Materials used and sterilization of glass wares
The materials used were funnels, Whatman No. 1 filter paper, measuring cylinder, plain bottles, paper tape, mulin cloth, beaker, conical flask, foil paper, cotton wool, syringes, test tubes, test tube rack, petri dishes, inoculating loop, cork borer (6 mm), swab stick, gloves, bijou bottles, bowl, marker and membrane filter. All glasswares were sterilized for 40–60 min at 160 °C in a hot air oven.
Solvents used 90% ethanol and distilled water.
Diluent used dimethyl sulphur oxide (DMSO) and distilled water.
Equipment used electric blender, autoclave, incubators, refrigerator, rotary evaporator, weighing balance and spectrophotometer.
Collection of bacterial cultures and identification
The bacterial isolates used in this study were obtained from the Department of Microbiology at the Federal University of Technology. These isolates were previously isolated from surface water samples in Akure, Ondo State, Nigeria, and they include Escherichia coli, Bacillus substiles, Staphylococcus aureus, Streptococcus pyrogens, Corynebacterium sp. Enterococcus faecalis and Pseudomonas aeruginosa.
Collection and extraction of plant material
The Vernonia amygdalina leaf was collected in a farm settlement at Apatapiti, FUTA south gate, Akure, Ondo State. They leaves were authenticated at the department of Crop, Soil and Pest management in Federal University of Technology, Akure. Using the air-drying method, the leaves were air-dried for about three weeks and pulverized using an electric blender (Marlex Electrolyne IS: 250). 100% ethanol and cold water were used as the solvent for extraction. 453.5 g of the pulverized bitter leaf was soaked in 2250 ml (2.25 l) of ethanol. 453.5 g of homogenized Vernonia amygdalina was dissolved in 3500 ml (3.5 l) of cold water. The solutions was allowed to left for 72 h for proper extraction to occur. Using a muslin cloth or cheese cloth and filtered using No 1 Whatman filter paper, each of the solution were sieved; one after the other. The sieved solutions were collected in a beaker and concentrated in vacuo using rotary evaporator for solvent evaporation. The weight of the dried extracts was measured, and the percentage extract recovery was calculated.
$${\mathrm{Percentage}}\;{\mathrm{Extract}}\;{\mathrm{Recovered}} = \frac{{{\mathrm{dry}}\;{\mathrm{weight}}\;{\mathrm{of}}\;{\mathrm{extract}}\;{\mathrm{recovered}}\;{\mathrm{after}}\;{\mathrm{extraction}}}}{{{\mathrm{initial}}\;{\mathrm{dry}}\;{\mathrm{weight}}\;{\mathrm{of}}\;{\mathrm{plant}}\;{\mathrm{part}}}} \times 100\%$$
Colonial, morphological and biochemical characterization of bacterial isolates
Colonial characterization of bacterial isolates
The bacterial isolates were plated on suitable agar plates and were incubated for 18–24 h at 37 °C, and the growths on the plates were observed for their colonial characteristics.
Morphological characteristics of bacterial isolates
Using the Gram staining technique, the morphological characteristics of the cell wall of the isolates were observed. Gram-positive bacteria retained the colour of the primary stain which is purple. Examples of Gram-positive bacteria are species of Bacillus, Clostridium, Staphylococcus, Streptococcus, Bacillus. Gram-negative bacteria stain red or pink, which is the colour of the counter stain (safranin) (Leboffe and Michael 2014).
Biochemical characterization of bacterial isolates
This was done to give more definite and distinguishing characteristics of the isolates. The bacterial isolates were confirmed by subjecting them to biochemical characterizations. The tests include Gram staining, indole, catalase, motility, oxidase, coagulase test, urease test, sugar fermentation test and spore staining test.
Oxidase test
A flamed inoculating loop was used to pick a colony of freshly grown organism 18–24-h-old growth on a culture plate. A smear was then made on a filter paper and moistened with the oxidase reagent (2–3 drops of 1% tetra methyl-p-phenylenediaminedihydrochloride, (Kovac’s formulation) or dimethy-p-phenylenediaminedihydrochloride (Gordon and McLeod’s reagent). A colour change of blue was observed at the site of inoculation within 10–30 s which indicates a positive result. Pseudomonas is oxidase positive (Fawole and Oso 2001).
Motility test
This was done to know whether the organism is able to move from one place to another. A 24-h old broth of the isolate was placed on a cover slip and covered with the corner of the cover slip; the slide was inverted immediately and was examined under high power and oil immersion lenses (Fawole and Oso 2001).
Coagulase test
This was done to know the organism that have coagulase enzyme. The slide was marked into two sections after which a loop of normal saline (0.85%) NaCl in aqueous solution was dropped on the slide; a small amount of the organism (24-h cultured bacteria) was then emulsified in each drop to get a homogenous suspension; a drop of blood plasma was then added to one of the suspensions and stirred for 5 s after which was then observed after few minutes, clumping near re-emulsified mixture indicated coagulase-positive result (Tortora et al. 2016).
Urease test
The isolates were cultured in a medium that contains urea and an indicator phenol red. The urease-positive isolates break down urea into ammonia and carbon dioxide. Which releases ammonia, the medium becomes alkaline and is indicated by the change of colour of the medium to pink or red. Urease gradient was prepared in a bijou bottle. The test organism was inoculated into the urease slope and incubate for 24 h at 37 °C. Colour change was observed. Colour change to pink-red is an indication that the test organism is urease positive (Winn et al. 2006).
Catalase test
On a grease free slide, a drop of hydrogen peroxide was added using dropping pipette. With the aid of an applicator stick, a pure colony of the organism was picked from the cultured plate and drop on the hydrogen peroxide. A production of effervescence was observed which is characterized by the production of bubbles, indicating that the organism is catalase positive. However, the absence of bubbles of gas signifies a catalase-negative test organism (Fawole and Oso 2001).
