Penjelasan Praktikum GEH

ERITRASMA

PENJELASAN PRAKTIKUMSISTEM GASTROENTEROHEPATOLOGIMICROBIOLOGY DEPARTMENTFACULTY OF MEDICINE HASANUDDIN UNIVERSITYMAKASSAR 2014

Oleh:

dr. Ammar Abdurrahman Hasyim, S.Ked.

1PENDAHULUANMICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

Specimen : Depend on symptoms and timingUrine, blood, pus, spinal fluid, sputum, or stool Gram stainingCultures (Blood Agar, EMB, Mac Conkey, SS Agar, TCBS)Biochemical Tests (TSI, SIM, VP, Methyl Red, citrate, Urea, KH test)Nucleic acid methods (PCR, hybridization, blots)Antibody mediated (ELISA, slide/latex agglutination, widal reaction, tagged fluorescence, titer)Diagnosing in Microbiology2Escherichia coli Shigella dysenteriaeSalmonella typhiiVibrio cholera

BAKTERI YANG AKAN DIAMATI

MICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

3Kegiatan PraktikumMICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

Mengamati morfologi bakteri Enterobactericea pada medium agar dan interpretasinya.Mengamati karakteristik bakteri Enterobactericea pada Triple Sugar Iron Agar (TSIA) dan interpretasinya.Mengamati hasil tes-tes biokimia bakteri Enterobactericea dan interpretasinya.Mengamati medium transport dan medium enrichment yang digunakan untuk spesimen yang disuspek mengandung bakteri Enterobacteriacea.

Meliputi:4Mac Conkey MediumNutrient AgarSS AgarTCBS Agar

MEDIUM AGARMICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

5Mac Conkey Medium MacConkey MediumUntuk isolasi dan diferensiasi basil enterik Gram-negatif.Komposisi: Enzymatic Digest of Gelatin 17 g Enzymatic Digest of Casein 1.5 g Sumber nitrogen dan vitamin bagi bakteriEnzymatic Digest of Animal Tissue 1.5 g Lactose 10 g Melihat kemampuan bakteri memfermentasikan laktosa PINK COLOURBile Salts Mixture 1.5 g Crystal Violet 0.001 g Agen selektif, menghambat bakteri cocci gram (+)Sodium Chlorida 5 g Mempertahankan lingkungan osmotikNeutral Red 0.03 g Zat warna dasarAgar13.5 g dan Final pH: 7.1 0.2 at 25 oC


Medium dasarE. coli6Nutrient AgarMICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

Nutrient AgarMedia kultur dasar yang digunakan untuk pertumbuhan bakteri, budidaya organisme yang spesifik dengan memperkaya dengan serum atau darah dan juga digunakan untuk memeriksa kemurnian sebelum tes biokimia atau serologi.Komposisi (@ Gms/litre): Peptic digest of animal tissue 5.000Beef extract 1.500Yeast extract 1.500Sodium chloride 5.000 Mempertahankan lingkungan osmotikAgar 15.000Final pH ( at 25C) 7.40.2Sumber nitrogen, karbon, dan vitamin untuk pertumbuhan bakteri .

Medium dasarE. coli7Salmonella-Shigella AgarMICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

Medium dasar SS AgarMedia selektif diferensial yang digunakan untuk mengisolasi spesies Salmonella dan Shigella dari bakteri patogen lainnya.Komposisi (@ Gms/litre): Peptic digest of animal tissue 5.000Sebagai sumber nutrisi esensial Beef extract 5000Lactosa 10.000 fermentasi karbohidratBile salts mixture 8.500 Sodium thiosulphate 8.500 Selektif menghambat pertumbuhan bakteri gram (+)Brilliant green 0.00033Sodium citrate 10.000 Ferric citrate 1.000 bereaksi dengan H2S membentuk precipitat hitamNeutral red 0.025Agar 15.000Final pH ( at 25C) 7.0 0.28Salmonella-Shigella Agar


