rapid microbiological methods

ISO Standard 6579ISO Standard 6579

Detection of SalmonellaDetection of Salmonella

Day1

BPLS Agarmodified24-48 h /35-37°C

Any otherSalmonella

Agar

Day2

Day3

Day4

Day5

Day6

For confirmation take from each plate 5 suspected colonies and streak on Nutrient agar 18 - 24 h / 35 - 37 °C

Biochemical / Serological Confirmation TestsTSI / Urea / Lysin / -Gal / VP / Indol

10 ml BPW to 100 ml SC Broth

24 h at 35 - 37 °C 48 h

0.1 ml BPW to 10 ml RVS Broth

24 h / 42 °C

BPLS Agarmodified24-48 h /35-37°C

Any otherSalmonella

Agar

BPLS Agarmodified24-48 h /35-37°C

Any otherSalmonella

Agar

25 g/ml test samplein 225 ml BPW

16 - 20 h / 35 - 37 °C

FDA-Bacteriological Analytical Manual 8th Ed. 1995FDA-Bacteriological Analytical Manual 8th Ed. 1995Chapter 5. Salmonella method for raw flesh Chapter 5. Salmonella method for raw flesh

foods , highly contaminated foods and animal feedsfoods , highly contaminated foods and animal feeds

Day1

Day2

Day3

Day4

Day

5

Day6

Take from each plate suspected colony and streak on Triple Sugar Iron Agar ( TSI ) and Lysin Iron Agar ( LIA ) 24± 2 h at 35 °C

Further Biochemical / Serological Confirmation Tests

Bismuth Sulfite Agar

24± 2 h at 35 °C

Bismuth Sulfite Agar # 5418

24± 2 h at 35 °C

if negative after 24 h

Subculture from RV and TT Broth to a set of 3 plates each

1 ml Lactose Broth to 10 ml TT Broth

24 ± 2 h at 43 ± 0.2 °C

0.1 ml Lactose Broth to 10 ml RV Broth

24 ± 2 h at 42 ± 0.2 °C

25 g or 25 ml samplein 225 ml Lactose Broth

22 - 26 h / 35 °C

XLD Agar

24 ± 2 h at 35 °C

Hektoen Agar

24 ± 2 h at 35 °C

FDA-Bacteriological Analytical Manual 8th Ed. 1995FDA-Bacteriological Analytical Manual 8th Ed. 1995Chapter 5. Salmonella method Chapter 5. Salmonella method for other foods thanfor other foods than

raw flesh foods , highly contaminated foods andraw flesh foods , highly contaminated foods andanimal feedsanimal feeds

1 ml Lactose Broth to 10 ml TT Broth

24 ± 2 h at 35 °C

1 ml Lactose Broth to 10 ml SC Broth

24 ± 2 h at 35 °C

Day1

25 g or 25 ml samplein 225 ml Lactose Broth

22 - 26 h / 35 °C

Day2

Day3

Day4

Day5

Take from each plate suspected colony and streak on Triple Sugar Iron Agar ( TSI ) and Lysin Iron Agar ( LIA ) 24± 2 h at 35 °C

Day6

Further Biochemical / Serological Confirmation Tests

Bismuth Sulfite Agar

24± 2 h at 35 °C

Bismuth Sulfite Agar # 5418

24± 2 h at 35 °C

if negative after 24 h

Subculture from SC and TT Broth to a set of 3 plates each

XLD Agar24 ± 2 h at 35 °C

Hektoen Agar

24 ± 2 h at 35 °C

Stomacher

Kultur mediaDi dalam kapsul

Cara Pembuatan

Suspendi

ng HeatingDigesting

Homogenating for autoclave

Homogenating after autoclave

Cara Pembuatan

Suspendi

ng HeatingDigesting

Homogenating for autoclave

Homogenating after autoclave

Cara Pembuatan

Suspendi

ng HeatingDigesting

Homogenating for autoclave

Homogenating after autoclave

Cara Pembuatan

Suspendi

ng HeatingDigesting

Homogenating for autoclave

Cara Pembuatan

Suspendi

ng HeatingDigesting

Homogenating for autoclave

Homogenating after autoclave

Cara Pembuatan

Suspendi

ng HeatingDigesting

Homogenating for autoclave

Homogenating after autoclave

Kata Chromogenic berasal dari kata Chronos yang berarti warna dan genesis yang berarti pembuatan.

Jika disatukan mungkin kata chromogenic berasal dari kata proses pembuatan warna atau proses pembuatan pigment.

Media mikrobiologi Chromogenik adalah media mikrobiologi yang digunakan untuk membedakan satu mikroorganisme dengan yang mikroorganisme lain dengan adanya perubahan warna yang jelas dengan bantuan substate chromogenic.

