478
B. Bairagi and D. Nath
treated; Group 4: normal + thermal wound (untreated); Group 5: normal + thermal
wound + SSD treated; Group 6: normal + thermal wound + AgNPs treated. Silver
nanoparticle was given at the rate of 15 mg/kg body weight one time to each mouse.
Thermal wound was developed with hot water. The wounds were covered with
proper dressing prepared by SSD ointment and different concentrations of silver
nanoparticles. After 5th, 10th, and 15th days of treatment, the skin samples were
collected for histological analysis and PCR technique to see the cytokine levels
(Bhagavathy and Kancharla 2016).
2.6 Reverse Transcription and Amplification
Skin tissue was homogenized and processed for isolation of RNA. Reverse transcription and real-time polymerase chain reaction were done by preparing the complementary cDNA using TaqMan reverse transcription with MultiScribe reverse transcription as per manufacturer’s protocol. Then the reaction mixture was taken to
thermal cycling in a real-time PCR system with the conditions: after incubating with
hexamer at 25 °C for 12 min, the reverse transcription was continued for 58 min at
37 °C. Then the temperature was increased to 95 °C for 6 min to stop the reaction
and to get the final cDNA product. cDNA product was stored at −20 °C until used
for amplification.
2.7 Antibacterial Activity Testing
Wound swab was collected and bacterial colonies were cultured on nutrient agar
plates. Nutrient agar was mixed with water (28 gm/L) and then slightly heated to
dissolve the agar. The dissolved agar was poured into the Petri dishes. After 3–4 h, the
agar plates were ready to culture the bacterial samples. After inoculating the plates
with bacterial samples, the plates were kept in BOD incubator at 37 °C. Within 24–
48 h, bacterial colony formation was observed. After proper growth of the bacterial
colonies on agar plates, each type of colony was isolated and separately cultured on
agar medium. To get a pure culture of each bacterial sample, 3–4 agar plate transfer
was done. After getting pure culture, all the bacterial samples were identified by
16S rDNA-based molecular technique. DNA was isolated and evaluated on agarose
gel. DNA sample was amplified by PCR technique and then subjected to Sanger
sequencing. Then bidirectional DNA sequencing reaction of PCR amplicon was
carried out. Consensus sequence of 16S rDNA was generated from forward and
reverse sequence data using proper software. The 16S rDNA sequence was used to
carry out BLAST alignment search tool of NCBI Genbank database.
The bacterial colonies on agar plates were used to measure the zones of inhibition
with synthesized nanoparticles in comparison with antibiotics and standard synthetic
drugs.
B. Bairagi and D. Nath
treated; Group 4: normal + thermal wound (untreated); Group 5: normal + thermal
wound + SSD treated; Group 6: normal + thermal wound + AgNPs treated. Silver
nanoparticle was given at the rate of 15 mg/kg body weight one time to each mouse.
Thermal wound was developed with hot water. The wounds were covered with
proper dressing prepared by SSD ointment and different concentrations of silver
nanoparticles. After 5th, 10th, and 15th days of treatment, the skin samples were
collected for histological analysis and PCR technique to see the cytokine levels
(Bhagavathy and Kancharla 2016).
2.6 Reverse Transcription and Amplification
Skin tissue was homogenized and processed for isolation of RNA. Reverse transcription and real-time polymerase chain reaction were done by preparing the complementary cDNA using TaqMan reverse transcription with MultiScribe reverse transcription as per manufacturer’s protocol. Then the reaction mixture was taken to
thermal cycling in a real-time PCR system with the conditions: after incubating with
hexamer at 25 °C for 12 min, the reverse transcription was continued for 58 min at
37 °C. Then the temperature was increased to 95 °C for 6 min to stop the reaction
and to get the final cDNA product. cDNA product was stored at −20 °C until used
for amplification.
2.7 Antibacterial Activity Testing
Wound swab was collected and bacterial colonies were cultured on nutrient agar
plates. Nutrient agar was mixed with water (28 gm/L) and then slightly heated to
dissolve the agar. The dissolved agar was poured into the Petri dishes. After 3–4 h, the
agar plates were ready to culture the bacterial samples. After inoculating the plates
with bacterial samples, the plates were kept in BOD incubator at 37 °C. Within 24–
48 h, bacterial colony formation was observed. After proper growth of the bacterial
colonies on agar plates, each type of colony was isolated and separately cultured on
agar medium. To get a pure culture of each bacterial sample, 3–4 agar plate transfer
was done. After getting pure culture, all the bacterial samples were identified by
16S rDNA-based molecular technique. DNA was isolated and evaluated on agarose
gel. DNA sample was amplified by PCR technique and then subjected to Sanger
sequencing. Then bidirectional DNA sequencing reaction of PCR amplicon was
carried out. Consensus sequence of 16S rDNA was generated from forward and
reverse sequence data using proper software. The 16S rDNA sequence was used to
carry out BLAST alignment search tool of NCBI Genbank database.
The bacterial colonies on agar plates were used to measure the zones of inhibition
with synthesized nanoparticles in comparison with antibiotics and standard synthetic
drugs.