ISOLATION AND IDENTIFICATION OF …mnurcholis.lecture.ub.ac.id/files/2013/04/2_Isolasi-dan...Isolation •If an individual bacterial cell is separated from other cells and has space

ISOLATION AND IDENTIFICATION OF

MICROORGANISMS

Mochamad [email protected]

The 5 I’s of Culturing Microbes

1. Inoculation – introduction of a sample into a container of media to produce a cultureof observable growth

2. Isolation –separating one species from another

3. Incubation – under conditions that allow growth

4. Inspection5. Identification

Isolation• If an individual bacterial cell is separated

from other cells and has space on a nutrient surface, it will grow into a mound of cells - a colony.

• A colony consists of one species.

• A colony consists of millions cells

Insert figure 3.2Isolation technique

Streak Plate

Spread Plate

Pour Plate

• Isolation techniques include:

Insert figure 3.3Isolation methods

Media: Providing Nutrients in the Laboratory

Media can be classified according to threeproperties:1. Physical state – liquid, semisolid and solid

2. Chemical composition – synthetic (chemically defined) and nonsynthetic(complex)

3. Functional type – general purpose, enriched, selective, differential, anaerobic, transport, assay, enumeration

Media: Providing Nutrients in the Laboratory

• Most commonly used media:–nutrient broth – liquid medium

containing beef extract and peptone–nutrient agar – solid media

containing beef extract, peptone and agar

Media: Providing Nutrients in the Laboratory

• Synthetic – contains pure organic and inorganic compounds in an exact chemical formula

• Complex or nonsynthetic – contains at least one ingredient that is not chemically definable

• General purpose media- grows a broad range of microbes, usually nonsynthetic

• Enriched media- contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes

• Selective media- contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes

• Differential media – allows growth of several types of microbes and displays visible differences among desired and undesired microbes

Media: Providing Nutrients in the Laboratory

Miscellaneous Media

• Reducing medium – contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria

• Carbohydrate fermentation medium –contains sugars that can be fermented, converted to acids, and a pH indicator to show the reaction; basis for identifying bacteria and fungi

Insert figure 3.10Differential media

Incubation, Inspection, and Identification

Incubation – temperature-controlled chamber at appropriate temperature and atmosphere– microbe multiplies and produces macroscopically

observable growth

Inspection – observation; macroscopic and microscopic– pure culture – grows only single known species of

microorganisms

– mixed cultures – hold two or more identified species or microorganisms

– contaminated culture – once pure or mixed culture that has unwanted microbes growing

Incubation, Inspection, and Identification

Identification – macroscopic and microscopic appearance, biochemical tests, genetic characteristics, immunological testing

IDENTIFICATION Morphology of Colony

1. Shape 2. Elevation

point

small

Medium

large

circular

irregular

spindle

filamentus

rhizoid

IDENTIFICATION Morphology of Colony

3. Surface 4. Marginhalus mengkilap

kasar

berkerut

kering

entire

lobate

undulate

serate

filamentus

Growth of Microbes on Agar Medium

Agar Slant Agar

Specimen Preparation for Optical Microscopes

• Wet mounts and hanging drop mounts – allow examination of characteristics of live cells: motility, shape, and arrangement

• Fixed mounts are made by drying and heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts.

Staining

Dyes create contrast by imparting a color to cells or cell parts.

• Basic dyes - cationic, with positive charges on the chromophore

• Acidic dyes - anionic, with negative charges on the chromophore

• Positive staining – surfaces of microbes are negatively charged and attract basic dyes

• Negative staining – microbe repels dye, the dye stains the background

Staining

• Simple stains – one dye is used; reveals shape, size, and arrangement

• Differential stains – use a primary stain and a counterstain to distinguish cell types or parts (examples: gram stain, acid-fast stain and endospore stain)

• Special stains – reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains

22

Insert figure 3.27Types of stains

IDENTIFICATION(Metabolic Testing)

Triple Sugar Iron (TSI) :• Identifying Gram negative enteric Bacili based on

fermentation glucose, lactose, sucrose, and H2S production.

• Alkaline slant & alkaline butt (K/K) : nonfermenter

• Alkaline slant and acid butt (K/A) : glucose fermentation

• Acid slant & acid butt (A/A) : glucose, sucrose, lactose fermenter

• K/A/gas + H2S = glucose fermentation, gas n H2S production

IDENTIFICATION(Metabolic Testing)

Lysine Iron Agar (LIA) :• Determine wheteher a bacterium decarboxylates or

deaminates lysine and form H2S.