Indole test
The test organism was inoculated into peptone water and incubated at 35–37 °C for 24 h. After incubation, few drops of the broth culture into a test tube. About two drops of Kovac’s reagent was added and shaken. The appearance of red ring at the uttermost layer indicates that the organism is indole positive (Fawole and Oso 2001).
Methyl red test
This test is used to check for the production of acid by bacterial isolates. The remaining broth from the culture grown for the Voges–Proskauer test is used to perform the methyl red test. A few drops of methyl red solution were added to the culture. An immediate red reaction signifies acid fermentation. A yellow colour is negative (Fawole and Oso 2001).
Sugar fermentation test
This was done to know the sugars a particular organism can ferment. This test was done by preparing a medium containing 100 ml of nutrient broth and 1 g fermentable sugars such as glucose, sucrose, galactose, maltose and lactose after which 0.02 g of phenol red was then added as an indicator. Eight millilitres (8 ml) from each of this sugar solution was transferred into 5 test tubes each with Durham tubes in inverted form making sure that bubbles are not present in the Durham tubes. It was then sterilized in an autoclave at 121 °C for 15 min. After sterilization, each tube containing the sugars was inoculated with one organism and was incubated at optimum temperature of 25 °C for 3 days, acid production was observed by a change in colour from red to yellow and this indicated the utilization of the sugar by the isolate resulting in the production of acid; gas production by the organism was indicated by gas in the Durham’s tube (Fawole and Oso 2001).
Gas chromatography–mass spectroscopy (GC–MS) analysis
Using a gas chromatography–mass spectrometry analysis, the chemical components and their percentage of abundance in the extracts were evaluated. The extracts were analysed using a Varian GC–MS equipment (Varian 4000 mass spectrometer, USA) together with a mass spectrometer (MS) 3800 and equipped with Agilent MS capillary column (30 m × 0.25 mm, i.e., film thickness). The conditions for the analysis operation were as follows: a starting temperature of 35 °C which was steadily raised to 95 °C at a rate of 3 °C/min for 10 min, it was increased to 270 °C at a steady rate of 10 °C/min and a final temperature of 300 °C at a steady rate of 3 °C/min that was maintained for 3 min; column flow rate of 0.8 mL/min; carrier helium at a flow rate of 1.0 mL/min; split ratio 10:1; the ionization voltage of 70 eV; run time 67.5 min; and sample injection volume was 1 µL solution of extract (5 mg/mL). The components were identified by comparing the obtained spectra with those on the NIST 05a library database, and the percentages of abundance were determined with the total ion chromatogram. 1 mL sample was prepared by diluting the extract with analytical absolute ethanol at a ratio of 1:20 (w/v). The sample and replicate were continuously injected as one batch in random order to discriminate technical from biological variations. Additionally, the prepared pooled samples were used as quality controls (QCs), which were injected at regular intervals throughout the analytical run to provide a set of data from which the repeatability can be assessed.
Sterility proofing of extracts
The sterility proofing of the extracts was done by introducing 2 ml of the extract into 10 ml of Muller Hinton broth and incubated at 37 °C for 24 h. The absence of turbidity or clearness of the broth after the period of incubation signifies the presence of a sterile extract.
Reconstitution of extracts
The different concentrations of the extract (i.e., 25 mg, 50 mg and 100 mg) to be used were weighed and dissolved in 1 ml of diluted DMSO (dimethylsulphoxide) and a membrane filter was used to sieve out any contaminants in the extract.
Antibacterial testing of bacterial isolates
Antibacterial activities of the plant extracts were determined using the agar well diffusion method. Different concentrations of 25 mg/ml, 50 mg/ml, 100 mg/ml of the extracts were used for the bioassay. After incubation, zones of inhibition formed in the medium were measured in millimetre (mm) diameter. Ciprofloxacin (0.15 g/ml) was used as standard antibacterial agent for positive control. Using Mueller Hinton agar, 15.2 g of the agar was measured and dissolved in 400 ml of water (prepared according to manufacturer’s specification) in conical flask which was shaken to homogenize the mixture. The mixture was cocked and labelled appropriately and autoclaved at 121 °C for 15 min. After autoclaving, the media was brought out and allowed to cool; on a sterile work bench, petri dishes were arranged and the cooled media was poured into the dishes (10 ml per plate) under aseptic condition. After pour plating, the media was allowed to solidify. Using a swab stick, an already prepared 18–24 h nutrient broth of the organisms was swabbed across the agar plate and labelled accordingly. The swabbed plates were left for 15 min before boring holes in it (about 3–4 holes per plate) using a sterile cock borer (6 mm) and the wells were labelled according to the concentrations of the extracts to be used. The extracts (of different concentrations) and the controls were introduced into the wells until they were full using a sterile syringe to which a membrane filter was attached to and the plates were incubated afterwards at 37 °C for 24 h and zones of inhibition were checked for and measured and recorded.
Determination of minimum inhibitory concentration of leaf extracts of V. amygdalina
The MIC of isolates was carried out using tube dilution technique. McFarland turbidiometric standard (106 cfu/ml) was used to standardize the concentration of test coliforms. A tube containing 9 ml of nutrient broth was seeded with 0.9 ml of the extract with a loopful of the test organism previously diluted to 0.5 McFarland turbidiometric standard. After incubation for 24 h at 37 °C, the tubes were then examined for microbial growth by observing the turbidity with a spectrophotometer.
Data analysis
Data obtained were subjected to Analysis of Variance (ANOVA) using Statistical Package for Social Science (SPSS) version 17.0, and means were separated according to Duncan’s Multiple Range Test (DMRT) at 5% probability level.