Potato dextrose agar medium

The impact of NPs on the mycelial growth of a specific pathogen was established using the mycelium inhibition technique on potato dextrose agar (PDA, 2%) with certain modifications [23 ]. To make the final concentration of conjugated nanoparticles as 0.5, 1.0, and 1.5 ppm, autoclaved potato dextrose agar medium (Hi-Media, Mumbai, India; temp. 40 °C) was placed onto separate sterile Petri plates (90 mm × 15 mm) and allowed to harden. A 5.0 mm mycelial disc was removed from a seven-day-old test pathogen culture and placed in the center of the test Petri dish, where it was incubated at 28 ± 1 °C under constant monitoring. Growth was measured after four days for three replications, and the treated plates were compared to the control plates (without nanocomposites) to calculate the percent suppression of mycelia by NPs using an earlier approach [24 (link)].

where dc is the control mycelial diameter and dt is the mycelial treatment diameter.

Antimicrobial screening is an important method of analysis of the inhibitory effects of compounds against microorganisms [64 (link)]. There are a few laboratory methods available to evaluate the antimicrobial activity of a compound. The agar dilution or disc diffusion method is the most common [65 (link)]. Antimicrobial screening of ZnO NPs (50 µl dose and concentration 150 µg disc⁻¹) was assessed against various bacterial and fungal strains. Three gram-positive bacterial strains such as Staphylococcus aureus (cars-2), Bacillus megaterium (BTCC-18) and Bacillus cereus (carsgp-1), as well as two fungal strains, namely Aspergillus niger (carsm-3) and Trichoderma harzianum (carsm-2), were used in the agar well diffusion method, similar to our previous report [66 (link)]. Mueller–Hinton agar (HiMedia, India) was used to form an agar medium to culture bacteria and potato dextrose agar medium (HiMedia, India) was used to culture fungal strains. As standards, Ceftriaxone (10 µl) was used for bacterial strains and amphotericin-B (10 µl dose and 50 µg disc⁻¹) was used for fungal strains [66 (link)]. After placing the sample in a culture medium, the discs were incubated for 24 h at 37°C for bacteria and 48 h at 26°C for fungi. The antimicrobial activity was determined by measuring the zone of inhibition (ZOI).

The LCEO (By Samia^®, Cotia, Brazil) was acquired in a market located in Maringa City, Parana State, Brazil, in a single lot (number: 217). Ethanol was purchased from Synth^® (Diadema, Brazil), and potato dextrose agar medium (PDA) was obtained from Himedia^® (Mumbai, India). β-CD, Tween-80, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), L-glutamine, phosphate-buffered saline (PBS) and dimethyl sulphoxide (DMSO) were obtained from Sigma-Aldrich^® (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Invitrogen^® (New York, NY, USA). All reagents were of analytical grade.

In this study, as-purchased
analytical grade chemicals were used in the experimentation process.
Zinc chloride (Thomas Baker (Chemicals) Pvt. Ltd.) purity 98%, zinc
acetate dihydrate (Sisco Research Lab (SRL)), zinc nitrate hexahydrate
(Merck life sciences pvt.ltd.), diethylene glycol (Sisco Research
Lab (SRL)), silver nitrate (Thomas Baker (Chemicals) Pvt. Ltd.) purity
99.8%, sodium hydroxide pellets (ACROS organics) and methylene blue
(Sisco Research Lab (SRL)), rose bengal (Molychem), and potato dextrose
agar medium (Himedia, Mumbai, India) were commercially purchased.
In this study, all the solutions were prepared in double distilled
water (DDW).

Sustainable production of camptothecin was investigated in the two highest yielding endophytes isolated in the study. Freshly isolated strains (stored as 25% glycerol stocks) were streaked separately as slants made of potato dextrose agar medium (5 ml) (HiMedia, Mumbai) and incubated at 28 °C for 7 days with an initial pH of 5.6. These culture slants were used as inoculum for generating suspension and were considered as the first generation (subculture cycle) slants in the study. After 7 days, single mycelia from the first generation slant was picked and streaked on to the new slants with fresh medium (5 ml of potato dextrose agar) under similar conditions. These were considered as the second generation slants. Similarly, subsequent generation slants (up to 12th) were created after every 7 days of subculture on to fresh medium.
Suspension cultures from each generation slants were initiated as described earlier. After 7 days of growth, the slant-wash with 5 ml of saline (0.9% NaCl) was used as inoculum (2% v/v) for suspension culture. After 8 days of the cultivation period, the shake-flask cultures were harvested (in triplicate) for the estimation of biomass and camptothecin production.