• Alkaline slant & acid butt (K/A) = glucose fermentation

• Alkaline slant & alkaline butt (K/K) =lysine decarboxylation or no fermentation

• Red slant and acid butt (R/A) = lysine deamination and glucose fermentation

IDENTIFICATION(Metabolic Testing)

Tryptone Broth :• Medium is a pancreatic digest of casein

• High tryptophane content

• Differentiate culture that produce or do not produce indole

IDENTIFICATION(Metabolic Testing)

MR-VP (Methyl-red Voges-Proskauer) :• Contain glucose, peptone, some salts

• Discriminate between organisms using mixed acid pathway and butylene glycol pathway

• Methyl red test detect strong acids formation

• Voges Proskauer test detect acetoin formation

DETEKSI BAKTERI PATOGEN PADA PRODUK

PANGAN

Mochamad [email protected]

Deteksi Escherichia coli

Deteksi Salmonella spp.

Deteksi Coliform & E. coli

Deteksi Staphylococcus aureus

Escherichia coli

Karakteristik :Enterobacteriaceae

Gram (-)Flagellated rod-shaped

Facultative anaerobOxidase (-)Indole (+)Citrate (-)

Ferments glucoseProducing acid & gas

Optimum 35-37oC

Karakteristik Escherichia coli

• E. coli includes a large number of physiologically diverse strains.

• Tipe patogen : E. coli O157:H7

• Suhu pertumbuhan optimum 35-37oC

• 93-95% E. coli strain produce β-D-glucoronidase, except E. coli O157:H7

• E. coli O157:H7 produce cytotoxic factors known as verotoxins (Shiga-like toxin)

Gejala Keracunan

• Gejala keracunan : muntah, diare, yang paling berbahaya hemorrhagic colitisinclude severe cramping and bloody diarrhea

• Produk pangan yang sering tercemar E. coliyaitu :

Daging dan Ikan

Sayuran dan Buah

Air

Tipe Deteksi E. coli

KONVENSIONAL

RAPID DETECTION

CULTURAL

BIOCHEMICAL

SEROLOGICAL

IMMUNOLOGY

PCR Method

Deteksi Escherichia coli metode Kultur

Media :

• Non selective & non differential : Tryptic Soy Agar (TSA) for use in SPCs

• Selective & Differential : VRBA + MUG (4-methylumbelliferyl-β-D-glucoronide)

Mekanisme :

• β-D-glucoronidase cleavage MUG linkage produce a fluorescent product under UV light

PROSEDUR KERJA DETEKSI Escherichia coliMetode Kultur

• Ambil sampel ± 25 g scr aseptis

• Dilusi dng 225 g larutan pepton steril

• Dihomogenisasi dalam stomacher selama 2 menit

• Dilusi s/d pengenceran 10-6.

• Pipet o,1 ml ke dalam petri berisi media VRBA + MUG metode spread plate

• Inkubasi suhu 32oC selama 48 jam

Deteksi Escherichia coli

Deteksi Salmonella spp.

Deteksi Coliform & E. coli

Deteksi Staphylococcus aureus

Enterobacteriaceae

Facultative anaerob

Rods shapeGram (-)

Salmonellosis Karakteristik Salmonella

spp

• Diarrhea Salmonellosis

• Unique serotype : flagellar protein antigens (H), cell wall polysaccharide antigens (O), capsular polysaccharide antigen (Vi)

• Strain berbahaya : Salmonella typhi Cause of typhoid fever

Salmonella spp

• Prepare sample in appropriate, non selective media : Tryptic Soy Broth (TSB), Lactose Broth (LB) 37oC for 24 h

Detection

• Transfer into enrichment broth, : Tetrathionate Brilliant Green (TGB), Selenite Cystine Broth, incubate 37oC for 24 h

• Streak to selective-differential media : HE, XLD, BS, HE : blue-green colonies with or w/o black center XLD : pink colonies with or w/o black center BS : brown-gray, black colonies, sometimes with

metallic sheen

• Pick 2 characteristic Salmonella colonies from each plate and inoculate one TSI and LIA slant for each colony

Detection

• Observe TSI and LIA slants. Inoculate tryptone broth, MRVP broth and simmons citrate agar from + TSI slants

