3. MIC dan MBC

Laboratory Evaluation of Antimicrobial Agent : MIC and

MBC

Tiana Milanda

Definitions

Biocidal : bactericidal, virucidal, sporicidal and fungicidal are an agency which kills bacteria, viruses, spores and fungi

Bacteriostatic and fungistatic are an agency which ihibits the growth of the bacteria and fungi.

Definitions

When evaluating the activity of bacterial agents, the terms MIC and MBC are commonly used.

MIC (Minimum Inhibitory Concentration) refers to the minimum concentration of an antimicrobial agent that inhibits growth of the microorganism

MBC (Minimum Bactericidal Concentration) or MFC (Minimum Fungicidal Concentration) refers to the minimum concentration of an antimicrobial agent that kills the microorganism

MIC and MBC are generally recorded in mg/L or µg/L

Definitions The MIC and MBC are often near or equal in value

within one or two dilutions If the ration MBC/MIC ratio is 32 or greater, the term

tolerance is used. Tolerance is a term that implies the ability of some

bacterial strains to survive, but not grow at levels of antimicrobial agent that should normally cidal

Tolerance may be realated to the Eagle phenomenon

(paradoxical effect), where increasing concentration of antimicrobial result in less killing rather than the expected increase in cidal activity

Evaluation of potentiql chemotherapeutic antimicrobial

Test involving potential chemotherapeutic agents (antibiotics) invariably have as their focus determination of MIC

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MINIMUM INHIBITORY CONCENTRATION (MIC)

Penentuan MIC diperlukan untuk menentukan dosis minimum (MEC) suatu antimikroba secara in vitro. Dosis minimum diperlukan untuk menentukan dosis terapi suatu antimikroba secara in vivo.

MIC MEC Dosis Dosis Dosis Dosis LD50 LD100

terapi maksimum toksis letal



Dosis minimum (MEC/Minimum Effective Concentration) : konsentrasi terkecil yang masih dapat menghambat pertumbuhan mikroba dalam tubuh atau dosis minimum in vivo.

MEC diperoleh dari penentuan MIC, dimana MEC = 2-4X MIC.

Dosis maksimum adalah konsentrasi terbesar untuk terapi, tanpa mengakibatkan kerusakan pada jaringan.

Dosis terapi adalah rentang antara MEC dan dosis maksimum.



Dosis toksis adalah dosis yang menyebabkan kerusakan sel atau jaringan tubuh.

Dosis letal adalah dosis yang menyebabkan kematian (pada binatang percobaan). Dosis yang menyebabkan kematian 50% disebut LD50 (lethal dose 50). Sedangkan LD100 adalah dosis yang menyebabkan kematian 100%.

Penentuan dosis maksimum, dosis toksis dan dosis letal dilakukan melalui uji toksisitas (pada Mata Kuliah Farmakologi).


Nilai MIC sangat tergantung pada :1. Jenis antimikroba2. Bakteri ujinya Untuk menentukan MIC, harus digunakan

galur bakteri uji yang belum pernah kontak

(sensitif) terhadap antibiotik tersebut.


Test for bacteriostatic activity : tube dilution method (MIC cair), agar dilution method (MIC padat) and E-tests

All employ chemically defined media at pH 7.2-7.4

Tube Dilution Method

Diencerkan dgn pelarutnya (lihat Farmakope) dan air suling steril dalam labu ukur

1 ml

1 ml

1 ml 1 ml

Antibiotik berbentukcairan

9 ml air suling steril

kocok

Antibiotik berbentukpadat digerus, lalu ditimbang teliti

1 ml 1 ml

1 ml NB biasa

kocok

1 ml NB double strenght

1 ml buang + 1 ose bakteri

A B C

a b c

Tube dilution method

Doubling dilutions, usually in the range 0.12-256 mg/L, of the antimicrobial under test are prepared in a suitable broth medium

A volume of log phase cells is added to each dilution to result in a final cell density of around 5 X 105 CFU/mL (CFU = colony forming unit = jml koloni mikroba)

Dilution tests require a number controls : sterility control (negative control) and growth control (positive control)

Tube Dilution Method After incubation at 35-37oC for 18-24 hours, the

concentration of antimicrobial contained in the first clear tube is read as the MIC

Tube Dilutions Method

PENGAMATAN TABUNG

a b c

KEKERUHAN - - +

MIC terletak pada tabung terakhir yang beningContoh : MIC terletak pada tabung b dengan konsentrasi 7 g/ml

MEC : 2-4 X MIC : (2 X 7 g/ml) sampai (4 X 7 g/ml) : 14 – 28 g/ml

Agar Dilution Method

Dilution tests agar also be carried out using a series of agar plates containing known antimicrobial concentration

Appropriate bacterial suspensions are inoculated onto each plate and the presence or absence of growth is recoded after suitable incubation

In case of solid media, agar plates of defined thickness (approximatelly 3 mm)


Diencerkan dgn pelarutnya (lihat Farmakope) dan air suling steril dalam labu ukur

