<i>Flash microbiocide</i>: A Rapid and Economic Method for Determination of MBC and MFC

doi:10.4236/ajps.2013.44104

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American Journal of Plant Sciences, 2013, 4, 850-852

http://dx.doi.org/10.4236/ajps.2013.44104 Published Online April 2013 (http://www.scirp.org/journal/ajps)

Flash microbiocide: A Rapid and Economic Method for

Determination of MBC and MFC

Camila Hernandes*, Juliana da Silva Coppede, Bianca Waléria Bertoni, Suzelei de Castro França,

Ana Maria Soares Pereira

Department of Biotechnology, University of Ribeirão Preto, Ribeirão Preto, Brazil.

Email: *camila.hernande[email protected]

Received February 24th, 2013; revised March 28th, 2013; accepted April 4th, 2013

cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Although nowadays there are methods for determining the Minimal Bactericidal Concentration and Minimal Fungicidal

Concentration, it is indispensable that the establishment of innovative methodologies could be more practical and

cheaper. The new methodology Flash microbiocide is an assay in which one aliquot from 96 well plate of Minimal In-

hibitory Concentration test is transferred to another plate containing different culture medium. The correspondence with

the reference methods described in the National Committee for Clinical Laboratory Standards (NCCLS-CLSI) docu-

ment M26-A was achieved, denoting the efficiency of this fast and simple method.

Keywords: Antimicrobial Activity; Minimal Inhibitory Concentration; Minimal Bactericidal Concentration; Minimal

Fungicidal Concentration; Methodology

1. Introduction

The determination of Minimal Inhibitory Concentration

(MIC) is sufficient to indicate the ability of a compound

to inhibit microbial replication. However, for patients

with immunosuppressive infections or inflammatory dis-

eases, such as osteomyelitis, it is necessary to determine

the Minimal Bactericidal Concentration (MBC) and Mi-

nimal Fungicidal Concentration (MFC) to accurately de-

termine the dosage of the antimicrobial agent to be pre-

scribed, which will contribute to the success of the treat-

ment [1-3].

Although there are numerous antibiotics available in

the market, with defined ranges of MIC and MBC, the

tolerance and resistance shown by microorganisms de-

mands the development of new antimicrobial agents as

well as new methodologies to precisely quantify the mi-

crobicidal activity of the new pharmaceuticals [3,4].

Nowadays, the techniques for determining bactericidal

or bacteriostatic action include MBC, time-kill assay and

serum bactericidal titer (SBT). However, those tech-

niques present problems related to the interpretation of

results and reproducibility [5], moreover they are expen-

sive and require a long execution time. Thus, it is neces-

sary that the establishment and standardization of new

and low-cost methods are capable to overcome the limi-

tations of the existing techniques.

The aim of this work is to establish a new method for

determining MBC and MFC which is rapid, easily inter-

preted, reproducible and inexpensive.

2. Materials and Methods

2.1. Actives

Ampicillin (Sigma Aldrich®), Gentamycin sulphate (Ou-

rofin o®), Amphotericin B (Sigma Aldrich®), lyophilized

aqueous extract of stem bark of Stryphnodendron ad-

stringens (barbatimão), and Lippia velutina essential oil,

with the latter being a non-commercial active extracted at

the Biotechnology Unit of the University of Ribeirao

Preto.

2.2. Preparation of the actives

Ampicillin (64 μg·mL−1), Gentamycin sulphate (64 μg·mL−1),

Amphotericin B (32 g·mL−1), S. adstringens extract (2

mg· mL −1), L. velutin a essential oil (40 μL·mL −1 dissolved

into 10% Tween 80 (Dinâmica)).

2.3. Microorganisms

Bacillus subtilis ATCC 6633, Escherichia coli ATCC

*Corresponding author.

Flash microbiocide: A Rapid and Economic Method for Determination of MBC and MFC 851

25922, Pseudomonas aeruginosa ATCC 9027, Staphy-

lococcus aureus ATCC 6538, Candida krusei ATCC

6258 and Aspergillus niger ATCC 16404.

2.4. Antimicrobial Susceptibility Testing

Firstly, the Minimal Inhibitory Concentration (MIC) was

determined by microdilution method following CLSI

guidelines [6-8].

2.5. Determination of Bactericidal and

Fungicidal Activity

The Minimal Bactericidal Concentration and Minimal

Fungicidal Concentration were determined following

CLSI guidelines [3] with the objective to compare the

results obtained using the new method.

