Chapter 2: In vitro antimicrobial activity of extracts from plants used traditionally in
2.5. Materials and methods
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into sterile plastic Petri dishes and allowed to gel. After gelling of the agar, the Petri dishes were kept at 4 °C.
The Kwik-stikT M unit was removed from 2-8 °C storage and allowed to equilibrate to room temperature. The middle of the ampoule cap of the Kwik-stikT M unit was pinched to break the ampoule and that was followed by release of the hydrating fluid which was then allowed to flow through the swab shaft and into the bottom portion of the unit containing the gelatine pellet. The bottom portion of the pellet was crushed by pinching it repeatedly to form a homogeneous suspension. The fluid-hydrated swab was saturated and transferred to Petri dishes with MH agar (Klebsiella pneumoniae and Staphylococcus aureus) or Middlebrook 7H10 agar supplemented with 0.5% or 0.2% glycerol and 10% OADC (Mycobacterium aurum A+ and Mycobacterium tuberculosis H37Ra). For each bacterial strain, the same swab was used repeatedly to streak the inoculated area. The inoculated agar plates were then incubated for 24 h (Klebsiella pneumoniae, Staphylococcus aureus), or 72 h (Mycobacterium aurum A+) at 37 °C to allow bacterial colonies to grow. For Mycobacterium tuberculosis H37Ra, the agar plates were incubated in a 5% carbon dioxide incubator at 37 °C for four weeks.
After incubation, the bacteria were kept as stocks in sterile cryovials (Greiner, Germany) containing glycerol solution for Klebsiella pneumoniae and Staphylococcus aureus. Whereas Mycobacterium aurum and Mycobacterium tuberculosis H37Ra stocks were kept in sterile Middlebrook 7H9 broth supplemented with a 0.5% glycerol and 10% OADC supplement.
The cryovials with stock cultures were then stored in an ultra-freezer at -70 ºC until required for antibacterial assay.
2.5.4. Subculturing for bioassays
Bacterial stock strains were streaked and sub-cultured in sterile MH agar (for Klebsiella pneumoniae and Staphylococcus aureus) or Middlebrook 7H10 agar supplemented with 0.5%
glycerol and 10% OADC supplement (for Mycobacterium aurum and Mycobacterium tuberculosis). The plates were incubated for 24 h for Klebsiella pneumoniae and Staphylococcus aureus, 72 h for Mycobacterium aurum and 3 to 4 weeks for Mycobacterium tuberculosis H37Ra to allow bacterial colonies to develop. When the bacterial colonies had developed, the plates were then stored at 4 °C to prevent further bacterial growth. The same
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procedure was followed to sub-culture stocks on a monthly basis in order to maintain viability.
2.5.5. Antibacterial activity using microdilution
The minimum inhibitory concentrations (MIC) of plant extracts were determined using the microdilution method in 96 well microtitre plates against Staphylococcus aureus and Klebsiella pneumoniae as described by ELOFF (1998a). The extracts were dissolved in 10%
dimethylsulfoxide (DMSO) except for aqueous extracts that were dissolved in sterile distilled water. The test organisms (Staphylococcus aureus and Klebsiella pneumoniae) were cultured in MH broth for 24 h at 37 °C. Neomycin was used as the positive control whereas broth (negative), 10% DMSO, and water (solvent controls) were used as negative and solvent controls, respectively. For antibacterial testing against Staphylococcus aureus and Klebsiella pneumoniae, one hundred microlitres of water were added in each well of the 96 well microtitre. One hundred microlitres of solvent controls and test samples were added to the first wells of the column at an initial concentration of 100 mg/ml of extracts and 50 mg/ml for the positive control and then two-fold serially diluted down the wells. The optical density of Staphylococcus aureus and Klebsiella pneumoniae was adjusted with MH broth to match that of McFarland standard, equivalent to 108 colony forming units/ml (CFU/ml) at 600 nm. One hundred microlitres of the diluted culture were added to all the wells. The microtitre plates were then incubated for 24 h at 37 °C. After incubation, 40 𝜇l of p-iodonitrotetrazolium violet (INT) indicator was added to evaluate growth inhibition. The results were observed following 30 min incubation at 37 °C. The lowest concentration containing clear wells were considered as the MIC values. The assay was repeated twice with two replicates per assay.
2.5.6. Antimycobacterial activity using the resazurin microplate assay
The resazurin microplate assay (REMA) and broth microdilution method as described by JADAUN et al. (2007) modified by GREEN et al. (2010), to determine the antimycobacterial activity of the extracts, were performed against Mycobacterium tuberculosis H37Ra and Mycobacterium aurum A+. Mycobacterium tuberculosis H37Ra was subcultured in Middlebrook 7H9 broth supplemented with 10% OADC and 0.2% glycerol, and then incubated at 37 ºC for 4 weeks in 5% carbon dioxide until logarithmic growth was
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reached. Turbidity equivalent to that of McFarland’s No. 1 standard solution for Mycobacterium tuberculosis H37Ra was achieved by mixing the culture with a sufficient volume of sterile supplemented Middlebrook 7H9 broth. The test inoculum was obtained by further dilution (1:20) of the suspension with the same culture medium to approximately 6×106 CFU/ml immediately prior to use. Mycobacterium aurum A+ was subcultured in Middlebrook 7H9 broth supplemented with 10% OADC and 0.5% glycerol, and incubated for 72 h at 37ºC. The optical density of Mycobacterium aurum A+ was adjusted to 0.125 at 550 nm using Middlebrook 7H9 broth enriched with 0.5% glycerol and 10% OADC growth supplement. Rifampicin and streptomycin were used as positive controls whereas broth (negative), 10% DMSO and water (solvent) were used as negative and solvent controls, respectively. The extracts were dissolved in 10% DMSO to a concentration of 100 mg/ml and maintained at room temperature for 1 h to assure their sterilisation. For antimycobacterial testing, 100 µl of Middlebrook 7H9 broth supplemented with 0.5% glycerol, and 10% OADC were then added in each well. Solvent controls and test samples (100 µl) were added to the first wells of the column starting with a concentration of 100 mg/ml of extracts and 50 mg/ml for the positive control and then two-fold serially diluted down the wells. One hundred microlitres of the adjusted bacterial cultures were added to all the wells. The organic and aqueous extracts from each plant were assayed in duplicates. Each microplate with Mycobacterium tuberculosis H37Ra as a test organism was incubated for 5 days at 37 ºC in a 5% carbon dioxide atmosphere, while the microplates with Mycobacterium aurum A+ were incubated for 72 h at 37 ºC. After incubation, 32 µl of freshly prepared filter sterilised 0.002% resazurin solution was added to each growth control well only. For Mycobacterium tuberculosis H37Ra, the microplates were incubated again at 37 ºC in a 5% carbon dioxide atmosphere for 24 h. When a color shift from blue to pink was observed in the growth control sample, 32 µl of resazurin solution was added to each of the remaining wells, and the microplate was further incubated for 24 h. For Mycobacterium aurum, the results were observed after 30 min of incubation after adding the indicator. A well-defined pink colour was interpreted as positive bacterial growth, whereas a blue colour indicated the absence of growth.