isolation and identification of microorganisms · isolation and identification of microorganisms...
TRANSCRIPT
![Page 2: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/2.jpg)
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
![Page 3: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/3.jpg)
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
![Page 4: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/4.jpg)
Insert figure 3.2Isolation technique
![Page 5: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/5.jpg)
Streak Plate
Spread Plate
Pour Plate
• Isolation techniques include:
![Page 6: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/6.jpg)
Insert figure 3.3Isolation methods
![Page 7: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/7.jpg)
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
![Page 8: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/8.jpg)
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
![Page 9: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/9.jpg)
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
![Page 10: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/10.jpg)
• 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
![Page 11: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/11.jpg)
![Page 12: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/12.jpg)
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
![Page 13: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/13.jpg)
Insert figure 3.10Differential media
![Page 14: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/14.jpg)
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
![Page 15: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/15.jpg)
Incubation, Inspection, and Identification
Identification – macroscopic and microscopic appearance, biochemical tests, genetic characteristics, immunological testing
![Page 16: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/16.jpg)
IDENTIFICATION Morphology of Colony
1. Shape 2. Elevation
point
small
Medium
large
circular
irregular
spindle
filamentus
rhizoid
![Page 17: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/17.jpg)
IDENTIFICATION Morphology of Colony
3. Surface 4. Marginhalus mengkilap
kasar
berkerut
kering
entire
lobate
undulate
serate
filamentus
![Page 18: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/18.jpg)
Growth of Microbes on Agar Medium
Agar Slant Agar
![Page 19: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/19.jpg)
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.
![Page 20: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/20.jpg)
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
![Page 21: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/21.jpg)
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
![Page 22: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/22.jpg)
22
Insert figure 3.27Types of stains
![Page 23: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/23.jpg)
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
![Page 24: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/24.jpg)
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
![Page 25: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/25.jpg)
IDENTIFICATION(Metabolic Testing)
Tryptone Broth :• Medium is a pancreatic digest of casein
• High tryptophane content
• Differentiate culture that produce or do not produce indole
![Page 26: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/26.jpg)
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
![Page 28: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/28.jpg)
Deteksi Escherichia coli
Deteksi Salmonella spp.
Deteksi Coliform & E. coli
Deteksi Staphylococcus aureus
![Page 29: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/29.jpg)
Escherichia coli
Karakteristik :Enterobacteriaceae
Gram (-)Flagellated rod-shaped
Facultative anaerobOxidase (-)Indole (+)Citrate (-)
Ferments glucoseProducing acid & gas
Optimum 35-37oC
![Page 30: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/30.jpg)
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)
![Page 31: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/31.jpg)
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
![Page 32: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/32.jpg)
Tipe Deteksi E. coli
KONVENSIONAL
RAPID DETECTION
CULTURAL
BIOCHEMICAL
SEROLOGICAL
IMMUNOLOGY
PCR Method
![Page 33: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/33.jpg)
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
![Page 34: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/34.jpg)
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
![Page 35: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/35.jpg)
Deteksi Escherichia coli
Deteksi Salmonella spp.
Deteksi Coliform & E. coli
Deteksi Staphylococcus aureus
![Page 36: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/36.jpg)
Enterobacteriaceae
Facultative anaerob
Rods shapeGram (-)
Salmonellosis Karakteristik Salmonella
spp
![Page 37: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/37.jpg)
• 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
![Page 38: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/38.jpg)
• 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
![Page 39: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/39.jpg)
• 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
![Page 40: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/40.jpg)
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
![Page 41: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/41.jpg)
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
![Page 42: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/42.jpg)
Deteksi Escherichia coli
Deteksi Salmonella spp.
Deteksi Coliform & E. coli
Deteksi Staphylococcus aureus
![Page 43: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/43.jpg)
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)
![Page 44: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/44.jpg)
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
![Page 45: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/45.jpg)
Deteksi Staphylococcus aureus
Baird Parker Medium
Mannitol Salt Agar
Brain-heart Infusion BrothCoagulase plasma with EDTA
![Page 46: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/46.jpg)
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.
![Page 47: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/47.jpg)
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%)
![Page 48: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/48.jpg)
• 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.
![Page 49: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/49.jpg)
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
![Page 50: ISOLATION AND IDENTIFICATION OF MICROORGANISMS · ISOLATION AND IDENTIFICATION OF MICROORGANISMS ... Deteksi Escherichia coli Deteksi Salmonellaspp. Deteksi Coliform & E. coli Deteksi](https://reader031.vdocuments.us/reader031/viewer/2022022804/5c99cb0b09d3f2a06c8b51be/html5/thumbnails/50.jpg)
Terima kasih