9TCBS AGAR

Medium dasarVibrio cholera Tiosulfate Citrate Bile salts SucroseUntuk isolasi dan media pertumbuhan Vibrio cholerae dan vibrio enteropatogen lainnya yang disebabkan oleh keracunan makanan.Komposisi (Gms/Litre): Proteose peptone 10.000 Yeast extract 5.000Sodium citrate 10.000Oxgall 8.000Sodium thiosulphate 10.000Ferric citrate 1.000Sucrose 20.000 Fermentasi karbohidrat dalam metabolisme Vibrio cholerae (yellow)Sodium chloride 10.000 Mempertahankan keadaan osmotikBromo thymol blue 0.040Thymol blue 0.040Agar 15.000 dan Final pH ( at 25C) 8.60.2MICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

Sumber nitrogen, vitamin B kompleks, dan nutrisi pertumbuhan bagi bakteriMenghambat bakteri gram positif dan bakteri koliformSumber sulfur dan melihat produksi hidrogen sulfidaIndikator pH medium10TRIPLE SUGAR IRON AGAR MICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

Untuk diferensiasi bakteri berdasarkan fermentasi dekstrosa, laktosa, dan sukrosa dan adanya produksi H2S (hidrogen sulfida).Interpretasi:c. Medium dasar1. Slant: Alkali, Butt: Netral2. Slant: Alkali, Butt: Asam3. Slant: Alkali, Butt: Netral, H2S (+)4. Slant: Asam, Butt: Asam, Gas (+)5. Slant: Alkali, Butt: Asam, Gas (+)6. Slant: Asam, Butt: Asam, H2S (+) 11TES-TES BIOKIMIAMICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

Sulfur Indole Motility (SIM)MRVPCitratUreaGlukosa, Laktosa, SukrosaMannitol

12Produksi H2S pada SIMHow to Perform Test: Stab SIM media with inoculating needle.Property it tests for: This test is used to help differentiate species of the family Enterobacteriaceae. This test is used to determine the ability to reduce sulfur into H2S.Media and Reagents Used: SIM media contains the sulfur containing amino acid, cysteine, sodium thiosulfate, & peptonized iron or ferrous sulfate.Reading Results: H2S will react with the iron or ferrous sulfate and produce a black precipitate. A positive result has a black precipitate present and a negative result has no black precipitate.


Indole TestHow to Perform Test: Inoculate Tryptone broth with inoculating loop.Property it tests for: This test is performed to help differentiate species of the family Enterobacteriaceae. It tests for the bacteria species ability to produce indole. Bacteria use an enzyme, tryptophanase to break down the amino acid, tryptophan, which makes by-products, of which, indole is one. Media and Reagents Used: Tryptone broth contains tryptophan. Kovacs reagentcontains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcoholyellow in color.Reading Results: Kovacs reagent reacts with indole and creates a red color at the top part of the test tube.


Motility TestHow to Perform Test: Stab motility media with inoculating needle.Property it tests for: This test is done to help differentiate species of bacteria that are motile.Media and Reagents Used: Motility media contains tryptose, sodium chloride, agar, and a color indicator. Reading Results: If bacteria is motile, there will be growth going out away from the stab line, and test is positive. If bacteria is not motile, there will only be growth along the stab line. A colored indicator can be used to make the results easier to see.


(+)(-)(+)Methyl Red/ Voges Proskauer (MR/VP)How to Perform Tests: Inoculate 2 glucose broths with inoculating loop. After 48 hours of incubation, add a few drops of MR to one tube, and VP reagents to the other tube.Properties they test for: Both tests are used to help differentiate species of the family Enterobacteriaceae. MRtests for acid end products from glucose fermentation. VPtests for acetoin production from glucose fermentation.Media and Reagents Used: Glucose BrothMethyl Red indicator for acidVoges Proskauer reagentsA: 5% Alpha-Naphthol, & ethanol, B: Potassium Hydroxide, & Deionized Water.MICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

MR/VP lanjutan....Reading Results: MR a + result is red (indicating pH below 6) and a result is yellow (indicating no acid production)VPA + result is red after VP reagents are added (indicating the presence of acetoin) and a result is no color change.

