Determining the effects of Erythromycin on CHO and Ltk- cell lines.
Antibiotics are a type of antimicrobial used in the treatment and prevention of bacterial infection. They may either kill or inhibit the growth of bacteria. Antibiotics are an important aspect of medicine as they can treat both mild conditions such as acne as well as potentially life-threatening conditions such as pneumonia (Voet et al 2012). Examples of antibiotics include Hygromycin, Tetracycline, Penicillin and Erythromycin.
The aim of this experiment was to see the effects of the antibiotic Erythromycin on two different cell lines. The cytotoxicity of this antibiotic was also evaluated. The cell lines used in this experiment were Chinese hamster ovary (CHO) cells and Mouse L cells lacking thymidine kinase (Ltk-).
Erythromycin is in the macrolide class group of drugs and is useful for the treatment of a number of bacterial infections including infections of the Streptococcus, Staphylococcus, and Haemophilus species. It has a similar antimicrobial spectrum to Penicillin and is often prescribed to patients who have an allergic reaction to Penicillin. Erythromycin is a bacteriostatic antibiotic that seems to inhibit growth of bacteria at high concentrations (Voet et al 2012). It works by binding to the 50S subunit of bacterial 70S rRNA complex which then inhibits protein synthesis (Griffiths et al 2000). Structural and functional deformities lead to the bacteria not replicating thus leading to inhibition of growth.
Chinese hamster ovary (CHO) cells are a cell line derived from the ovary of the Chinese hamster. CHO cells are one the most common mammalian cell lines used in industrial production of therapeutic antibodies and are used to study genetics, toxicity screening and gene expression (NCBI 2010). In this experiment, CHO cells were used to test the cytotoxicity, which is the quality of being toxic to cells, of Erythromycin. Mouse L cells lacking thymidine kinase (Ltk-) (NCBI 1997), were also used in the experiment so a comparison could be made between the effects of Erythromycin on the two different cell lines. The data book value of the toxic concentration of Erythromycin on these cell lines is 5µM (5×10^-6 M).
My hypothesis is that both cell lines will show no growth at Erythromycin concentrations of 5µM and above as this concentration is toxic to these cells. Concentrations ranging from 1-8µM of the antibiotic were used on both cell lines to test the effectiveness and cytotoxicity of the antibiotic. My prediction is that at 1-4µM concentrations of the antibiotic, the effectiveness of the antibiotic preventing bacterial growth in the cell cultures will increase in ascending order. I also predict that there will be no cell growth at 5-8µM as this concentration is toxic to the cells. Finally, a control was also used to see if the effects of the antibiotic are truly significant. This control was growing cell cultures only in growth media with no antibiotic present. I predict that there will be minimum to no growth in this culture as it will be vulnerable to bacterial infections.
Pilot study.
A pilot study was carried out first to find out which dilution of cells to use in the real experiment. The target was to aim for around 50,000 cells per well as this would give a significant confluence of cells to measure and observe the effectiveness, if any, of the antibiotic.
Firstly, the cells were counted. This was done using a haemocytometer and a microscope. 100µl of each cell lines were placed in to two separate labelled tubes. 100µl of Trypan blue dye was then added to both samples. After mixing, 100µl of the samples were loaded into the haemocytometers and observed through the microscope. Viable and dead cells were counted on all four corners of the haemocytometer and an average was taken of the viable cells.
CHO cell count.
| Corner square | Viable cells | Dead cells | Total cells |
| 1 | 36 | 0 | 36 |
| 2 | 42 | 1 | 43 |
| 3 | 40 | 2 | 42 |
| 4 | 45 | 0 | 45 |
| Mean | 41.5 |
Table 1: table showing cell count of viable and dead CHO cells.
CHO cells dilution calculation:
41.5 X (1×104) X 2 = 830,000 cells/ml
1/6 dilution is 8×105/6 = 130,000 cells/ml
1/12 dilution is 8×105/12 = 60,000 cells/ml
1/24 dilution is 8×105/12 = 30,000 cells/ml
After observing cell cultures of all three dilutions under an inverted microscope, the 1/12 dilution which showed 60,000 cells/ml was chosen as this produced the best colony and confluence of CHO cells.
