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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 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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