ALTERNATIF PENGGUNAAN KERTAS SARING SEBAGAL PENGGANTI KERTAS CAKRAM PADA UJI RESISTENS] BAKTERI Aeromonas sp. TERHADAP AMPISILIN DAN KLORAMFENIKOL ‘Eulis Reni Sundari "Fakcalias Derikaman dan Ta Kelautan, Universias Padjadjaran Ti. Raya Bandung Stmedan KML], Jatinmngor, Kabupaten Sumedang, Jena Barat 45363 © Enull: eulisdmpadacid Abstrak Sam ini sxdapar boryak pocaltion womans nf raioandi alee mmapmalens enateda dic dition (os Kitby Bouse) Preap penngien din maeds difuri edaish mamampatins [ress rxiven yang ‘lsh diber perialnon sanyers aniibelear, dongan komsentraci becbeda pada mondin jong telah Gioia orgs din akon dou seca mascot, Hal ook omomnjakios Iobeeuim eras carom mening waiingm parks cigmaien babe lain wbegai slmeuif penned ken alr ape surodepercan Sasi yang bar dn efaif Topum dlslulasnys penalries ni néala mendzpmiss thesan! poner karst uarng totage pengeami eros calram dalam sii sisters Saltart rremons sp. tukadp ampisiin das Hocemfsciko!. Pooslitios i dilabsio ped Guba Novambor 2021 ‘Walaupun kondisi penting untuk pemeriksazn in vitro telah distandarksn, namun tidsk ada kondisi in vitro yang mengambarken kondisi yang sama dengan keadaan in viva tempat yang sebenamya bakteri tersebut menginfeksi, bengan demikian ada beberapa faktor yang memegang peranan penting dari pasien disamping hal-hal yang dapat mempengaruhi hasil uji kepekaan yang. telah iperhitungken pada metode uji. Fsktor tersebut antara lain, yaitu: Difusi antimikroba pada sel dan jaringan hospes ‘Protein serum pengikst antimikrabs ‘Gangguan dan interaksi obat ‘status daya tahan dan sistem imun pasien ‘Mengidap beberspe penyakit secara bersamaan Virulensi dan patogenitas bakteri yang sntekei ‘7. Tempat infeksi dan keparahan penyakit” vee op o Isi Terdapst beberaps prinsip daser pemeriksaan uji_kepekaan —terhadap ‘antimikroba, antara lain? 1. Merupakan metode yang. langsung mengukur aktivitas satu atau lebih -antimikroba terhadap inokulum bakteri. 2. Merupakan metode yang secara langsung mendeteksi keberadsan mekanisme resitensi spesifik pada inokulum bakteri_ 3. Merupakan metode khusus untuk mengukur interaksi antara mikroba dan cantimikroba” Kemampuan —antimikraba_ dalam melawan bakteri dapat diukur menggunakan matode yang biasa dilakukan, yaitu: 1. Metode Dilusi Metode dilusi terdini dari dua teknik pengerjaan, yaitu teknik dilusi perbenihan cair dan teknik cilusi agar yang bertujuan untuk penentuan aktivitas antimikroba secara uantitatif, antimikroba dilarutkan kedalam media ager atu kaldu, yang kemudian ivanami bakteri yang akan cites, Setelah diinkubsasi semalam, konsentrasi terendah yang dapat manghambat perumbuhanbakteri disebut dengan MIC [minimal inhibitory concentration). Nilai MIC dapat pula dibandingkan dengan konsentrasi_ obat yang didapat di serum dan cairan tubuh lainnya ‘untuk mendapatkan perkiraan respon klinik?7 a. Dilusi perbenihan cair Dilusi perbenihan cir terdiri dari makrodilusi dan mikrodilusi. Pada prinsipnya pengerjaannya sama hanya berbeda dalam volume. Untuk makrodilusi volume yang igunakan lebih dari 1 mi, _sedangkan mikrodilusi volume yang digunakan 0,05 ml sampai 0,1 ml Antimikraba yang digunakan sdisediskan pada berbagai macam pengenceran biasanya dalam satuan g/ml, konsentrasi bervariasi tergantung jenis dan sifat antibiotik, misalnya sefotaksim untuk uji kepekaan terhadap Streptococcus pneumonia, pengenceran tidak melebihi 2 g/ml, sedangkan untuk Escherichia coli pengenceran sdilskuken pada 16 ug/ml atau lebih. Secora umum untuk penentuan MIC, pengenceran antimikroba dilakukan penurunan konsentrasi setengahnya misainys mulai dari 16, 8, 4, 2, 2, 0,5, 0,25 ug/ml konsentrasi terendah yang menunjukkan hambatan perturnbuhan dengan jelas baik dilihat secara visual atau alat semiotomatis dan otomatis, disebut dengan konsentrasi. daya hambat minimum/MIC (minimal inhibitory concentration) *# ue units [ venume | wemors || msret 2083] 20a ‘So a Jorma of Pret Anh) 2-79 this review we focused on the use of antimicrobial testing ‘methods far the in vitro investigation of extracts and pure drugs as potential antimicrobial agents. “Alter the revolution in the "gokdenera", when almost all groupe ‘of impartant antibiotics (tetracyclines, cephalosperins, aminoghy= ‘cosides and macrolides) were discovered and the main problems of chematherapy were solved in the 1960s, the history sepeats self nowadlays and these exciting compounds are in danger of losing their eficacy because of the increase in microbial resistance (1) Curent ts impacts consierable with treatment ures associated with mulidrugeresistant bacteria and