MICROBIAL ANALYSIS
A.) Total microbial count
B.) Pathogen detection
A.) TOTAL MICROBIAL COUNT
- Pour plate method by serial dilution:
Take 2 test tubes and label them A & B and 6 petriplates (3 for SCDA &3 for SDA) and label the petriplates as 10ˉ¹, 10ˉ², 10ˉ³.
Pour 9ml buffer solution (NaCl / Peptone water) in each test tube.
Crush the tablets in pestle motar and make it in powder form.
Take 99ml of Soyabean Casein Digest Medium (Tryptone Soya Broth) in borosilicate glass bottle and add 1 gm of tablet powder sample to it to make 1:100 dilution and mix well.
Pipette out 1ml of this dilution with the help of sterile micropipette and open the lids of empty sterile petriplate labeled 10ˉ¹ and dispense into the middle of each petriplate aseptically and then close the lid of petriplate. Transfer 1ml of tablet sample sample to the test tube labeled A to make 1:10 dilution and mix well. Take 1ml of sample from test tube A and dispense aseptically into the petriplate labeled 10ˉ². Transfer 1ml of sample from test tube A to test tube B and mix well. Then take 1ml of sample from test tube B and dispense the sample into the petriplate labeled 10ˉ³. Remove the cotton plug from the flask of molten agar (SCDA & SDA) and pass the rim of opened flask through the flame of Bunsen burner. Open the lid of petriplate containing the sample and pour 20-25ml sterile Soyabean Casein Digest Agar (for TAMC) and Sabouraud Dextrose Agar (for TYMC) cooled at 45ºC into the petriplates. Close the lid of petriplates and mix thoroughly by gently swirling the plates & allow solidifying the media at room temperature.
Use aseptic technique throughout the procedure
Invert and incubate the SCDA plates at 30- 35ºC for 3-5 days (for TAMC) and SDA plates at 20-25ºC for 5-7 days (for TYMC). Also incubate the Soyabean Casein Digest Medium containing capsule sample for 24 hours at 30- 35ºC temperature for pathogen detection test.
After incubation examine the SCDA plates for total bacterial count and SDA plates for total yeast and mold count. Take maximum from the two plates and calculate the no. of CFU/ml of water sample.
B.) PATHOGEN DETECTION
- Detection of Escherichia coli:
Streak a loopful culture of enriched pre-incubated Soyabean Casein Digest Medium containing capsule sample on the surface of sterile MacConkey Aagr plate. Incubate the plates at 30-35 for 18-72 hours. After incubation presence of brick red colonies on MacConkey agar indicate the presence of E.Coli. Further confirmation is done by gram’s staining.
- Detection of Salmonella:
Transfer 0.1 ml of enriched Soyabean Casein Digest medium to 10 ml of Rappaport Vassiliadis
Salmonella enrichment broth and incubate at 30-35 for 24-48 hours. Growth of green colonies with blank centre and in 48 hours the colonies become uniformly black colonies surrounded by a dark zone and metallic sheen indicates the possibility of presence of Salmonella.
If subcultured on plate Xylose-Lysine Deoxycholate Agar and incubate at 30-35 for 24-48 hours. Well developed red colonies with or without black centre indicates the possibility of presence of Salmonella. Further confirmation is done by gram’s staining.
- Detection of Pseudomonas aeruginosa:
Streak a loopful culture of pre-incubated Soyabean Casein Digest Medium (containing capsule sample) on the surface of Cetrimide Agar medium and incubate at 30-35 for 18-72 hours. A greenish colored colony indicates the possibility of presence of P.aeruginosa. Carry out the further conformation by gram’s staining. Further confirmation done by oxidase test.
- Detection of Staphylococcus aureus:
Streak a loopful culture of pre-incubated Soyabean Casein Digest medium (containing capsule sample) on the surface of Mannitol Salt Agar plates. Incubate the plates at 30-35 for 18-72 hours. Examine the plates after incubation. If upon examination of incubated plates none of them contains yellow colonies with yellow zones the sample meets the requirements for the absence of Staphylococcus aureus. If growth occurs further confirm by streak the colonies on the surface of Baired Parker agar plates black, shinny colonies surrounded by clear zones indicates the presence of Staphylococcus aureus. Further confirm by gram’s staining.
- Detection of Asperigillus brasiliensis:
Streak a loopful culture of enriched fluid Soyabean Casein Digest medium on the surface of Sabooraud Dextrose Agar plates. Incubate the plates at 20-25 for 18-72 hours.
Growth of black color colonies may indicate the presence of Asperigillus brasiliensis.
Further confirm by microscopic examination.
- Test for Candida albicans:
Streak a loopful culture of enriched fluid Soyabean Casein Digest medium on the surface of Sabooraud Dextrose Agar plates. Incubate the plates at 20-25 for 18-72 hours.
Growth of white colonies may indicate the presence of Candida albicans.
