Production of a Standard Curve Between Absorbance and Direct Bacteria Account - Lab Report Example

Protocol for the production of a standard curve between Absorbance, and direct bacteria account: Materials and procedures First, prepare 6 nutrient broth cultures by adding 20 ml of nutrient broth to each of 6 of the centrifuge tubes. 2- Secondly, add a colony of E. coli to each of three of the centrifuge tubes. However, it is important to aseptic a loop by flaming before transferring a colony of E. coli to centrifuge tube each time. 3- Next, add a colony of staphylococcus aureus to each of three of the centrifuge tubes. However, it is important aseptic a loop by flaming before transferring a colony of to staphylococcus aureus centrifuge tube each time. 4- After that, incubate at 37C in a shaker incubator for 24 to 48 hours. So that bacteria would be in log phage. 5- Then, put 6 cultures in Centrifuge Machine and spin 20 minutes at 3000 RPM. 6- Afterward, remove 15 ml supernatant, leaving 5 ml in each tube. 7- Pool the three E. coli cultures in one tube. Also, pool the staphylococcus aureus cultures in one tube. 8- Put spectrophotometer to 595nm. 9- Add 1m of nutrient broth to plastic curvette and blank spectrophotometer with nutrient broth. 10- Add 1 ml E. coli culture to plastic curvette and record at 595nm. 11- Still to do dilutions by adding nutrient broth until the number arrive around 750 and recorded at 595nm. 12- Still to do dilution for the last number to be recorded (750) by adding nutrient broth until the number arrive around 550 to 450 and recorded at 575 nm. 13-To find next dilution, take 0.4 and divide by the last number do you have for example, 550 and multiply by 3 (0.4/ 0.55  3) and the number do you have minus from 3 and the number is nutrient broth. 14- Repeat the dilation in concentration 0.3, 0.2, 0.1, 0.05 15- Repeat with S. aureus and record at 595 nm. 16- Put all the tubes on ice because it holds bacterial growth. 17- Make 3 dilutions 1:10², 1:10³ and 1:10 by adding 900μl distilled water and 100μl from concentration. Then add 100μl from 1:10² to 1:10³. Next, add 100μl from 1:10³ to 1:10 ∆ 1.0 = 10²,10,10,10,10,10,10 0.8= 10,10,10 0.5=10,10,10 0.4=10³,10, 10 0.3=10³, 10, 10 0.2=10², 10³, 10 0.1=10, 10², 10³ 0.05=10, 10², 10³ 18- After that, add 50μl to nutrient agar plate and spread in the plate by using sterile glass spreader. 19- Afterward, for each concentration you have three nutrient agar plates. 20- Incubate at 37C for 24 hours. 21- Next, count the number of colonies on the plate. (If the numbers on plate is more than 50 colonies, record as "To many to count" and if less than 50 colonies record in lab book. 22- Then, take the average for the colonies for each dilution. 23- To work out CFU for each culture concentration, use the dilution that gives highest accurate number. For example, (1:1 is 96.5 colonies, the 1:10 is 11 colonies and 2:10 as the highest CFU/ ml. 24- To obtain CFU/ml= CFU  dilution factor  20 25- Plot a curve on Excel program to describe the bacterial growth curve from the absorbance values obtained. Methods: MIC (Measure Inhibition Zone) 1- Prepare eight nutrient broth cultures by adding 25 ml of nutrient broth to each of eight of the centrifuge tubes. 