it has become 2 ‘plobal concern to public health (2.2). For this reasan, discavery of new antibiatics is an exrlusively important abjective. Natural products are sill ene of the major sources af new drug molecules teday. They are derived fram prokaryotic bacteria, eukaryotic microorganisms, plants and ‘various animal organisms. Microbial and plant products occupy the majar part af the antimicrabial compounds discovered until now [4], Plants and other natural sourees can provide a huge range of ‘complex and structurally diverse compounds. Recently, many te searchers have focused on the investigation of plant and microbial microbial effect of these natural products, the comparison bee ween results is often difficult, because of the use of different none standardized approaches inoculum preparation techniques, inoculum size, growth medium, incubation conditions and ence points determination. The fact that a plant extract exhibits antimicrobial activity is of interest, but this preliminary part of data shawld be and allow researchers to compare results, avoiding work in which researchers use the antimicrobial activity investigation only 2 2 ‘complement to a phytachemical study. “Avvariety of laboratory methods can be used to evaluate or semen the in vitro antimicrobial activity of an extract or 2 pure ‘compound, The most known and basic methods ave the diskedife fusion and brath or agar dilution methods Other methods are used expecially for antifungal testing, such as poisoned food technique. To further study the antimicrobial effect of an agent in depth, time-kill test and flow cytofluorometric methods are re- ‘commended, which provide information om the nature of the ine hibitony effect (bactericidal er bacteriostatic) (timesdependent or “concentrationsdependent} and the cell damage inflicted ta the test ‘ . Goring ta the new attraction eo the properties of new antie ‘microbial products lke cornbating rwultidrugeresistant bacteria, it is important ta develop a better understanding of the current methods available far screening andjor quantifying the ante microbial effect of an extract or a pure compound far its applica- tions im human health, agriculture and environment. Therefore, in this review, the techniques for evaluating the in vitro antimicrobial activity were discussed in detail, 2 Diffusion methods 241. Agar dishediffusion method Agar dikediffusion testing developed in 1240 [3], is the official method used in many clinical microbiology laboratories far ts tine antimicrobial susceptibility testing. Nowadays. many accepted and approved standards are published by the Clinical and Lae bboratory Standards institute (CLSE} for bacteria ancl yeasts testing [9.10]. Although not all fastidious bacteria can be tested accurately by this method, the standandization has been made to test cestain ‘Bstidious bacterial pathogens like strepiococe, Haemophilus ine foensae, Hoemophilus paranfivensar. Metsera gonorthsese andi ‘Neisseria meningitidis, using specific culture media, various ine “ ae ; TS f aunibos typing tool based on the sexisfance pheantype of the microbial strain tested, its outcoenes alzo guide clinicians in the. selection of intial empiric treatments, and antibiotics used for tmentous fung’ [1s The culture medium, inoealim size and (bation conditions are mentioned in Tablet [15]. ‘Tae above-mentioned advantages of this method, mainly sim plicity and low cost, have contribered to ts common ws forthe Sniumcrobial screening of plant extracts, esential a and other drugs [20523] 22, Antimierabil gradient method (Eeest) ‘The antimicrobial method combines the principle of luton methods with that of diffusion methods in order to de= termine the BC value. it is based oa the possibility of creating 2 this technique. in the procedure, a simp impregnated ‘creasing concentration gradient af the antimicrobial agent fram ‘one end tothe other is depasited on the agar surface, previousty antifungals and antimeycobactesials (24), MIC value is determined at the intersection of the strip and the grawih infubition ellipse. It 1 simple to implement: thus, it is routinely used to meet the de= ‘mands of clinicians. However, Btest® strips cost about $2=3 each.Komunikasl Pendeh (Shorr Comarsidestion) Wine — Absevit Annan Ai Pease dee Ref Cae Caden sigoreskan pada scluruh permukaan media NA pada cawan per, Media NA yang diimokulasi dengan bakteri ji dibuat tiga uhang sumuran dengan menggunakan lubang tips. Pads tiap cxwan petri dan pada. tiap nbang ditambabhan 130 yl masing- smasing larutan up. Caan petri diimkubsi sclama 