2.6. New Method for Determining Bactericidal

and Fungicidal Activity: Methodology

“Flash”

The new method for determining MBC and MFC, was

carried out with material collected from 96 well plate

used for establishing the MIC of agents. Aliquots of 10

and 100 μL were transferred to well plates containing

different culture media (Mueller Hinton for bacterial

strains and RPMI for fungal strains). The plates were

then incubated for 24 or 48 hours according to require-

ments for each strain. Bactericidal and fungicidal activi-

ties having minimum concentration showed no microor-

ganism growth. Data were compared with those from

assays based on CLSI [3] (Figure 1).

3. Results

With regard to the bacterial strains, 100% reproducibility

was observed for E. coli and P. aeruginosa, whereas dif-

ferences in MBC and MFC were found for S. aureus and

C. krusei, despite were not significant (1 well only) (Ta-

ble 1). For the strain B. subtillis, data obtained for MBC

(CLSI method) and the Flash microbiocide method were

equivalent, although the MIC was inferior. When A. ni-

ger was used, both methods were not found to be effi-

cient as they showed reproducibility.

Since obtained data indicated that there is no differ-

ence in reproducibility when using 10 or 100 μL aliquots

(data not shown), the aliquot of 10 μL was standardized

for containing minimal concentration of antibiotic to be

transferred to the new culture medium.

4. Discussion

Obtained results were similar to those reported in the

protocol endorsed by CLSI [3], ensuring the use of the

Flash microbiocide method as an effective technique for

determining microbicidal activity.

The main advantage of this innovative method is that it

was standardized for determining not only the MBC and

MFC of commercial substances but also of extracts and

essential oils of plants. Additionally, it reduces runtime

testing and decreases costs by 60% if compared to the

M26-A methodology recommended by the CLSI.

Another improvement achieved by Flash microbiocide

is the use of small-volume aliquots (10 μL) transferred to

fresh culture medium, reducing the chances of antim-

icrobial agents influence the results, considering that dif-

ferent authors have reported that the permanence of the

antimicrobial agents existing in the aliquots transferred to

the culture medium interferes with the determination of

MBC and MFC [5,9,10].

The already mentioned advantages permit to affirm

that using the Flash microbiocide method it is possible to

safely evaluate the minimum bactericidal concentration

and the minimum fungicidal concentration of actives to

be used in the development of innovative antimicrobial

drugs. Moreover, the methodology may be introduced in

the laboratory routine associated with antibiograms, to

help in the treatment of and immunocompromised pa-

tients infected with microorganisms resistant to common

therapy.

Figure 1. Scheme representing the assay procedures to eva-

uate the MBC and MFC of actives. l

Flash microbiocide: A Rapid and Economic Method for Determination of MBC and MFC

852

Table 1. Evaluation of antimicrobial activity of antibiotics and vegetal actives by preconized method CLSI and with the new

methodology Flash microbiocide.

Bacillus subtilis

ATCC 6633

Escherichia coli

ATCC 25922

Pseudomonas

aeruginosa ATCC

9027

Staphylococcus

aureus ATCC 6538

Aspergillus niger

ATCC 16404

Candida krusei

ATCC 6258

Actives

MIC MBC1 MBC2MIC MBC1 MBC2MIC MBC1MBC2MIC MBC1MBC2MIC MFC1 MFC2 MIC MFC1MFC2

Ampicillin

(g·mL−1) 0.5 0.2 0.2 4.0 4.0 4.0 1.02.02.00.52.02.0- - - - - -

Gentamycin

(g·mL−1) 1.0 2.0 2.0 0.5 1.0 1.0 2.0 2.0 2.0 8.0 8.016.0- - - - - -

Amphotericin

B (g·mL−1) - - - - - - - - - - - - 1.00.5 0.25 2.0 2.04.0

S.adstringens

extract

(g·mL−1)

250 n.d. n.d. 500 n.d. n.d. 250 n.d.n.d.500 n.d. n.d. n.d. n.d. 125 0.6 12562.5

L. velutina

essential oil

(L·mL−1)

0.3 0.6 0.6 0.3 0.3 0.3 0.3 0.3 0.30.3 1.2 1.20.1 <0.07 0.6 0.6 0.60.6

MIC = minimal inhibitory concentration; MBC1 = minimal bactericidal concentration (CLSI); MBC2 = minimal bactericidal concentration (Flash ); MFC1 =

minimal fungicidal concentration (CLSI); MFC2 = minimal fungicidal concentration (Flash microbiocide); (−): not assayed; (n.d): not determined (>1000

g·mL−1); assays MBC2 and MFC2 were performed with aliquotes of 10 µL from MIC.

5. Acknowledgements

This work was supported by University of Ribeirão Preto

(UNAERP), FAPESP 2010/07970-7 and Ourofino.

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