2- Add a colony of E. coli ATCC 15766 to each of four of the centrifuge tubes. 3- Add a colony of staphylococcus aureus ATCC 157293 to each of four of the centrifuge tubes, 4- Incubate the colony at 37C in a shaker incubator to culture them overnight. Therefore, bacteria would be in log phage. 5- Spin cultures at 6000 PRM for 10 minutes. 6- Remove supernatant, leaving 5 ml in each tube. 7- Resuspend pellet in the remaining supernatant. 8- Add 1ml of sterile nutrient broth to disposable plastic curvette and blank spectrophotometer. 9- Add 1ml E.coli culture to plastic curvette and record A600 (make sure A600 ˃ 0.5). 9- Make dilution of silver compounds (WI, WII, WIII, WIV, W V) 1PPM, 0.1PPM, 0.01ppm, 0,001 PPM, 0,0001PPM. The final volume is 3ml. I Ag+= 2.3PPM→ 3 ÷ 2.3= 1.304 Ag+ (1.695 water). II Ag+= 5.8PPM→ 3 ÷ 5.8= 0.517 Ag+ (2.482 water). III Ag+= 2.4 PPM→ 3 ÷ 2.4= 1.25 Ag+ (1.75 water). IV Ag+ = 5.4PPM→ 3 ÷ 5.4= 0.555 Ag+ (2.444 water). V Ag+ = 7.0 PPM→ 3 ÷ 7.0 = 0.428 Ag+ (2.571 water). 10- Soak four filters in each solution. 11- Spread 100μl of culture onto nutrient Agar plates. 12- Remove excesses liquid from filters and place on plate. 13- Allow to dry for 5 minutes. 14- Incubate overnight at 37C. 15- Measure inhibition zone. Results, The result was positive, because the S. aureus and E.coli grow around the filter paper, so the measure inhibition zone for each compound of sliver with different concentration is zero. As a result, it means that 1PPM of each compound of silver does not work for killing bacteria. Autoclave &non-autoclave: There are 2 method of silver delivery A- Silver spread in the plate ( autoclave & non-autoclave): 1- Prepare eight nutrient broth cultures by adding 25 ml of nutrient broth to each of eight of the centrifuge tubes. 2- Add a colony of E. coli ATCC 15766 to each of four of the centrifuge tubes. 3- Add a colony of staphylococcus aureus ATCC 157293 to each of four of the centrifuge tubes. 4- Incubate the colony at 37C in a shaker incubator for overnight culture. Therefore, bacteria would be in log phage. 5- Spin cultures at 6000 PRM for 10 minutes. 6- Remove supernatant, leaving 5 ml in each tube. 7- Resuspend pellet in the remaining supernatant. 8- Add 1ml of sterile nutrient broth to disposable plastic curvette and blank spectrophotometer. 9- Add 1ml E.coli culture to plastic curvette and record A600 (make sure A600 ˃0.2). 10- Add 1ml S.aureus culture to plastic curvette and record A600 (make sure A600 ˃0.2). 11- Make 1 ppm solution of each compound (autoclave &non-autoclave). I Ag+= 2.3PPM→ 3 ÷ 2.3= 1.304 Ag+ (1.695 water). II Ag+= 5.8PPM→ 3 ÷ 5.8= 0.517 Ag+ (2.482 water). III Ag+= 2.4 PPM→ 3 ÷ 2.4= 1.25 Ag+ (1.75 water). IV Ag+ = 5.4PPM→ 3 ÷ 5.4= 0.555 Ag+ (2.444 water). V Ag+ = 7.0 PPM→ 3 ÷ 7.0 = 0.428 Ag+ (2.571 water). 12- spread 100μl of each onto plate. 13- also spread 100μl water (-ve control). 14- for each compound + water on one plate spreads 50 μl of 0.2 OD culture. 15- incubate at 37 and see if ther any growth after 24/48 hours. B- Silver with culture: ( autoclave & non-autoclave): 1- Make over night culture for E.coli & s.aureus. 