• Perform indole, methyl red, Voges-Proskauer tests, Observe Simmons citrate result

Biochemical Reactions

OrganismsTSI*

(Slant)

LIA**

(Slant)H2S Gas Indole MR VP Citrate

Citrobacter A/A K/A +/- + +/- + - +

E. coli A/A K/K - + + + - -

Edwardsiella K/A K/K +/- + + + - -

Enterobacter A/A K/A - + - - + +

Klebsiella A/A K/K - + - - + +

Morganella K/A R/A - + + + - -

Proteus A/A R/A +/- + +/- + - +/-

Providencia K/A R/A - - + + - +

Salmonella K/A K/K + + - + - +

Serratia A/A K/A - + - - + -

Shigella K/A K/A - - +/- + - -

*K=Alkaline (red), A=Acid (yellow or black [acid+H2S])** K=Alkaline (purple), no fermentation or lysine decarboxylation, A=Acid (yellow), R=deamination rx

Biochemical Reactions

• H2S : black color, is produce from ferrous sulfate in acidic environment.

• Gas (CO2 & H2) is indicated by cracks or separation in the agar.

• Indole : (+) = red color observed at the top of the broth (Kovac’s reagent is added to grawth in Tryptone broth.

• MR : (+) = red color mixed acid metabolism

• VP : (+) = pink to ruby red color (addition naphtol & KOH acetoin production

• Citrate : (+) = color change from green blue

Deteksi Escherichia coli

Deteksi Salmonella spp.

Deteksi Coliform & E. coli

Deteksi Staphylococcus aureus

Staphylococcus aureus

Present in skin & nasopharinx area of human & animal

Growth in danger zone temperature of food (5-60oC)

Optimal temp ; 18-40oCGrow at lower Aw (<0,85)

Can utilize mannitol

Produce Staphylococcal enterotoxins (SEs)

Intoxication : vomit & diarrhea (without fever)

Intoxication : 4-12 h after consumption of food contaminated SEs

Level S. aureus > 106 CFU/g sufficient to produce enough SEs toxin

1

23

4

567

8

910

Foods commonly associated with SEs : deli meats (ham), deli salads (ham, chicken, potato, cream puff)

Property S. aureus S. epidermidis S. hyicus S. intermediusCoagulase + - w +

Heat-stable (thermo)

nuclease

+ - + +

Yellow pigment + - - -

Hemolysis + v - +

Mannitol fermentation

anaerobically

+ - - -

Characteristics of Selected StaphylococcusSpecies

(+) = positive (v)= variable(-) = negative (w) = weak reaction

Deteksi Staphylococcus aureus

Baird Parker Medium

Mannitol Salt Agar

Brain-heart Infusion BrothCoagulase plasma with EDTA

Prosedur

• Siapkan sebanyak 10 g sampel daging, kemudian masukkan ke dalam kantong plastik steril.

• Tambahkan sebanyak 90 ml larutan pengencer (larutan garam) steril.

• Homogenisasi sampel dengan larutan pengencer dengan alat stomacher selama 2 menit.

• Lakukan seri pengenceran sampai dengan pengenceran 10-5.

• Sebanyak masing-masing 0,1 ml ditanam ke dalam petridish berisi media Baird-Parker atau MSA secara spread plate.

• Inkubasi dilakukan pada suhu 37oC selama 24 jam.

Deteksi S. aureus

• Baird-Parker agar : black, shiny, convex colonies surrounded by clear

zone.

Other species may produce gas or less shiny black colonies.

Selective agent : lithium chloride & potassium tellurite

• Mannitol Salt Agar : Turn medium a bright yellow color .

pH Indicator (phenol red), differential agent (mannitol), selective agent (NaCl 7,5%)

• Coagulase Plasma Test : Lyophilized rabbit plasma + EDTA.

EDTA was added as an anticoagulant

Coagulase : enzyme that coagulate plasma from human, horses, rabbits and other animals

Coagulase bind with fibrinogen in the plasma, causing it to coagulate.

If coagulation (clotting) occurs within 6 hr indicate that coagulase is produced by culture.

REFERENCES• Talaro KP. 2012. Foundation in Microbiology

sixth edition. McGraw-Hill Company

• Yousef AE and Carlstrom C. 2003. Food Microbiology: A Laboratory Manual. John Wiley and Sons, Inc. New Jersey

• McLandsborough L. 2005. Food Microbiology Laboratory. CRC Press LLC. Boca Raton

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