Penggunaan Chromogenic substrate seperti ONPG, X-Gal, X-Glu bersama dengan selektifitas dan sensitifitas media merupakan dasar dari prinsip sederhana dari media mikrobiologi chromogenic.

Enzimatic Reaction

ß-D-Galactosidase– 95 % of Coliforms

ß-D-Glucuronidase–94-97 % of E.coli

Tryptophanase– 99 % of E.coli are Indole positive

Table : Summary of possible enzyme activities, chromogenic substrates, and selectivity system for microorganisms

Species Enzyme Substrate Selective Agents

Bacillus cereus β-glucosidase, specific Phospholipase C indoxyl-β-glucopyranoside, indoxyl-

myo-inositol-1-phosphate

polymyxin B

Campylobacter n/a n/a deoxycholate, cefoperazone,

amphotericin B

Candida Βacetylgalactosaminidase, alkaline phosphatase indoxyl-N-acetyl-β-D-glucosaminide,

indoxyl-phosphate

Chloramphenicol, Gentamicin

Clostridium perfingens β-glucosidase (plus sucrose fermentation) indoxyl-β-D-glucoside D-cycloserine, polymixin B

Coliforms/E. coli β-glucuronidase,

β-galactosidase

indoxyl-β-glucuronide, Indoxyl-β-

galactoside

bile salts, tergitol 7R, SDS,

novobiocin, cefsulodin

Cronobacter (E.sakazakii) α-glucosidase indoxyl-α-D-glucoside deoxycholate, crystal violet,

sodium thiosulfate

E. coli O157 β-glucosidase, α-galactosidase indoxyl-β-D-glucuronide, indoxyl-α-

galatoside

bile salts, SDS, crystal violet,

potassium tellurite, novobiocin,

cefixime

Enterococci β-D-glucosidase indoxyl-β-glucoside sodium azide, polysorbate 80

Extended Spectrum β-Lactamase

Enterobacteria (ESBL)

β-D-glucosidase indoxyl-β-glucoside cefpodoxime, cefotaxime,

ceftazidime

Klebsiella β-D-ribofuranosidase, β-D-glucosidase indoxyl-β-D-ribofuranoside,

indoxyl-β-D-glucoside

bile salts, SDS, carbenicillin

Listeria spp. β-glucosidase indoxyl-β-glucoside lithium chloride, ceftazidime

amphotericin B, nalidixic acid,

polymyxin B

L. monoctogenes Phosphatidylinositol-specific

Phospholipase C, β- glucosidase

indoxyl-β-glucoside, indoxyl-

myo-inositol-1-phosphate

lithium chloride, ceftazidime,

amphotericin B, nalidixic acid,

polymyxin B

Pseudomonas β-Alanyl arylamidase 7-Amido-1-pentyl-

phenoxazin-3-one

cetrimide

Salmonella α-galactosidase, lipase indoxyl-α-galactoside, indoxyl-fatty acid ester sodium deoxycholate

MRSA (Methicillin Resistant

Staphylococcus aureus)

α-glucosidase indoxyl-α-D-glucopyranoside methicillin, high concentration of sodium

chloride

Staphylococcus aureus α-glucosidase, phosphatase,

deoxyribonuclease

indoxyl-α,D-glucoside, phenolphthalein

phosphate, indoxyl-phosphate

tellurite, lithium chloride

Streptococci β-glucuronidase indoxyl-β-glucuronide sodium azide

UTI (Urinary Tract Infections) β-glucosidase,

β-galactosidase

indoxyl-β-glucopyranoside,

indoxyl-β-galactoside

Vibrio β-glucosidase, β-galactosidase indoxyl-β-glucopside, indoxyl-β-galactoside,

indoxyl-β-galactoside

high concentration of sodium chloride,

sodium thiosulphate, sodium citrate,

sodium cholate

VRE (Vancomycin Resistant

Enterococci)