2- Make 0.5 OD stock of culture. 3- Make Ag+1ppm final concentration on 0.2 O.D culture for E.coli and S.aureus. For E. coli : (0.2 ÷0.506)500= 197.6 μl 217.3 Ag+ (197.6 culture) (85.1water). =2.3 ÷I Ag+= 2.3 ppm→500 Ag+ (197.6 culture) (216.2 water).86.2 =5.8 ÷II Ag+= 5.8 ppm→500 .3 Ag+ (197.6 culture) (94.1 water). 208 =2.4 ÷III Ag+= 2.4 ppm→500 92.5 Ag+ (197.6 culture) (209.9 water). =5.4 ÷ IV Ag+= 5.4 ppm→500 71.4 Ag+ (197.6 culture) (231.0 water). =7.0 ÷ V Ag+= 7.0 ppm→500 For S.aureus : (0.2 ÷ 0.505)500= 198.0 μl 217.3 Ag+ (198.9 culture) (84.7water). =2.3 ÷I Ag+= 2.3 ppm→500 Ag+ (198.0 culture) (215.8 water).86.2 =5.8 ÷II Ag+= 5.8 ppm→500 .3 Ag+ (198.0 culture) (93.7 water). 208 =2.4 ÷III Ag+= 2.4 ppm→500 92.5 Ag+ (198.0 culture) (209.5 water). =5.4 ÷ IV Ag+= 5.4 ppm→500 71.4 Ag+ (198.0 culture) (230.6 water). =7.0 ÷ V Ag+= 7.0 ppm→500 4 - Leave it for one hour. 5- Spread 50μl on agar plate. 6- Incubate at 37 and see if there is any growth after 24/48 hours. Result: For E.coli and S.aureus the result is positive because bacteria grow on the plate. So, the silver does not work. Materials and method: (liquid cultures assays): - 1- Prepare eight nutrient broth cultures by adding 25 ml of nutrient broth to each of eight of the centrifuge tubes. 2- Add a colony of E. coli ATCC 15766 to each of four of the centrifuge tubes. 3- Add a colony of staphylococcus aureus ATCC 157293 to each of four of the centrifuge tubes, 4- Incubate at 37C in a shaker incubator for overnight culture. Therefore, bacteria will be in log phage. 5- Spin cultures at 6000 PRM for 10 minutes. 6- Remove supernatant, leaving 5 ml in each tube. 7- Resuspend pellet in the remaining supernatant. 8- Add 1ml of sterile nutrient broth to disposable plastic curvette and blank spectrophotometer. 9- Add 1ml E.coli culture to plastic curvette and record A600 (make sure A600 ˃ 0.5). 10- For each Ag+ compound (WI, WII, WIII, WIV, and WV) and +Ve control take 10 ml dilution. 11- Prepare 17 autoclave from liquid culture tubes 0.13 g from nutrient broth. The final volume is 10 ml. 12- Dilate each concentration on Ag+ until 1ppm WI Ag= 2.3ppm → 10 ÷ 2.3 = 4.3 Ag (5.7 water). WII Ag= 5.8ppm →10 ÷ 5.8 = 1.7 Ag (8.3 water). WIII Ag= 2.4ppm → 10 ÷ 2.4 = 4.2 Ag (5.8 water). WIV Ag= 5.4ppm → 10 ÷ 5.4 = 1.9 Ag (8.1 water). WV Ag= 7.0ppm → 10 ÷ 7.0= 1.42 Ag (8.6 water). 13- Add 50 ml of E.coli foe each different compounds of Ag+. 14- Add 50 ml of S. aureus foe each different compounds of Ag+. 15- For each compound and control, make 1:1, 1:102, 1:103for 0 time, 4 time. 16- For each compound and control, make 1:102, 1:103, 1:104, 1:105, 1:106 for 24 hours time interval. 17- Do time serious at the effective concentration and take simple at 1 hour, 4 hours and 24 hours. 18- Spread plate. 50 μl of circled dilutions above on nutrient agar plates. 19- Spread 50 μl of – ve control (no bug) controls. 20- Incubate over night culture at 37 C. 21- Count colonies. 