- Results Strain Test Control Titration Difference Td Enzyme Activity 44 6 Normal S 44 9 Aureus Resistant S 43 5 Aureu 1 (48.87 KiB) Viewed 65 times
- Results Strain Test Control Titration Difference Td Enzyme Activity 44 6 Normal S 44 9 Aureus Resistant S 43 5 Aureu 2 (48.87 KiB) Viewed 65 times
- Results Strain Test Control Titration Difference Td Enzyme Activity 44 6 Normal S 44 9 Aureus Resistant S 43 5 Aureu 3 (74.26 KiB) Viewed 65 times
- Results Strain Test Control Titration Difference Td Enzyme Activity 44 6 Normal S 44 9 Aureus Resistant S 43 5 Aureu 4 (56.9 KiB) Viewed 65 times
PROCEDURE (A flowchart for the assay is available in Figure 2) 1. Test the cultures of S. aureus and E. coli provided for the presence of B-lactamase as described below. In addition to the culture samples, you are provided with supernatant from an ultrasonicated culture of E. coli. Ultrasonication disrupts the bacterial cells and releases B-lactamase into the supernatant. Test all three samples for B-lactamase and compare the results obtained. The enzyme assay is based on the reaction of iodine with the penicilloic acid formed by the action of the B-lactamase on the lactam ring. lodine does not react with the intact penicillin molecule. For each sample: 1. Pipette 5.0 ml of benzyl penicillin (2.5 mg/ml in 0.1 M phosphate buffer pH 5.9) into each of two vials, labelled test and control. Place both vials in the 30°C waterbath. 4 tubes 2 At time zero, add 1 ml of culture sample to the test vial only and mix. 2 res. 2 sens. 3 Incubate at 30°C for 15 minutes. (N.B. a longer incubation period maybe required if the enzyme activity is low). 4. At the end of the incubation, add 5.0 ml of standard iodine (5mM) to both the test
and control vials. 5 Immediately after that, add 1 ml of culture sample to the control vial only (the control estimates iodine binding by the organic material present in the culture and medium). 6 Leave the vials to stand for 5 minutes to allow the iodine to react with the penicilloic acid. 7. Add a few drops of 1% starch solution before beginning the titration. Swirl the culture to mix completely. Starch gives a deep blue colour in the presence of free iodine. This colour will disappear when the endpoint is reached. & Titrate both samples (starting with the test vial) with standard sodium thiosulphate (5mm). The titration is considered complete once the sample turns colourless. 9 Calculate the B-lactamase activity of the sample in terms of moles penicillin degraded per hour per ml of culture. TD (units = mol/h"/ml-') Enzyme Activity = 6) **) 610**) ( Where TD T titration difference (ml) between test and control time of incubation (min) Volume of culture assayed (ml) V