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A total of 4 soil samples were obtained from the desert environment in the Kingdom of Saudi Arabia from foure different areas Thumamah, Al-Kharj, Al-Madina and Taif. The samples were taken from up to a depth of 15 cm, after removing approximately 3 cm of the soil surfaces. The soil samples were placed in sterile poly ethylene bags, closed tightly to avoid external contamination, labeled and transported to the laboratory where they were air dried at room temperature (25oC) for 1 week and then stored in sterile plastic bags until required (Figure 3.1.1.a).
Isolation of actinobacteria from soil using the serial dilution technique:
Serial dilution technique (Kelley & Post, 1982) was used to isolate actinobacteria from the soil samples. In this technique 1g (gram) of each air dried soil sample was suspended in a tube containing 10 ml (milliliters) of sterile distilled water, each tube was then caped and stirred for 1minute using a vortex mixer (VTX-3000L) to form a stock solution, from this stock 1ml of soil suspension was transferred to another tube containing 9 ml of sterile distilled water and as before the tube was also capped and stirred to form the first dilution. The steps above were repeated for all soil samples making a total of 3 dilutions for each sample 10-1, 10-2 and 10-3.
TWA (tap water agar) media (Ara et al, 2012c) was prepared containing [22g agar (WINLAB, UK) and 1L tap water] (See appendix 1). Approximately 0.1 ml inoculum of each of the three dilutions was added to a plate containing the media mentioned above. The inoculum was equally distributed all over the plate using a sterile glass rod. Antibacterial nor were antifungal agents used in preparing this media. The plates were incubated at 30oC for 2 weeks (Figure 3.1.2.a).
Actinobacteria colonies enumeration (Colony count)
After incubation for two weeks the actinobacteria colonies were examined by the naked eyes then the numbers of colonies from each sample were determined by the means of colony count technique (Jeffrey, 2008). In this technique the numbers of actinobacteria were counted for each dilution plate and then were divided by the inoculum amount plated and multiplied by the dilution factor.
Isolates purification on yeast ââ‚¬" starch agar media
YSA (Yeast Starch Agar) media (Ara et al , 2012a) was prepared containing [2g yeast extract (Oxoid, UK), 10g starch (AVONCHEM, UK), 15g Agar (WINLAB, UK) and 1L distilled water] (See appendix 1). About 50 actinobacteria isolates were picked up from the TWA plates and transferred to the YSA media plates using the Mossel ecometric streaking method (Singh and Agrawal 2003) in order to obtain a single colony from each isolate for purification purposes .The YSA plates were incubated at 30oC for one week. The single colonies were then cultured on fresh YSA media plates and incubated again at 30oC for one week. This step was repeated until the pure strains were obtained.
The fifty (50) actinobacteria isolates were chosen for preliminary screening of antimicrobial activity and these were coded as ( M1, M2, M3, M4, M5, M6, M7, M8, M9, M10, M11, M12, M13, M14, M15, M16, M17, M18, M19, M20, M21, M22, M23, M24, M25, M26, M27, M28, M29, M30, M31, M32, M33, M34, M35, M36, M37, M38, M39, M40, M41, M42, M43, M44, M45, M46, M47, M48, M49 and M50.From these strains twenty (20) were chosen for further study based on their antibacterial activity results (Figure 3.3.1.a).
Color grouping of the isolates (streaking on oatmeal agar media)
OA (Oatmeal agar) media was used for color group determination (Nonomura, 1988). The media contained [30g oatmeal (Quaker), 15g agar (WINLAB, UK) and 1L distilled water] (See appendix 1). The OA medium was inoculated by streaking to determine the color groups of the selected actinobacteria isolates. All plates were incubated at 30oC for 1 week. After the incubation period, colony colors of the isolates were visually determined using the ISCC-NBS color chart (Kenneth, 1958).
Preservation in 30% glycerol
Before farther investigation, the pure strains had to be preserved to insure that they will not be lost. 30% glycerol (Ozgur et al., 2008) was used in the preservation process which contained [30ml glycerol (WINLAB, UK) and 70ml distilled water] (See appendix 1). The glycerol was autoclaved and then distributed into 1.5 ml sterile eppendorf tubes up to half then blocks were made in the YS agar plates containing each actinomycetes strain. About 3 blocks of each strain was added to the tubes then were tightly caped and gently shaken in order to coat the blocks with the glycerol. All tubes were stored at -80oC in the central lab, King Saud University.
Test for secondary metabolites production
Cultivation of actinobacteria for secondary metabolites production
Each actinobacteria isolate was inoculated into a flask (250 ml) containing 100 ml of SGY (Starch Glucose Yeast) broth (Ara et al 2002, Ara 2003). the composition of the broth (per liter) was [10g starch soluble (AVONCHEM,UK), 10g glucose (FLUKA, UK), 10g glycerol (WINLAB, UK), 2.5g corn flower (RIYADH FOOD), 5g peptone (BIOCHEMICAL,UK), 2g yeast extract (WINLAB, UK) , 3g CaCo3 (WINLAB, UK) and 1L distilled water] (See appendix 1). The inoculation in SGY broth is shown in (Figure 3.6.1.a).
Secondary metabolites extraction:
The anti-bacterial compounds were recovered from the broth by solvent extraction method (Westley et al., 1979; Liu et al., 1986). Methanol (PANREAC, E.U) was added to the flasks containing the mixture of strain and broth in the ratio of 1:1 (v/v). The flasks were then returned to the shaker for 3 extra days which after the contents were filtered to separate the mycelium from the liquid (Figure 3.6.2.a). The filtrate was transferred in to a hot air oven for the methanol to evaporate to dryness. Two drops of distilled water was added to the residue obtained and was mixed well. The crude extract acquired was used to determine the antimicrobial activity.
Secondary metabolites activity
The test organisms that were used in this study were reference strains were as follows:
Gram-positive bacteria: Staphylococcus aureus ATCC 13076, Bacillus subtilis ATCC 6633, Shigella sonnei ATCC 11060. Gram-negative bacteria: Escherichia coli ATCC 2592, Pseudomonas aeruginosa ATCC 27583, Salmonella suis ATCC 13076. Fungus: Candida albicans ATCC 10231
Pathogenic microbial growth
For the antimicrobial activity test, the seven pathogenic microbes selected for this experiment was first cultured by adding a loop full of each of the desired microorganisms to tubes containing 9 ml of NB (Nutrient broth) then incubated at 37ÂÂ°C for 24 hours (Mutaz and Shahida. 2010) to form a working stock. The overnight cultures were used for the antimicrobial activity of the actinobacterial extracts obtained earlier in this study. In addition; MHA (Mueller Hinton Agar) media was prepared containing [38g Mueller Hinton agar (OXOID, UK) and 1L distilled water] (See appendix 1) and poured into petri dishes.