22- To obtain CFU/ml= CFU20dilution factor. Results For 0 time and 4 times, the results are positive because the E.coli and S.aureus grow and it is difficult to account colonies on the plate. small piece of silver 10 ppm A small piece of 7ppm silver has been collected from the Department of Chemistry. Cut it into 4 small pieces and prepare plates and spread 50μl on E.coli and S.aureus and put each small piece in one plate and incubate for 24 hours at 37C. Result: The bacteria growth has been shown in all 4 plates. MIC (Measure Inhibition Zone) 1ppm is not sufficient; therefore, try using high concentration for each compound of silver. There should be WII, WIII, WIV, WV and WVII there. WVII is an equivalent of WI only done on a different date and should have the same starting concentration WI. There were no sufficient WI stocks left to concentrate so exchanged it for WVII. Start with volume (150ml) and multiply with starting concentration by the number. WI= WVII(2.3ppm) 150 ÷ 14.5= 10.34, 10.342.3= 23.8ppm. WII (5.8ppm) 150 ÷ 22= 6.8, 6.85.8= 39.54ppm. WIII (2.4ppm) 150 ÷ 37.5= 4, 42.4= 9.6ppm. WIV (5.4ppm) 150 ÷ 14.4= 10.41, 10,415.4= 56.24ppm. WV(7.0ppm) 150 ÷ 25.4= 5.905, 5.9057.0= 41.33ppm Try doing the filter paper experiment again with higher concentrations of silver: try using the highest concentration for all 5 silver solutions: 20ppm, 10ppm, 5ppm.only make up 2ml of solution to immerse filter paper in. So, the solutions stretch further. From the solutions you put your filter papers in, the filters wont absorb it all, take 50μl and spread it on a plate, let it dry and then spread E. coli and S. aureus. WI= WVII(2.3ppm) 20÷ 23.7=0.4802= 1.68Ag+( 0.32 water). WII (5.8ppm) 20÷ 39.4= 3.44 2= 0.51Ag+( 1.49 water). WIII (2.4ppm) 5 ÷9.6 =0.52 1 = 0.52 Ag+( 0.48 water). WIV (5.4ppm) 20÷ 56.2=0.3552= 0.71Ag+( 1.29 water). WV(7.0ppm) 20÷ 41.3=0.482= 0.96Ag+( 1.04 water). Incubate overnight at 37C. - Measure inhibition zone for filter paper and see if there is any growth after 24 hours for spread plate. Result: - For MIC Measure inhibition zone The bacteria growth has been shown on all inhibition zones except WVII & WV for E.coli and S. aureus. For E. coli:- concentration 20ppm 10ppm 5ppm WVII 2ml 1 ml 0.5 ml WV 2ml 1 ml 0.3 ml For S.aureus:- concentration 20ppm 10ppm 5ppm WVII 2ml 1 ml 0.5 ml WV 2ml 1 ml 0.3 ml For spread plate The bacteria growth for all plates in all Ag+ compounds on 20ppm, 10ppm, 5ppm. MIC & spread plates Silver Nitrate (108 PPM AgNo3):- 1- Culture S.aureus and E.coli overnight and incubate at 37C. 2- Dilute AgNo3 until 20ppm, 10ppm, 5ppm and 1ppm. 3- Make 2ml final volume from AgNo+3 (2ml= 370ml AgNo+3 and 1630ml water). 4- Immerse filter paper in different dilutions. 5- Spread 100μl of culture on to nutrient Agar plates. 6- Remove excesses liquid from filters and place on plate. 7- Allow them to dry for 5 minutes. 8- The filters wont absorb it all, take 50μl and spread it on a plate, let it dry, and then spread E. coli and S. aureus 9- Incubate overnight at 37C. - 10- Measure inhibition zone for filter paper and see if there any growth after 24 hour for spread plate. For spread plate The bacterial growth for AgNO+3 on 20ppm, 10ppm, 5ppmand 1ppm. References Humberto H. Lara, 2009, “Bactericidal effect of silver nanoparticles against multidrug resistant bacteria” Retrieved November 5, 2009 from http://www.springerlink.com/content/g32341574x6q1k51/ Read More