Study area and sample processing. Five strawberry samples were collected from different super shop markets available in Dhaka city during the time span of January 2016 to February 2016. After aseptical collection of samples, they were transferred immediately to the laboratory for microbial analysis by following the procedures proposed by American Public Health Association. For both microbiological analysis and antimicrobial activity, strawberry samples were processed separately as skin and core. The skin and core separately were homogenized in normal saline followed by serial dilution up to 10-6.
Microbiological analysis. 0.1 ml suspension of strawberry skin suspension and core suspension was introduced onto nutrient agar (NA) and Sabouraud dextrose agar (SDA) media for enumerating total viable bacteria (TVB) and Total Fungal Count (TFC), respectively. For identification and enumeration of specific pathogenic bacteria both skin and core suspension was added and spread onto MacConkey agar, mannitol salt agar (MSA) and cetrimide agar media which can detect Escherichia coli, Staphylococcus spp. and Pseudomonas spp., respectively. SDA plates were incubated at 25oC for 48 to 72 hours. The other plates were incubated at 37oC for 24 hours. Eschericha coli or Klebsiella spp. can be identified by distinguishable pink colony on MacConkey agar, Staphylococcus spp. by their yellow colony on MSA and Pseudomonas spp. by their greenish colored colony on cetrimide agar.
Analysis for the presence of VBNC bacteria. For detection of Salmonella spp. 01 ml homogenized sample of strawberry skin and strawberry core was inoculated into 9 ml selenite cystine broth (SCB) for enrichment at 37oC for 6 hours. For detection of Vibrio spp., both samples from skin and core were inoculated into 9 ml alkaline peptone water (APW) by following the same procedure as for Salmonella spp. After enrichment, the SCB and APW media containing strawberry samples were serially diluted up to 10-6. After that samples were inoculated onto Salmonella-Shigella (SS) agar and Thiosulfate Citrate Bile Salt Sucrose (TCBS) agar and incubated at 370C for 24 hours. Black centered colonies on the SS agar is indicative for the presence of Salmonella spp. and the large, yellow colonies on the TCBS agar is indicative for the presence of Vibrio spp. Biochemical tests were performed to ensure the identity of the isolates.
Study of antibiotic resistance of the isolates. To determine the antibiotic susceptibility of the detected isolates from the strawberry samples, normal saline was subjected to add fresh isolates to get bacterial suspensions. After appropriate turbidity was attained, these suspensions were inoculated and spread over the Muller- Hinton agar plates using cotton swabs. Then some selected antibiotic discs were placed and incubated at 37oC for 24 hours to get the zone of inhibition. Antibiotics used in this study of antibiotic resistance traits, are, Ampicillin (10µg), Amoxicillin (10µg), Nalidixic acid (30µg), Erythromycin (15µg), Chloramphenicol (10µg), Gentamicin (10µg), Kanamycin (30µg), Streptomycin (10 µg), Vancomycin (30µg), Ciprofloxacin (5µg), Ceftriaxone (30µg).
Determination of antibacterial activity of strawberry. To determine the antibacterial activity of the strawberry samples, agar well diffusion method was used. At first some selected bacterial isolate (Pseudomonas spp, Bacillus spp, Salmonella spp, E. coli, Staphylococcus spp., Listeria spp., Klebsiella spp. and Vibrio spp.) were taken to make their suspensions in normal saline. Then the bacterial suspensions were introduced evenly onto Mueller-Hinton agar (MHA) media using cotton swab. Then holes were made using a cork borer on the MHA to introduce strawberry samples in it with a positive (antibiotic disc-Gentamicin -GEN, 10µg) and negative control (normal saline). After incubation at 37oC for 24 hours, plates were observed for the presence of clear zone indicating the antibacterial activity.