Chapter # 3 Material and Method

The present study was conducted at the Microbiology & Molecular Biology Laboratory (MMBL), Institute of Pure and Applied biology, Bahauddin Zakariya Univesity, Multan, Pakistan. In this study ager overlay method is used to produce bacteriocins from E.coli strains against Staphlococcus aureus.

3.1 Study area and sample collection:-

The main focus of this study is to determine the effect of colicins produced from E.coli on the staphylococcus aureusinhibition. 

·         Sample size:-

Total 20 samples were collected.

·         Sampling technique:-

All culture was maintained on MacConkey’agar plates.

3.2 MacConkey Medium:-

3.2.1 Media preparation:-      

Powdered media (50g) was suspended in one liter of distilled or deionized water in a conical flask. Mixed well and heat with frequent agitation until media was dissolved. Sterilized in a autoclave at 121ºC(15 lbs) for 20 minutes. Media was allowed to cool at 46 - 48ºC and than 20 mL media was poured into petri dishes. Plates with media were allowed to solidified and later inverted the dishes to avoid excess moisture on the surface of the medium.

3.3 Isolation of specimens:-

Single colony of the E.coliwas isolated from all collected cultures and purified at MMBL,BZU on new MacConkey ager plates by single colony streaking techniques. Than the plates were incubated at 37ºC

3.4 Trypton soy broth preparation:-

Powdered trypton soy broth 300g was suspended in one liter of distilled or deionized water in a conical flask. Mixed well and then 2-3 mL of it was poured in to test tubes. Sterilized the test tubes in a autoclave at 121ºC (15 lbs) for 20 minutes. Broth was allowed to cool at 46 - 48ºC. Take a single colony of E.coli with a loop and then add this to the single test tube of broth. After that incubate it overnight.

3.5 Soft ager preparation:-

3 g of powdered trypton soy broth with 0.8g of ager was suspended in 100mL of distilled or deionized water in a conical flask. Mixed well and heat it with frequent agitation than 2-3 mL of it was poured in to test tubes.Sterilized the test tubes in a autoclave at 121ºC(15 lbs) for 20 minutes. Soft ager was thanallow to cool at 46 - 48ºC.

3.6 Trypton soy medium preparation:-

Powdered trypton soy broth of 3g with 1.5g of agar was suspended in one liter of distilled or deionized water in a conical flask. Mixed well and heat with frequent agitation until media was dissolved. Sterilized in aautoclave at 121ºC (15 lbs) for 20 minutes. Media was allowed to cool at 46 - 48ºC and then 20 mL media was poured into petri dishes. Plates with media were allowed to solidify and later inverted the dishes to avoid excess moisture on the surface of the medium.

3.7 Methodology:-

3.7.1 Ager overlay method:-

The test tube of trypton soy broth with E.coli strain was taken and with the help of pipet 100 micro liter was poured on the trypton soy medium plate. Than the soft ager was melt in boiling water with slightly agitation. When it melt than poured it on prepoured media plate. When the plate was solidified than after 20-30 minutestake the single colony of staphylococcus aureus and stab inoculated it on the plates. And incubate it at37ºC for 24hrs. After that we observed both the colicin was produced and the zone of inhibition occur around the staphylococcus aureus colony or not.

Mackey’s Media plate

Streaking technique of e coli

                                                          Trypton soy media

Trypton soy broth

Trypton soy broth with grown E.coli