Ltk- cell count.
| Corner square | Viable cells | Dead cells | Total cells |
| 1 | 28 | 0 | 28 |
| 2 | 29 | 2 | 31 |
| 3 | 24 | 0 | 24 |
| 4 | 35 | 4 | 39 |
| Mean | 30.5 |
Table 2: table showing cell count of viable and dead Ltk- cells.
Ltk- cells dilution calculation:
30.5 X (1×104) X 2 = 600,000 cells/ml
1/6 dilution is 6×105/6 = 100,000 cells/ml
1/12 dilution is 6×105/12 = 50,000 cells/ml
1/24 dilution is 6×105/12 = 25,000 cells/ml
After observing cell cultures of all three dilutions under an inverted microscope, the 1/12 dilution which showed 50,000 cells/ml was chosen as this produced the best colony and confluence of Ltk- cells.
Methods
Stage 1- Setting up the well plates.
- 1ml of cell culture was mixed with 5ml growth media in a tube to produce a 1/6 dilution.
- 5ml of this 1/6 dilution tube was taken and mixed with 0.5ml growth media in a separate tube. This tube is the 1/12 dilution that is required.
- 1ml of this 1/12 dilution was added to each well of 4×6 well plate. (9 wells required including control.)
- The required amount of Erythromycin was then added to each well. (Calculations and distribution of antibiotic shown below.)
- Finally, growth media was added to all wells to take the total volume in each well to 2ml.
- (Control plate only contained cells and growth media, no antibiotic.)
Distribution of antibiotic concentration:
Figure 1: diagram showing the distribution of antibiotic concentration in each well
Calculation of Erythromycin stock solution:
734g/l = 1M
734mg/ml = 1M
0.734mg/ml = 1mM
3.67mg/5ml = 1mM
0.367mg/0.5ml = 100µM (This was used as the stock solution.)
| Concentration of antibiotic stock solution | Well no. | Volume of antibiotic solution added (µl) | Volume of growth media added (µl) | Concentration | Final concentration after adding to wells that already contain 1ml of solutions |
| 10 µM | 1 | 200 | 800 | 2 µM | 1 µM |
| 10 µM | 2 | 400 | 600 | 4 µM | 2 µM |
| 10 µM | 3 | 600 | 400 | 6 µM | 3 µM |
| 10 µM | 4 | 800 | 200 | 8 µM | 4 µM |
| 10 µM | 5 | 1000 | 0 | 10 µM | 5 µM |
| 100 µM | 6 | 120 | 880 | 12 µM | 6 µM |
| 100 µM | 7 | 140 | 860 | 14 µM | 7 µM |
| 100 µM | 8 | 160 | 840 | 16 µM | 8 µM |
Table 3: table showing how desired antibiotic concentrations were derived from the stock solution.
All steps mentioned above were carried out four times in total to produce duplicates for both cell lines. Plates were stored away for a week.
Stage 2- Staining the well plates and observing cell lines.
- Observed each well under the inverted microscope, noting down the confluency and appearance of the cells.
- Inverted the plates over the sink to remove the growth medium from all the wells and washed cells gently using distilled water.
- Added crystal violet stain to each well and after waiting 5 minutes, tipped the plates over and washed gently with water again.
- Observe plates under the inverted microscope again, noting down appearance of the cells.
Stage 3- Measuring absorbance values
- 100µl was taken from each well, including all duplicates, and inserted into a 96-well plate.
- Plate inserted into a spectrophotometer which gave out absorbance values for each well.
Results
As seen in Table 4, confluency of CHO cells in all plates were at 100%, meaning all wells were covered with CHO cells at maximum coverage. When further observing the well plates using a computer linked to the inverted microscope, it could be seen on the image displayed that there was in fact 100% coverage, as shown in Figure 2. Both replicates of the CHO cell line showed maximum coverage across all Erythromycin concentrations tested. Both control values also showed 100% confluence unexpectedly.
The Ltk- cell line, however, did produce a variety of cell confluences after being treated with different concentrations of Erythromycin. As seen in Tables 6 and 7, Ltk- cell confluency at 5µM of Erythromycin, which was deemed toxic, had confluences of 0-10%. However, at 6-8µM of Erythromycin, which is above the toxic concentration, had greater cell growth than at 5µM. Figure 4 shows Ltk- cells that have lost shape and are deforming at this toxic concentration. Whereas, in Figure 3, at 2µM antibiotic concentration the cells seem to have their shapes intact.