1 ml

1 ml

1 ml 1 ml

Antibiotik berbentukcairan

9 ml air suling steril

kocok

Antibiotik berbentukpadat digerus, lalu ditimbang teliti

1 ml 1 ml

A B C

A B C19 ml NA bersuhu 40-50C

Goyang2kan, lalu biarkan membeku


1 2 3

- Bagi permukaan dasar cawan menjadi 4 bagian- Gores setiap bagian dengan 1 ose bakteri uji berbeda yang berumur 18-24 jam ( 4 jenis bakteri : a, b, c dan d)

Buat kontrol positif dan negatifKontrol positif : NA + 1 ose bakteri uji berbeda yang berumur 18-24 jamKontrol negatif : NA

+ - 1, 2, 3, kontrol positif dan negatif diinkubasi 37C 18-24 jam

Amati pertumbuhan koloni pada cawan petri 1, 2 dan 3 Dibandingkan cawan petri kontrol positif dan negatif.



PENGAMATAN CAWAN PETRI

1 2 3

a b c d a b c d a b c d

PERTUMBUHAN KOLONI

- - + - + - + - + + + -

MIC terletak pada cawan petri terakhir yang tidak tampak pertumbuhan koloni

Contoh : - Untuk bakteri a : MIC terletak cawan petri 1 - Untuk bakteri b : MIC terletak cawan petri 2 - Untuk bakteri c : MIC terletak sebelum cawan petri 1 - Untuk bakteri d : MIC terletak pada atau sesudah cawan petri 3


Kelebihan Agar Dilution Method/MIC padat : Dapat digunakan untuk menentukan MIC

dari suspensi zat antibiotik yang keruh sulit untuk membedakan kekeruhan yang disebabkan zat antibiotik atau oleh pertumbuhan bakteri uji.

Dapat digunakan untuk menentukan MIC zat antibiotik terhadap beberapa bakteri uji sekaligus.

E tests The E (Epsilometer) tests is

performed in a nylon strips that have a linear gradient of antimicrobial lyophilized on one side. On the other side are a series of lines and figures denoting MIC values

The nylon strips are placed antimicrobial side down on the freshly prepared bacterial lawn

Afer incubation, MIC is determined by noting where the ellipsoid (pear shaped) inhibition zone crosses the strips

Problematic bacteria

The standard methodology may fail to detect the resistant phenotype.

This due to variety of factors including heterogenous expression of resistance, poor agar diffusin of the antimicrobial and slow growth of cells

Expression is enhanced at lower temperatures , at higher salt concentration and more incubation time

Minimum bactericidal concentration (MBC)

MBC testing is required for evaluation of novel antimicrobials

The MBC is the lowest concentration (in mg/L) of antimicrobial that results in ≥ 99.9 % killing of the bacterium under test

MBC are determined by spreading 0.1 mL volume of all clear (no growth) tubes from a dilution MIC test onto separate agar plates.

Minimum bactericidal concentration (MBC)

After incubation at 35-37oC for 18-24 hours, the numbers of colonies growing on each plate are recorded

The first concentration of drug that produced < 50 colonies after subculture is considered the MBC (the initial inoculum 5 X 105 CFU/mL)

Test for fungistatic activity

Tests for fungisatic activity have been based on the established bacterial techiques

The medium is different (SDA or RPMI plus 2% dextrose)

The inoculum density used is reduced (104 CFU/mL)

A lawn producing just separated/distinct colonies. Addition of methylene blue (0.5 µg/mL to media may improved the clarity of inhibition zone edges

More incubation times (72 hours for filamentous fungi)

Tests for fungicidal activity

About 20 µL from MFC test are subculture onto suitable growth medium from each clear tube

This plate are incubated until growth is evident on the growth control subculture.

MFC is the lowest drug concentration showing no growth or fewer than 3 colonies per plate to obtain 99-99.5% killing activity

Evalution of preservatives

Preservatives are widely employed in the cosmetics and pharmaceutical industries

The inhibitory or cidal activity of the preservative can be evaluated using an appropriate in vitro test system

Its continued activity when combined with the other ingredients in the final product must be estalished

Problems clearly exist with some product, where partitioning into various phase or one or more the components may inactive the preservative

Evalution of preservatives

In challenge test, the final preserved products is deriberately inoculated with a suitable enviromental microorganism which may be fungal, e.g candida or bacterial, e.g. S. aureus, E. coli, P. aeruginosa

Death or growth of the inoculum is the assesed using viable count techniques


Viable count techniques

+ bakteri/jamur

Pengenceran

Hitung koloni menggunakan alat coulter counter darimasing-masing cawan petri, kalikan denganfaktor pengenceran, lalu rata-ratakan. Hasilyang baik 30-300 koloni per cawan petri

Contoh :30.101 X 35.102 x 40.103

3=……mikroba/ml or CFU/mL

1 ml

1 ml 1 ml 1 ml

1 ml 1 ml

Sampel padat

Sampel kental

9 ml medium cair

kocok

20 ml medium padat

3. MIC dan MBC

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