-Fusidic acid (FC, 10ug) - fusidic acid is a narrow spectrum bacteria used to treat the infection on skin, this also works by blocking protein synthesis in a bacteria
-Methicilin (MT, 10ug) - these antibiotics are not inactivated by enzymes and so they are used to treat which other penicillins are incapable of countering.
-Novobiocin (NO, 5ug)- NO is effective against some Gram-positive and negative bacteria, but it is no longer used and have no medical reference.
-Streptomycin(S 10ug)- is almost used for the treatments of tuberculosis. This antibiotic binds on bacterial ribosome by interfering with it and inhibiting protein synthesis.
-Tetracycline (T, 25ug) - this is broad spectrum antibiotic. T works by blocking protein synthesis and cell Wall in bacteria.
Apparatus
- lab coat
- Nutrient/Macconkeys agar plate
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Bottle/test-tube containing 10cm3 E.coli
- 1 antibiotic multi-test discs
- 1 spreader
- Bunsen burner
- Match stick
- labelling tape/marker
-
10cm3 alcohol
- Pipettes
- 1 forceps
- Tissue paper
- Disinfectant solution
Planning/pilot method
- Put on lab coat before collecting apparatus, to prevent bacteria contamination on your clothes
- Close the windows, to prevent any micro-organism entering through the windows
- Wipe the bench top with disinfectant solution before starting the experiment; so that any previous contamination of microbes is washed away.
- Take a nutrient/macconkeys agar plates and label them on the bottom with the name of agar in it, the date and initial of the investigator so that it can be identified.
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Light the Bunsen burner using match stick and adjust it to blue flame, this is the hottest and is done to make sure to avoid contamination. Always remember to work near the Bunsen burner again to avoid contamination.
- Open the bottle with little finger on the other hand to avoid placing the lid on the bench.
-
Flame the neck of the bottle containing E.coli, for a few seconds by passing the opening through a Bunsen burner. This is done to produce an upward flow of air from the bottle so that any organism in the area will not fall into the bottle.
- Flame the neck of the bottle before putting the lid on, because transferring micro-organisms from one medium to another, may cause unwanted contamination
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Use sterile syringe to transfer 0.2cm3 of E.coli and place it the centre of agar plate.
- Use spreader that has been sterilised by dipping it in alcohol in a bottle and then flame the spreader in a Bunsen burner and allow it to burn and wait till it cools down.
- Spread the bacteria (E.coli) over the agar with sterilised spreader, until the bacteria is well spread over the agar.
- Sterilise forceps by flaming it in Bunsen burner for few seconds.
- Then use the sterilised forceps to place the antibiotic multi-test disc in the agar plate until flatly placed.
- Put the prepared plate away
- Wipe the bench top with a disinfectant solution, to kill unwanted micro-organisms on the tops.
- Wash Hands thoroughly with disinfectant before leaving the laboratory, to prevent the possibly of carrying E.coli on hands.
- Leave it in oven for 2-3 days
- Then measure the area of no growth of the bacteria
Method
- A lab-coat was put on before collecting any of the apparatus, to prevent bacteria contamination on clothes.
- Windows were closed, in order to prevent any micro-organisms entering through the windows.
- On the top of the bench was wiped with disinfectant solution before starting the experiment, so that any previous contamination of microbes was washed away.
- Nutrient agar plates was collected and then labelled on the bottom with the name of agar in it, the date and initial of the investigator so that it can be identified.
- By using match stick, the Bunsen burner was light and adjusted it to blue flame; this is the hottest and is done to make sure to avoid contaminations. Worked near Bunsen burner throughout the practical, again to avoid contamination.
- A bottle containing E.coli was opened with little finger on other hand to avoid placing the lid on the bench
- The bottle is then flamed at the neck for few seconds, by passing the opening through the Bunsen burner. This is done to produce an upward flow of air from the bottle so that any organism in the area will not fall into the bottle.
-
A sterilised syringe was used to transfer 0.2 cm3 of E.coli and placed in the centre of the agar plate.
- A spreader that has been sterilised was used by dipping it in alcohol in a container and then the spreader was flamed over a Bunsen burner and allowed to burn and waited till it cooled down.
- The sterilised spreader was used to spread the bacteria (E.coli) over the agar in the plate, until the bacteria was well spread over the agar
- Forceps were sterilised by flaming it over Bunsen burner for few seconds
- The sterilised forceps were used to place the antibiotic multi-test discs in the agar plate until flatly placed.
- Then the prepared plate was put away
- The bench top was wiped with tissue paper, after disinfectant solution was sprayed on the top of bench in order to kill unwanted micro-organisms on the bench top.
- Hands were washed thoroughly with disinfectant soap before leaving the laboratory, to prevent the possibility of carrying E.coli on hands.
- The prepared plates were left in an oven for 2 days
Then finally the area of no growth was measured
Health and safety
- Bunsen burner must be carefully used, especially when sterilising equipments, for example spreaders are made from glass so it must not left long on Bunsen burner, because it can melt
- In the experiment alcohol is used to sterilise the spreader this may flame, so it must be carefully used and put away from the Bunsen burner and lid must be tightly closed when not in use.