The graph in Figure 5 further proves that CHO cell lines were at 100% coverage as absorbance values were a lot greater than for those of Ltk- cell lines. This is because intensity of the staining is proportional to the number of cells still present in the wells.
CHO cell line 1:
| Antibiotic conc. in well (µM) | Confluency (%) | Amount of cells visible |
| 0 (control) | 100 | Lots of cells |
| 1 | 100 | Lots of cells |
| 2 | 100 | Lots of cells |
| 3 | 100 | Lots of cells |
| 4 | 100 | Lots of cells |
| 5 | 100 | Lots of cells |
| 6 | 100 | Lots of cells |
| 7 | 100 | Lots of cells |
| 8 | 100 | Lots of cells |
Table 4: table showing effects of different antibiotic concentrations on CHO cells.
CHO cell line 2:
| Antibiotic conc. in well (µM) | Confluency (%) | Amount of cells visible |
| 0 (control) | 100 | Lots of cells |
| 1 | 100 | Lots of cells |
| 2 | 100 | Lots of cells |
| 3 | 100 | Lots of cells |
| 4 | 100 | Lots of cells |
| 5 | 100 | Lots of cells |
| 6 | 100 | Lots of cells |
| 7 | 100 | Lots of cells |
| 8 | 100 | Lots of cells |
Table 5: table showing effects of different antibiotic concentrations on CHO cells.
Figure 2: image displayed on computer showing 100% confluency of CHO cells which was unexpectedly found in all wells.
Ltk- cell line 1:
| Antibiotic conc. in well (µM) | Confluency (%) | Amount of cells visible |
| 0 (control) | 100 | Lots of cells |
| 1 | 80 | Many cells |
| 2 | 60 | Not as many cells |
| 3 | 80 | Many cells |
| 4 | 60 | Not as many cells |
| 5 | 10 | Very few cells |
| 6 | 50 | Few cells |
| 7 | 40 | Few cells |
| 8 | 60 | Not as many cells |
Table 6: table showing effects of different antibiotic concentrations on Ltk- cells.
Ltk- cell line 2:
| Antibiotic conc. in well (µM) | Confluency (%) | Amount of cells visible |
| 0 (control) | 100 | Lots of cells |
| 1 | 80 | Many cells |
| 2 | 60 | Not as many cells |
| 3 | 70 | Many cells |
| 4 | 40 | Few cells |
| 5 | 0 | No cells |
| 6 | 20 | Very few cells |
| 7 | 40 | Few cells |
| 8 | 50 | Few cells |
Table 7: table showing effects of different antibiotic concentrations on Ltk- cells.
Figure 3: image displayed on computer showing the well containing 2µM of Erythromycin that found around 60% confluency of Ltk- cells.
Figure 4: image displayed on computer showing the well containing 5µM of Erythromycin that found around 10% confluency of Ltk- cells.
Figure 5: graph showing absorbance levels for both cell lines at different concentrations of antibiotic.
Discussion
The experiment clearly did not work for the CHO cell line as all wells of this cell line had maximum cell growth. This could suggest that the antibiotic Erythromycin is ineffective on this cell line. Or that the cultures have acquired bacterial infections instead. Other aspects that could have gone wrong for this part of the experiment include; not growing the cell cultures properly, not using the right dilution of cells and not using the right concentration of Erythromycin. In Figure 5, absorbance values were relatively high for all CHO cell cultures grown. This is because the intensity of staining left in the wells after removing all the solutions, reflects the number of cells that have adhered on to the walls of the wells. To improve this limitation, the experiment should be repeated for the CHO cell line to investigate if an error has occurred or if Erythromycin is indeed ineffective on CHO cell lines.
The results show that Erythromycin has been effective on Ltk- cell lines. Bacterial growth was inhibited in these cell cultures due to the fact that cell confluence increased as antibiotic concentration also increased up until the toxic concentration of 5µM. As seen in Tables 6 and 7, for both duplicates, Ltk- cells were significantly more confluent at 1-4µM of antibiotic concentrations compared to 5-8µM. This proves that Erythromycin is toxic towards Ltk- cells at concentrations of 5µM and above. This is further backed up by the comparison of Figures 3 and 4 as the cells look much healthier at the lower concentration of 2µM. At 5µM, the cells look to be degrading and losing components. This may be because the antibiotic has interrupted with protein synthesis of the cells and could have also damaged structural and functional properties of the cells, leading to cell death or less replication (Strachan et al 2010). This aspect proves how crucial it is to get the concentration of antibiotics used absolutely right in terms of using antibiotics as treatments for bacterial infections.
