogenes

Oxford agar

LIM

Umbrella-like motilility

ALOA agargreen-blue colonies

surrounded by an opaque halo

Detection of Vibrio cholerae in food(BAM online 2004)

25 g of sample+225 mL APW pH 8.6 (+ 2475 mL APW pH 8.6)*

35 o

C 6-8 h

1 Loop

Selective platingTCBS

35 o

C

18-24 h

TCBS

35 o

C 18-24 h

Typical colonies : smooth,yellow and slightly flattened colonieswith opaque center and translucent peripheries

Biochemical test

TSI Butt /slant acid /acidGas Negative H2

S Negative

LIM Lysinedecaboxylase PositiveIndole PositiveMotility Positive

Serological

testAgglutination test with antisera 01 and 0139

Enrichment

Polyvalent Ogawa and Inaba

35 o

C Over Night

1 Loop 42 ±

0.2 0

C 18 –

21 h*

TCBS

*

For raw oysters

25 g of sample +

225 mL Alkaline Peptone water (APW) pH 8.635 o

C 18-24 h

TCBS35 o

C 18-24 hTypical colonies : round, slightly turbid

and bluish-green colonies

TSI

Butt /slant acid / alkalineGas Negative H2

S Negative

LIM

Lysinedecaboxylase PositiveIndole PositiveMotility Positive

Pw 0% NaCl No growthPw 3% NaCl growthPw 8% NaCl growthPw 10% NaCl No growth

Biochemical test

Detection of Vibrio parahaemolyticus in food(BAM online 2004)

Vibrio cholerae : smooth,yellow and slightly fattened colonies with opaque center and translucent peripheries

Vibrio cholerae

Vibrio parahaemolyticus

Vibrio parahaemolyticus : round, slightly turbid and bluish-green colonies

Detection of Viable C.botulinum and Toxicity Testing for Food

Sample 1 -

2 g or mL

Pink suspicious colony( Lipase positive )

Continue page 2

Streak on modified BHI EY agar/orMc Clung -

Toabe EY agar

Incubate 35 -

30o

C 2 days

heat at 60o

C , 15 minCooked meat medium

Incubate at 30o

C , 1 -

2 days

Continue incubation ,at 30o

C , 5 -

7 days

Centrifuge12,000 rpm ,20 min at 4o

C

1

mL supernatant ( ปรับ pH 6.0 -

6.2 )

Biochem testand typing of toxin

Trypsinize with 0.25 mL of 0.5% trypsin (Difco 1 : 250)

at 35 -

37oC , 30 -60 min

Heat 100oC , 10 min

5 fold dilution with gelatinphosphate diluent pH 6.0

Inject 2 micewith 0.5 mL

Inject 2 micewith 0.5 mL

Observe micewithin 96 h.

alive

Observe mice within 96 h.

alivedied

Neutralization test for typing of toxin

(Continued)

Typical colonies

Expression of results and test report

25 g of sample + 225 bolton broth

Confirmation

Microaerobic

41.5

0C 44 ± 4 h

Detection of Campylobacter spp.(ISO 11272-1:2006)

Identification

Microaerobic

37 0C 4-6 hand then at 41.5 0C 44 ± 4 h

mCCD agar and Preston agar

or Skirrow’s agar

Enumerate greyish,often with a metallic sheen,flat and moist with a tendency to spread

< 150 colony

Expression of results and test report

Homogenize and sample( 50 g sample + 450 mL Phosphate buffer

1:10, 1:102, 1:103)

Confirmation

Microaerobic

41.5

0C 44 ± 4 h

Enumeration of Campylobacter spp. (ISO 11272-2:2006)

Identification

mCCD agar

0.1 ml

CONFIRMATION

Typical colonies

morphology motility

Micro-aerobic growth at 25oC (-)

Aerobic-growth at 41.5 oC (-)

Oxidase test (+)

Columbia blood agarMicroaerobic

41.5

0C 24 - 48 h

Curved

bacilli

spiralling “corkscrew”

motillity

Sample ( 2 x 10 ml, 2x 100 ml, 2 x 1000 ml )

Microaerobic

41.5

±10C 44 ± 4 h

Detection and Enumeration of termotolerant Campylobacter spp.

(ISO 17995:2005)

mCCD agar

0.1 mlMembrane filtration (0.45 μm)

Three filters in Preston broths Three filters in Bolton broths

Microaerobic

37

±

1

0C 44 ±4

h

Typical colonies

Aerobic-growth at 41.5 ±oC (-)

Micro-aerobic growth at 41.5oC (+)

microscopy

identification

Numbers of campylobacters present in sample as a function of the results obtain on the test volumes

Sample volume tested Semiquantitative estimate per 1000 ml

1000 ml 100 ml 10 ml

-

+

-

+

+

+

-

-

-

-

+

+

-

+

+

-

-

-

-

-

+

+

+

+

< 1 cfu a

≥

1

cfu but < 10 cfu

≥

1

cfu but < 10 cfu b

≥

10 cfu but < 100 cfu

≥

10 cfu but < 100 cfu b

≥

100 cfu

≥

100 cfu c

≥

100 cfu c

+ Test volume campylobacter-positive

- Test volume campylobacter-negative

a cfu = colony –

forming units.

b Depending on the number of campylobacters in the sample,the

probability of this atypical outcomr can be up to 10%

caused mere coincidence.

c This outcome can be seen when large numbers of competing microorganisms in the sample inhibit the campylobacters.

characteristic C.jejuni C.coli C.lari C.upsaliensisCatalase + + + -

or slight

Nalidixic acid S

a S

a R/S b S

Cephalothin R R R S

Hydrolysis of hippurate + - - -

Indoxyl acetate + + - +

Key : + = positive ; -

= Negative; S = sensitive; R = resistant

a An increase in the resistant to nalidixic acid of C.jejuni

and

C.coli

strains has been show

b Broth sensitive and resistant C.lari

strain exist.

Identification