CONVENTIONAL IDENTIFICATION OF

MICROORGANISMS

Mochamad NurcholisCholis_federer@yahoo.co.id

The 5 I’s of Culturing Microbes

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

2. Isolation –separating one species from another

3. Incubation – under conditions that allow growth

4. Inspection5. Identification

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

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

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.

Bacteria Shape & Size

• Coccus

• Bacillus

• Spirilium

• Vibrio

• Spirochetes

• 0.5 -1 µm x 2–5 µm

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

Perbedaan relatif sifat bakteri gram positif dannegatif

SIFAT Bakteri gram + Bakteri gram –

Komposisi dinding sel Kandungan lipid rendah (1-4%)

Kandungan lipid tinggi (11-22%)

Ketahanan thdp penisilin

Lebih sensitif Lebih tahan

Penghambatan oleh pewarna basa (mis. Violet kristal)

Lebih dihambat Kurang dihambat

Kebutuhan nutrien Kebanyakan spesies relatif kompleks

Relatif sederhana

Ketahanan terhadap perlakuan fisik

Lebih tahan Kurang tahan

Urutan pewarnaan gram serta reaksi yang terjadi danwarna yang terbentuk

Urutan pewarnaan Reaksi dan warna bakteri

Gram positif Gram negatif

Violet Kristal (VK), 1 menit

Sel berwarna violet Sel berwarna violet biru

Larutan Iodium (I), 1 menit

Terbentuk kompleks VK-I, sel berwarna violet biru

Terbentuk kompleks VK-I, sel berwarna violet biru

Pencucian dengan alkohol

Dinding sel mengalami dehidrasi

Pori-pori berkerut

Permeabilitas menurun

Kompleks VK-I tidak dapat keluar sel

Sel tetap berwarna violet biru

Lemak terekstraksi dari dinding sel

Pori-pori membesar

Kompleks VK-I tercuci keluar

Sel tidak berwarna

Safranin, 20 detik Tidak berpengaruh

Sel tetap berwarna violet-biru

Sel menyerap zat warna

Sel berwarna merah

LAPISAN DINDING SEL

GRAM POSITIF

• KAPSUL

• PEPTIDOGLIKAN

• ASAM TEIKOAT

• RUANG PERIPLASMA

• MEMBRAN SITOPLASMA

GRAM NEGATIF•KAPSUL•LIPOPOLISAKARIDA+ LIPOPROTEIN

•PEPTIDOGLIKAN

•RUANG PERIPLASMA•MEMBRAN SITOPLASMA

• Motility - movement

• Arrangement basis for classification

–Monotrichous; 1 flagella

–Lophotrichous; tuft at one end

–Amphitrichous; both ends

–Peritrichous; all around bacteria

Flagella

Flagella Type of Bacteria

PertumbuhanKapang

• Jamur filamen

• Filamen bercabang hifa

• Dinding sel hifa chitin

• Kumpulan hifamiselium( jamak : miselia)

• Ukuran : 2-10 µm x 1-2 mm

Pertumbuhan Hifa

mycelium

germinating

spore

hifa

Hyphae

Yeast / Khamir

• Unicellular fungi• P 1-50 µm x L 1-10 µm • Budding , Binary fission,

Budding Fission & sporulasi

• Slime CapsuleCandida

Saccharomyces

IDENTIFICATION(Metabolic Testing)

Catalase Test :• To detect the presence of the enzyme catalase

• Catalase enzyme is found in most bacteria

• Breakdown of 3% hydrogen peroxide (H2O2) with the release of free Oxygen

• Catalase (+) bubbles formation

• False (+) using catalase containing medium

(ex : blood agar medium)

• False (-) using old culture

IDENTIFICATION (Metabolic Testing)• Gas Production

IDENTIFICATION(Metabolic Testing)

Urease Test

• Inokulasi biakan mikroorganisme pada media urea agar miring

• Inkubasi pad suhu 350C selama 24 jam.

• Urease (+) terjadi perubahan warna dari kuning menjadi merah keunguan (Hadioetomo, 1985)

Fermentative Activities of Microorganisms

Fermentative Activities of Microorganisms

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 Indole Test :• Medium is a pancreatic digest of casein

• High tryptophane content

• Differentiate culture that produce or do not produce indole

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

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

Karakteristik Escherichia coli

Karakteristik Salmonella spp.

Karakteristik Staphylococcus aureus

Escherichia coli

Karakteristik :Enterobacteriaceae

Gram (-)Flagellated rod-shaped

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

Ferments glucoseProducing acid & gas

Optimum 35-37oC

Identifikasi Escherichia coli

KONVENSIONAL

RAPID DETECTION

CULTURAL

BIOCHEMICAL

SEROLOGICAL

IMMUNOLOGY

PCR Method

Karakteristik Escherichia coli

Karakteristik Salmonella spp.

Karkateristik 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

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 growth 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

Karakteristik Escherichia coli

Karakteristik Salmonella spp.

Karakteristik 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)

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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