Dew and acacia gum preparation 

Dew drops  is water in the form of water droplets that appears on thin, Dew drops are collected in the early morning from the alfalfa plant, collected by a sterile towel and squeezed into a sterilized glass bottle. Some elements, salinity, alkalinity and acidity are estimated. Glassware is kept in the refrigerator at a temperature until use in the experiments. Mixing for (Nat.ag.) is calculated amount of acacia gum with a size of dew drops to form an anti-bacterial paste. Concentration of dew preparation was used to 32 to 624uml/ml., also, for acacia gum was cleaned from impurities and then washed with sterile distilled water, and concentration of AG preparation was used from 32 to 624ug /ml. this solution was termed as natural agents of AG and incubated at 5 °C for 24h, The solution was centrifuged at 2500 x g for 20min at 5 °C.

Bacteria and Culture Conditions

Waterborne and foodborne bacterial isolate P. aeruginosa was collected from the wastewater treatment station, Egypt. P.aeruginosa is gram negative bacteria (G-) and aerobic (and at times facultative anaerobic), rod-shaped bacterium with unipolar motility. It has been identified as an opportunistic pathogen of both humans and plants. P.aeruginosa isolate was cultured in TTC tergitol 7 agar (solid media) for 24 hours at 37˚C and cultured in nutrient broth (liquid media) with continuous agitation at 100 rpm.  P. aeruginosa grown at 37°C for 24 hours in nutrient broth (NB) with  shake-flasks at 60 rpm and TTC7with tregetol agar  for solid media. 1ml of bacterial broth was removed, and bacteria were washed twice at 6000rpm for 3 minutes in deionized water to remove any growth media. 20 µl of the bacterial suspension was added to 1 ml of the relevant solution in the presence or absence of natural agent (0.5–10%). Samples were then incubated for 20 min before being subjected to further washing and centrifugation at 6000 x g for 6 min to remove excess natural agents for sizing, and morphology analyses.

Minimum inhibition and bactericidal concentration on P. aeruginosa

For the MIC and MBC assayed, MDAG was serially diluted with P. aeruginosa cell suspension (10⁷ CFU/ml) and was incubated at 37 °C overnight. 2 fold serial dilutions prepare of natural agents up to 7 row and last row negative control (NC) without bacteria in microdilution plate. Put the inoculum a few colonies from an agar plate P.aeruginosa, preparing a Mac Farland standard, and then diluting this standard into media. Distribute P.aeruginosa into the microdilution plate with serial dilution test of dew and acacia and incubate the microdilution plate. Plate a portion of each well on an agar media, incubate the agar and check for colonies to determine MBC and read the microdiluation plate to determine MIC value.

Evaluation of Antibacterial Activity of natural agents

Antibacterial activity of dew, Acacia gum and mixed dew with acacia gum (MDAG) against pathogenic bacteria was determined using the High Throughput Spot Culture Growth Inhibition Assay (HT-SPOTi) as described by (Danquah et al. 2016). Briefly, a double serial dilution of a stock solution of the test extracts and fractions (1000 μg/mL in 2% dimethyl sulphoxide, DMSO) was done in a PCR half-twisted plate to give a concentration range of 7.8–500 μg/mL. Each dilution aliquots (100 μL) were dispensed into corresponding wells of a 96-well plate and mixed with 200 μL of molten agar. The plates were stirred and spotted with 2 μl of a standard microbial suspension of P.aeruginosa. The plates were stand and closed for 20 min, and incubated at 37 °C for 24 h. The presence or absence of growth was determined by visual inspection compared to control wells.

Biofilm Inhibition Assay

The effect of natural agents separate and MDAG on biofilm formation by  P. aeruginosa was investigated using the microplate crystal violet stain retention method ( Abidi et al. 2014]. P. aeruginosa cultures prepared in nutrient broth (NB) were used for the assay. The test samples were solubilized in DMSO (2%) and reconstituted in NB to achieve a concentration range of 32–624 μg/mL. Aliquots of the microbial culture (180 μL) and the test samples (20 μL) were pipetted into a flat bottom 96-well microtiter plate and incubated at 37°C for 24 hours. Negative controls were included in the plate; the experiment was performed in triplicate. After microtiter plate incubation, the supernatant was captured and each well was washed with phosphate buffer saline (pH 7.2) to remove capture cells. The biofilms were fixed by drying at 50°C for 30 minutes and stained with 0.1% crystal violet aqueous solution for 10 minutes at 25° C. The wells were carefully washed with sterile water and the stain bound to cells solubilized with 95% ethanol. Absorbance was measured at 600 nm using an automated microplate reader. The mean absorbance was determined, and the percentage inhibition of biofilm was calculated as:

Biofilm inhibition% = ABS (control)-ABS(test)/ABS (control) *100         (Eq1)

For the biofilm formation quantification of viable cells assayed in plastic micro plates, this method was investigated by (Stepanovic et al. 2004). A 20 μl portion of broth culture of P.aeruginosa was added to each well of a 24-well plate which was incubated aerobically, with mild agitation at 70 rpm, for 72 h at 37 °C. Every 12 h, the bacterial cell replaced by fresh media nutrient broth supplemented by2% sucrose. Negative controls were obtained by incubating the micro plates with media without inoculum. After supernatant media removed , the biofilm was treated with AG and MDAG 64, 128, 256, 312 and 624 μg/ml. but,  in case of  treated with dew to bacterial biofilm concentration was 64, 128, 256, 312 and 624 μl/ml, at room temperature and without agitation. Same concentrations of natural agents were used as positive control. Following treatment, the extract was removed and the wells were gently washed twice with sterilized distilled water.