Methyl Red: left and right +

VP: left + and right


CitrateHow to Perform Test: Inoculate slant with inoculating loop. Property it tests for: This test is used to help differentiate species of the family Enterobacteriaceae. It is selective for bacteria that has the ability to consume citrate as its sole source of carbon and ammonium as sole nitrogen source.Media and Reagents Used: Simmons Citrate Agar contains sodium citrate (carbon source), ammonium ion (nitrogen source), & pH indicatorbromthymol blue.Reading Results: A + result is blue (meaning the bacteria metabolised citrate and produced an acid end product) and a result remains green.MICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

(+)(-)Hidrolisis UreaHow to Perform Test: Inoculate Urea broth with inoculating loop.Property it tests for: This test is done to determine a bacterias ability to hydrolyze urea to make ammonia using the enzyme urease.Media and Reagents Used: Urea broth contains a yeast extract, monopotassium phosphate, disodium phosphate, urea, and phenol red indicator.Reading Results: Urea broth is a yellow-orange color. The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a bright pink color, and is positive. If test is negative, broth has no color change and no ammonia is made.MICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

(+)(-)Glucose Fermentation & Gas ProductionHow to Perform Test: Inoculate broth with inoculating loop.Property it tests for: This test is done to help differnetiate species of the family Enterobacteriaceae. This tests for the bacterias ability to ferment glucose and produce gas and/or an acid end-product..Media and Reagents Used: Glucose broth contains beef extract, gelatine peptone, and glucose. A phenol red indicator is added to indicate an acid enproduct. A Durham tube is added to indicate gas production.ResultsA positive result for acid is yellow after indicator is added (indicating glucose fermentation)A positive result for gas is a bubble in the Durham tube.A completely negative result has no color change or reddish color and no bubble.MICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

Lactose FermentationHow to Perform Test: Inoculate lactose broth with inoculating loop.Property it tests for: This tests for the bacterias ability to ferment lactose.Media and Reagents Used: Lactose broth contains beef extract, gelatin peptone, and lactose. A phenol red indicator is added to indicate acid production from fermentation.ResultsA positive result is yellow after indicator is added (indicating lactose fermentation)A negative result will have no color change or will be redish.MICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

Sucrose FermentationHow to Perform Test: Inoculate sucrose broth with inoculating loop.Property it tests for: This test is done to help differentiate species of the family Enterobacteriaceae. This tests for the bacterias ability to ferment sucrose and production of acid end-productMedia and Reagents Used: Sucrose broth contains beef extract, gelatin peptone, and sucrose. Phenol red indicator is added to indicate an acid end-product.ResultsA positive result is yellow after indicator is added (indicating sucrose fermentation)A negative result has no color change or is reddish.MICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

Sugar Fermentation Tests

Tube 1: Negative acid /Negative gasTube 2A: Must incubate longer (ambiguous result)Tube 2B: Positive acid /Negative gasTube 3A: Positive acid/ Positive gasMICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

Escherichia coliCocco bacil, gram (-)Katalase (+)H2S (-) Aerob or could be facultative anaerobicThe major flora in human intestine Could be motile or nonmotileOksidase production (-)Glucose fermentation releasing gasNot using citrate as its energy source


Biochemical test of Escherichia coli TSI Slam : asam Butt : asamGas : (+)H2S : (-)SIM : Indol : (+)H2S : (-) Motility : (+) VP : (-) Citrat : (-)Urea : (-)MR : (+)MICROBIOLOGY DEPARTMENT FACULTY OF MEDICINE HASANUDDIN UNIVERSITY

Salmonella typhii TSI Slam : alkali Butt : asam Gas : (+/-)H2S : (+)SIM : Indol : (-)H2S : (+) Motility : (+) VP : (-) Citrat : (-/+)Urea : (-)MR : (+)


Shigella dysenteriae TSI Slam : alkali Butt : asam Gas : (-)H2S : (-)SIM : Indol : (-)H2S : (-) Motility : (-) VP : (+) Citrat : (-)Urea : (-)MR : (-)

Vibrio cholera Best enrichment medium : TCBS agar (Thiosulfate-Citrate-Bile-Sucrose Agar)Yellow colonies for vibrios due to oxidase reaction

TSI Slam : asam Butt : asam Gas : (-)H2S : (-)SIM : Indol : (-)H2S : (-) Motility : (-)


MEDIUM TRANSPORTMedium Carry and BlairMedium transport untuk spesimen feces dan swab rectal (Vibrio dan Entrobactericeae patogen).

Medium StuartUntuk transport bakteri basil gram positif dan negatif.


29MEDIUM ENRICHMENTMedium Ox BileMedium enrichment untuk Salmonella typhi dari spesimen darah.

Medium Air PeptoneMedium enrichment untuk spesies Vibrio dari spesimen feces.


30SYUKRON

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