α-glucosidase,

β-glucosidase,

β-galactosidase

indoxyl-α-glucopyranoside, indoxyl-β-

glucopyranoside, indoxyl-β-galactoside

vancomycin

Yeasts and Moulds β-N-acetylgalactosaminidase, β-

xylosidase

indoxyl-N-acetyl-β-D-glucosaminide, indoxyl-

β-D-xyloside

oxytetracycline

Table: HiMedia product line of chromogenic media according to organisms detected

Organisms Brand Cat. No. Media

Bacillus cereus HiMedia M1651 HiCrome™ Bacillus Agar*

E. coli HiMediaHiMedia M1295

M1488

HiCrome™ E. coli Agar B*

HiCrome™ ECD Agar with MUG*

E. coli & Coliforms HiMedia

HiMedia

HiMedia

HiMedia

M1300

M1293

M1294

M1826

HiCrome™ Coliform Agar*

HiCrome™ ECC Agar*

HiCrome™ ECC Selective Agar*

HiCrome™ Rapid Coliform Broth*

E. coli 0157:H7 HiMedia

HiMedia

HiMedia

HiMedia

M1574

M1575

M1598

M1340

HiCrome™ EC O157 Agar*

HiCrome™ EC O157:H7 Selective Agar, Base*

HiCrome™ Enrichment Broth Base for EC O157:H7*

HiCrome™ Mac Conkey Sorbitol Agar*

Thermotolerant E. coli HiMedia M1571 HiCrome™ m-TEC Agar*

Enterobacter sakazakii (Cronobacter spp.) HiMedia

HiMedia

MV1577

MV1641

HiCrome™Enterobacter Sakazakii HiVeg Agar*

HiCrome™Enterobacter Sakazakii HiVeg Agar, Modified*

Enterococci HiMedia

HiMedia

M1376

M1414

HiCrome™ Enterococci Broth*

HiCrome™ Enterococci Agar*

Enterococcus faecium HiMedia M1580 HiCrome™Enterococcus faecium Agar Base*

Klebsiella HiMedia MV1573 HiCrome™ Klebsiella Selective Agar Base*

Listeria HiMedia M1417 HiCrome™ Listeria Agar Base, modified*

Proteus, enteropathogenic gram-

positive organisms

HiMedia M1418 HiCrome™ UTI Agar, modified*

Salmonella HiMedia

HiMedia

HiMedia

HiMedia

HiMedia

M1393

M1633

M1634

M1296

M1466

HiCrome™ MM Agar*

HiCrome™ RajHans Medium, Modified*

HiCrome™ Salmonella Agar*

HiCrome™ Salmonella Agar, Improved*

Staphylococcus aureus HiMedia M1468 HiCrome™ Aureus Agar Base*

Methicillin-resistant Staph. aureus HiMedia M1674 HiCrome™ MeReSa Agar Base*

Vibrio HiMedia MV1682 HiCrome™ Vibrio Agar*

Yeasts and fungi HiMedia M1467 HiCrome™ OGYE Agar Base*

Table: HiMedia product line of chromogenic media according to organisms detected

Table : Biochemical Tests on Disks and StripsCat. No. Product Description

DD015-1VL Bacitracin Disks Used in the presumptive identification of group A β-hemolytic streptococci.

DD024-1VL Bile Esculin Disks For rapid detection of esculin hydrolysis in the presence of bile for differentiating group D streptococci from non-group

D streptococci.

DD040-1VL DMACA Indole Disks Used for the Indole test to determine the ability of an organism to split tryptophan into indole and α-

aminopropionic acid.

DD035-1VL Hippurate Disks Recommended in qualitative procedures to detect organisms possessing the enzyme hippurate hydrolase.

DD019-1VL Kovac’s Reagent Strips Used to determine the ability of microorganisms, primarily Enterobacteriaceae, to split indole from the tryptophan

molecule.

DD042-1VL Nitrate Reagent Used to detect an organism’s ability to reduce nitrate.

DD008-1VL ONPG Disks Used to detect the presence of β-galactosidase, an enzyme found in lactose-fermenting organisms.

DD009-1VL Optochin Disks Optochin (ethyl hydrocuprein hydrochloride) is inhibitory for pneumococcal growth whereas other streptococci show

good growth or a very small zone of inhibition.

DD018-1VL Oxidase Disks

DD036-1VL Sterile Disks Used to test a variety of antibiotics, carbohydrates, substrates, and antiseptics on bacteria in petri dishes. Soak a disk in a

solution or apply some solution on the disks.

DD022-1VL X + V Factor Disks Haemophilus species requiring both X and V factors exhibit growth only in the vicinity of the X +V factor disks.

DD021-1VL V-Factor Disks

What Does ATP Detect ?

Total Organic Living Soil :

-Microbial-Animal-Plant-Human

Information :

Total Microbial Contamination +

Total Food Debris

What Does ATP Detect ?

Our Website

www.AlatAlatLaboratorium.comToko Online Alat Laboratorium

www.AlatAlatLaboratorium.com/BlogPanduan Memilih Alat Laboratorium

www.AlatAlatLaboratorium.com/LaboratoriumMikrobiologiDaftar Peralatan Pembuatan Laboratorium Mikrobiologi

www.AlatAlatLaboratorium.com/MemmertInformasi Product Memmert di Indonesia

Telp/Sms : 0852-6727-7949.

E-mail : sales@AlatAlatLaboratorium.com.

Facebook : AlatAlatLaboratorium.

Twitter : Alat2Lab.Q+A

THANK YOU!