24 jm pada sub 37°C. Setels imkubasi selesai, slskukan pengamatan dan pengukuran zou hmbat dengan penggnrix, Kelman = daynhambat tEkclompakkan berdisarkan dizmciemya seperti pada tabel 1. Analisis Data Bata yang diperolch dinalisis secara, statistik dengan uji One Hay ANOVA dan uji Tukey menggunkan program SPSS BM 20, HASIL DAN PEMBAHASAN ‘Hasil uji organobeprik: dam ftokimia air perasan dan aie rebwsam dawn ealincing Hasil uji erganolepak air perasans dan rebusan duun calincing memiliki perbedaan sebugaimana tercamtum paca Tabel 2 Air perasan beranmna hija (Gambar 1. Rumus Pethitu yan Diameter Zona Hambat (Harti, 2015) {Colewlaniow farmuta of imbibition some igmoter) (Harti, 2015) ta dan mempunya aroma asam karen kandumgan sam oksilat pada daun calincing. Air rebusam dun calineing berwama kuning hingga oranye dengan roma seperti aroma teh. Rasa sepat paca air perasam don rebwsen disebubkan karena dawn calincing mengandung ianin (Dlimarths, 2008) Hasil fitckimia menunjukkan bubwa air perasan dan air rebusan dawn calincing smengandusg saponin, flavonoid, dan tanin {Garber 2 dan Tabet 3). Hail iii sesumi dengan basil review ofch Sharma dan Kumari (2014) dan hasil penebtion Kasmarini (2017) bahwa dan calincing = mengandung senyawa Aaveaoid, tanin, saponin, Senal, din berbagai am lemma. Abtivitas antibaktert Diameter sna hambat ait peraan dan air rebasan daun salincing pada berhagai kansenirasi C0, 0%, dan 604) terhndsp Soudan menunjublan perbedaan yang myats teretuma pads konseniras: 6066 (pelL05), sodanghan pada kenseniresi yang lebitt rendah (20% oan 0%) tidak berbedh nyata meskipun oda kecenderungan ameter zona humibst air rebusan lebih besar dari air perasan. ‘Tabel 1. Kategori diameter coma hamben (Surjowandojo er al, 2015) (The eawgary of mhiburion ome = Microbiology A Laboratory Manual ELEVENTH EDITION Cappuccino * Welsh ) Pearson‘Sulfadiazine (etionarniie) prAminobenzale acid coon NH, Pynmidine pana Me component (exsontial merabsatite) ‘sulfonamide ‘component tanarmetcta) Figure 42.1 Chemical similarity of sulfanitamnide and PABA preformed state. Therefore, these drugs have no competitive effect an human cells, The similarity between the chemical structure of the antimetabo- lite sulfanilamide and the structure of the essential metabolite PABA is illustrated in Figure 42.1. The Antibiotic Sensitivity Test Procedure eon nha (Once you have completed this experiment, you should understand 1. The Kirby-Bauer procedure for the evaluation of the antimicrobial activity of chemotherapeutic agents. Principle ‘The available chemotherapeutic agents vary in their scape of antimicrobial activity. Some have a limited spectrum of activity, being effective against only one group of microorganisms. Others exhibit broad-spectrum activity against a range of microorganisms. The drug susceptibilities of many pathogenic microorganisms are knows, but it is sometimes necessary to test several agents to determine the drug of choice. Astandardized diffusion procedure with filter paper discs on agar, known as the Kirby-Bauer method, is frequently used to determine the drug susceptibility of microorganisms lsalated from infectious processes. This method allows the 306 Experiment 42 vapid determination of the efficacy of a drug by nieasuring the diameter of the zone of inbubition that-resulis from diffusion of the agent into the media surrounding the dise. In this procedure, fitter-paper discs of uniform size are impregnated with specified concentrations of different antib- ‘ties and then placed on the suzface of an agar plute that has been seeded with the organism to bbe tested. The medium of choice is Mueller Hinton agar, with 2 pH of 7.2 to 7.4, which is poured into plates to a uniform depth of Sum and retriger- ated after solidification. Prior to use, the plates are transferred to an incubator at 37°C for 10 ta 20 minutes to dry off the moisture that develops ‘on the agar surface. The plates are then heavily inoculated with a standardized inoculum by means ‘of a cotton swab to ensure the confluent growth af the organism. The dises are aseptically applied to the surtace of the agar plate at well-spaced inter vals, Once applied, each disc is gently touched with asterile applicator stick to ensure its firm. ccantart with the agar surface. Following incubation, the plates are examined for the presence of growth inhibition, which ts indicated by a clear zone surrounding each dise i | : A measurement of the diameter of the zone ‘of inhibition in millimeters is made, and lis size Figure 422 Kirby-Bauer antibsiatic sensitivity test