Agar And Broth Preparation Biology Essay


Sunflower oil Helianthus annus L. Sweet almond oil Prunus Dulcis Grapeseed oil Vitis vinifera L. Rapeseed oil, Olive Extra Virgin oil Olea Europa L. All from essentially oils ltd. Parrafin oil from Sigma Aldrich. Wheatgerm oil(Titicum Vulgarie)from Groundnut oil and wheatgerm oil from local supermarket.

Candida albicans (oral strain 4) from clinical isolate the stock culture was maintained on Sabouraud dextrose agar.

The sabouraud dextrose agar (CM0041) and Sabouraud dextrose liquid medium( CM0147) obtained from Oxoid Ltd.(Oxoid Ltd. Basingstoke, Hampshire).

2.2 Agar and broth preparation

Sabouraud dextrose agar was prepared by dissolving 32.5g of the agar in 500ml of distilled water ,the solution was shaken well and put in the autoclave at 121°C for 15 minutes on the sterilization mode. Once removed from the autoclave the bottles were placed in a water bath set at 50°C to be cooled. Once cool enough to be held, the agar was poured into petri dishes with a lit Bunsen burner close by. The plates were left to solidify at room temperature. They were then stored in the chiller for further use.

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The Sabouraud dextrose broth was prepared by adding 10ml of water to 10 sterilized glass universals then 0.3g of the broth was added to each universal. They were vortexed and put in the autoclave at 121°C for 15 minutes on the sterilization mode. Once cool enough they were stored in the cupboard at room temperature.

Prior to each experiment, Sabouraud dextrose broths were inoculated with one to two ,24 hour old colonies of Candida albicans . One to two colonies of the species were taken from the agar plate and suspended into the broth and placed on a rotary shaker for 24 hours at 37°C.

2.3 Preparation of Candida albicans

Preparation of the yeast suspension was different in each assay according to the optimal concentration of the assay. Candida albicans was adjusted to give an optical density at 600nm between 0.9-1.1 OD. The spectrophotometer (Jenway 6305) was first calibrated with sterile water, the suspension was continuously diluted until the required optical density was reached.

2.4 Plate inoculation

100µl of Candida albicans suspension was pipetted on to Sabouraud dextrose agar plate and was inoculated on to the plate with a sterile spreader. Thin layers of Vaseline, which had been autoclaved at 121°C at 15 minutes was spread on the inside lid of the petri dish with a sterile swab and a square of sterile foil was added on top of this.

Cinnamon and lemongrass oil were each mixed with different carrier oils(Sunflower oil, rapeseed oil, grapeseed oil, walnut oil, groundnut oil, wheatgerm oil, sweet almond oil, extra virgin olive oil) using a 50:50 ratio.5ml of the carrier oil and 5ml of the essential oil were pipetted into a glass universal and vortexed to ensure proper mixing. Cinnamon and lemongrass oil were pipetted at different volumes (25µl, 10µl, 5µl, 1µl) on to the aluminium foil, which was first autoclaved for 15 minutes at 121°C and the plates were then incubated lid down at 30°C for 48 hours.

Control plates were prepared in parallel to ensure that viable organisms were present they were prepared in the same way without any carrier oil and essential oil as well as plates containing just the carrier oils to ensure no biological activity. Triplicate method was used. Zones of inhibition were measured using a petri dish plate which had been marked with 0.5cm-0.5cm squares on the surface. This was placed over the plate of the plate being tested and the squares were counted to provide a measurement of area. Percentage inhibition was calculated by ( Area of zone where no growth is observed /Total area of Candida growth ) - 100.

2.5 Textiles

Bandages were prepared by cutting into squares around 3cm-3cm squares they were placed on top of the foil. Then autoclaved on sterilization mode at 121°C for 15 minutes, to remove any finishing products used and all other residues from the surface. They were placed in drier as some of the fabrics were damp. The oil was then pipetted on top of this at the different volumes (25µl,10µl,5µl,1µl).

2.6 GC MS analysis

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The essential oils, lavender oil, geranium oil, innamon oil, lemongrass oil were analysed to find the components. The GC MS was carried out using Hewlett Packard Model 5890(manufacturer RESTEK) combined with mass spectrometer model Hewlett Packard 5972. The gas chromatogram was fitted with a capillary column 60 metres with an internal diameter of 0.25 mm, with helium as a carrier gas at a constant of 10 psi (1ml/min) .Carried out in Electron Ionisation mode. The electron ionization energy was set at 70eV .The mass scan range was set at between 50-550 amu. The oven temperature was set at 60 degrees with a temperature rate increase of 10 degrees/min to 300 degrees with a holding time of 3 minutes. The inlet temperature of GC was set at 280 degrees .The samples were analysed by Mass spectrometer at 300 degrees at a run time of 30 minutes.

1 µl of the sample was injected. The sample was prepared the needle was cleaned with Dichloromethane solution (DCM),2 drops of oil with 3ml pipette into glass tube, fill up quarter way up with DCM. Shake well.1µl of solution into GC MS machine, press run on computer after adding details of the oil. Clean needle between injections.

2.7 Statistical Analysis

Data was tested for normal distribution using the Anderson-Darling test. Data considered to be normal are presented as means ± SD. Raw data was analysed using One-Way ANOVA with post hoc comparisons using Tukeys test.2 t test used to show differences between two individual results. Associations were analysed using boxplots of data .The differences with PË‚0.05 was taken as critical significance value. All analysis was performed using Minitab version 16.0.