- Lab coats are worn to prevent any exposure of agar on clothes.
- Hands must be washed with disinfectant, after the experiment is finished to avoid any later consumption of the bacteria.
Results/implementing
In the practical, I used a ruler to measure the diameter of area of no growth for each dish (plate) and in order to get the average of the dishes, I added the readings of dish 1 and dish 2 together and multiplied the results of both dishes with 2 ( D1 + D2) and I recorded the results.
The pie and bar charts below contain the results average results of dish 1 and dish 2
Analysis/ Discussion
In the table of results above, erythromycin (E), tetracycline (T) and streptomycin (S) worked the best, all with average results above (4.00cm3), Erythromycin with average result of (4.40 cm3), tetracycline with average result of (4.25cm3) and streptomycin with average result of (4.20cm3) This could be the fact that streptomycin and tetracycline are broad spectrum, which means they can kill wide range of bacteria whether they are gram-positive or gram-negative. Novobiocin and fusidic acid also worked with the E.coli, novobiocin with average result (3.47cm3) and fusidic acid with average result (3.37cm3).
Variable
Novobiocin (NO) is effective against some Gram-positive and negative bacteria, although it is no longer in use and has no medical reference, but the fact that it works effectively against Gram-negative (such as E.coli used in the practical) could be why it worked with E.coli. Fusidic acid is a narrow spectrum but it worked with E.coli better than chloramphenicol which is broad spectrum antibiotic with average result of (2.85cm3). Methicilin didn’t work well on E.coli with average result of (1.40), this could the fact that methicin is a type of penicillin. And in this practical penicillin didn’t work at all, because penicillin works only with Gram-positive bacteria and the bacteria used in this practical E.coli is a Gram-negative and that’s why penicillin couldn’t work with E.coli at all.
There isn’t much difference between the readings of dish 1 and dish 2, because of the controlled variables between the two dishes, for example Novobiocin of dish 1 is 3.45cm3 and dish 2 is 3.50cm3 with difference of only 5cm3 and I presented the difference between the dishes. The difference between the dishes could be caused by human source of error, for example the clear zone of dish 2 overlapped and I used a ruler to measure the diameter of each dish instead of area.
My hypothesis which was that all antibiotic will kill bacteria effectively, did proved wrong in this practical, but I also said this will depend on the type of bacteria used for example Gram-positive and Gram-negative, since I used only Gram-negative bacteria in the practical, all broad spectrum antibiotics work well in the practical and Generally the average readings of broad spectrum antibiotics is 3.69cm3 and while average readings of narrow spectrum antibiotics is 2.29cm3, there is a difference of 1.40cm3, this shows that on whole that broad spectrum antibiotics is more effective with E.coli than the narrow spectrum and this proved my hypothesis.
Evaluation
I think my results are reliable and can be used by anyone, except the penicillin with anomalous result with (0.00cm3) and this could be caused the fact that penicillin only kills Gram-positive bacteria and E.coli is Gram-negative bacteria. I result of penicillin will change if I used gram-positive bacteria such as micrococcus.
I chose to measure the diameter of the clear zone instead of area, because the antibiotic tags had perfect circle of clear zone and measurable, but if I had irregular circles I would have measured area of clear zone using colony counter pen, which measure an area accurately.
Concentration of E.coli used in practical was unknown and haemocytometer could have been used to measure the concentration of the E.coli. This could be why some broad spectrum antibiotics didn’t work as expected like chloramphenicol, because may be the E.coli used was less concentrated, and may caused the inaccuracy of results from broad spectrum antibiotics would work better with bacteria. May be if the concentration of E.coli increased and the whole practical is repeated again, the results may change and antibiotics such as chloramphenicol with average result of 2.85cm3 would kill bacteria better.
In the experiment of some antibiotics overlapped, and because of this it was difficult to measure the area of no growth accurately and it could be measurements were done more than 24 hours. This can be avoided by placing each antibiotic disc on separate plate, so the overlapping of antibiotics will be well avoided.
May be I didn’t place the antibiotic multi-test disc in the agar plate flatly, and so the antibiotics didn’t had good contact with the E.coli.
Generally this practical can be improved with following extensions
- One could have looked at different types of bacteria, for example one could have used gram-positive bacteria and used penicillin instead of gram-negative bacteria.
- An effective antibiotic out of the eight antibiotics used in practical, could be used e.g. tetracycline which was most effective to the E.coli.
- One can also use two different types of bacteria for example gram-positive bacteria and gram-negative bacteria and compare them to find out which one is resistant or susceptible to antibiotics.
- Different antibiotics may work with different incubator temperature and may be if the temperature of the incubator is increased antibiotics such as chloremphenicol may work better with the E.coli.
Bibliography
Thomas.D.Brock, Michael .T. Madigan and Jack parker
Chapter 9 pages 349-355,
Biology of microbiology
Taylor, Stout and Green,
Chapter 15
Biology 1 & 2