Prophylactic potential of a Panchgavya formulation against certain pathogenic bacteria

Panchgavya preparation

The Panchgavya formulation used in this study was prepared using a method that was different from the one practiced traditionally (which yields a fermented preparation). Fresh cow dung and urine, sourced from a cow fed on cottonseed and sugarcane grass, were mixed thoroughly in a glass beaker. This mix was allowed to stand for 10 min and subjected to filtration through a muslin cloth (the traditional method does not involve filtration). To this filtrate, fresh cow’s milk and fresh curd was added, and mixed until a uniform mixture was formed. Finally, cow ghee was added to this mixture and mixed thoroughly.

Dung, urine, and milk were all sourced from a single cow. From the same batch of milk, curd and ghee were prepared. Cream of this milk was boiled for 30–40 min and filtered; the filtrate was taken as ghee. For curd preparation, one part of this milk was inoculated with previous batch of curd (prepared using milk from the same cow by adding few drops of lemon juice to the milk) followed by overnight incubation at room temperature.

The ratio of dung:urine:milk:curd:ghee in this preparation was 1:2:3:3:1. This Panchagavya mixture was then transferred to a copper vessel (covered with a muslin cloth) and allowed to rest for 30 min. This was followed by freeze-drying at -20 °C to convert the preparation in powder form, which was stored under refrigeration (4–8°C) until used for the microbiological experiments. When required for use, the Panchgavya powder was suspended in sterile distilled water to attain OD₆₂₅ = 0.10±0.01.

Test bacteria

Pathogenic bacteria used in this study included: Staphylococcus aureus (MTCC 737); beta-lactamase producing multidrug resistant strains of Chromobacterium violaceum (MTCC 2656) and Serratia marcescens (MTCC 97); multidrug resistant Pseudomonas aeruginosa; and Streptococcus pyogenes (MTCC 1924). P. aeruginosa was sourced from our internal culture collection. All other cultures were procured from MTCC (Microbial Type Culture Collection, Chandigarh, India).

In vivo assay

C. elegans worms (received gift from the Biology Division, Sophia College, Mumbai) maintained on NGM (Nematode Growing Medium; 3 g/L NaCl, 2.5 g/L peptone, 1 M CaCl₂, 1 M MgSO₄, 5 mg/mL cholesterol, 1 M phosphate buffer of pH 6, 17 g/L agar-agar; this medium was prepared by us using the listed ingredients purchased from Merck, Mumbai or HiMedia, Mumbai) agar plate with E. coli OP50 (LabTIE B.V., JR Rosmalen, the Netherlands) as food, were kept unfed 24h prior to being used for experiments.

These worms were fed with Panchgavya by mixing this formulation (100 µL) with M9 medium (800 µL) and placed in a 24-well plate (sterile, non-treated polystyrene plates; HiMediaTPG24) containing 10 worms per well. Duration of exposure of worms to Panchgavya was kept 24, 48, 72 or 96 h, followed by addition of pathogenic bacteria (100 µL of bacterial suspension with OD₇₆₄= 1.50). Appropriate controls i.e. worms previously not exposed to Panchgavya, but exposed to pathogenic bacteria; worms exposed neither to Panchgavya nor bacteria; and worms exposed to Panchgavya, but not to bacterial pathogens, were also included in the experiment. Incubation was carried out at 22°C.

Number of live vs. dead worms were counted every day for 5 days by putting the plate (with lid) under a light microscope (4X). Straight worms were considered to be dead. Plates were gently tapped to confirm lack of movement in the dead-looking worms. On the last day of the experiment, when plates could be opened, their death was also confirmed by touching them with a straight wire, wherein no movement was taken as confirmation of death.

Statistical analysis

Values reported are means of four independent experiments, whose statistical significance was assessed using t-test performed through Microsoft Excel (2013). P values ≤0.05 were considered to be statistically significant.