The experimental design of this investigation was good as it allowed for a valid comparison for the effects of Erythromycin. However, the results produced were not as expected to be. To improve this design, the range of different concentrations of antibiotics used could be increased i.e. using higher and lower concentrations as well as testing at smaller intervals i.e. testing the effects at 2.5-3.5µM of antibiotic. Also, other antibiotics such as Penicillin and Tetracycline could be used to compare the effectiveness of these antibiotics against Erythromycin. On the other hand, other cell lines can also be investigated to investigate if the effects of Erythromycin is different for different cell lines.
To conclude, the investigation was partially successful as some valid results were managed to be obtained. Calculating antibiotic concentrations required in the experiment proved difficult when having to factor in the dilutions already present inside the wells, but was successful in the end. Not getting decent results for the CHO cell line proved to be the only bad aspect of the experiment as it meant the aim of the experiment was not fully met. Overall, however, the investigation went well and it was found that Erythromycin was effective by preventing bacterial growth on Ltk- cell cultures. Also, it was found that an Erythromycin concentration of 5µM is indeed toxic to cell lines.
Word count: 1959 words
References
Griffiths, A., Miller, J., Suzuki, D., Lewontin, R., and Gelbart, W. (2000) An Introduction to Genetic Analysis. 7th edn. New York: W. H. Freeman
National Center for Biotechnology Information (2010) Cell engineering and cultivation of chinese hamster ovary (CHO) cells [online] available from < http://www.ncbi.nlm.nih.gov/pubmed/20210750>
National Center for Biotechnology Information (1997) Infection and Immunity [online] available from <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC421006/> [8 April 2015]
Strachan, T., Read, A. (2010) Human Molecular Genetics. 4th edn. New York: Garland Science, Taylor and Francis group LLC
Voet, D., Voet, J., and Pratt, C. (2012) PRINCIPLES OF BIOCHEMISTRY. 4th edn. Hoboken: Wiley
PAGE 1
Comment 1
And many more
Comment 2
I would put this paragraph in later
Comment 3
Good
Comment 4
You should ref er to the article you have looked at not to NCBI
Comment 5
Third person
PAGE 2
Comment 6
Third person
The prediction
Comment 7
We are not looking at bacterial growth!!
Comment 8
You don’t have to mention all of it in the introduction a short general hypothesis is ok.
The rest can be described in more detail I the results and discussion
Comment 9
This could go into the methods and the outcome in the results
Comment 10
Methods
PAGE 3
Comment 11
Methods
Comment 12
Results
Comment 13
Results
PAGE 4
Comment 14
The concentration of erythromycin used in the dif f erent wells
Comment 15
Term
PAGE 5
Comment 16
To detailed just mention that all the dif f erent erythromycin concentrations were done in
duplicates
Comment 17
Why bold?
Comment 18
You have to mention that the cells were f ixed with 4% f omaldehyde
Comment 19
Crystal violet was extracted f rom the wells using 200 ul of Ethanol
Comment 20
Transf ered
PAGE 6
Comment 21
Conf luency
Comment 22
Put the tables that show the results next to the text
Comment 23
Showed dif f erent cell densities
Comment 24
Show table here
Comment 25
Had changed morphology
Comment 26
What do you mean with def orming describe the morphology
Comment 27
No morphology change could be observed
Comment 28
Of viable cells that take up the stain
Comment 29
Conf luency is suf f icient
Comment 30
Formatting
PAGE 7
Comment 31
Objective used?
Comment 32
This interpretation goes not into the f ig legend
Comment 33
Again that is not a scientif ic value lots of cells etc.
Suf f icient to mention conf luency
PAGE 8
Comment 34
See comments above conf luency tell a cell biologist everything
Comment 35
Objective?
Image of LTK cells exposed to 2uM erythromycin af ter crystal violet stain using a 40x
objective
PAGE 9
Comment 36
Add the objective that has been used
Comment 37
Axis label
Comment 38
What are the numbers? Well number mention erythromycin concentrations or explain the
concentration in the f igure legend
Comment 39
Axis lable
PAGE 10
Comment 40
You could argue that CHO cell are less sensitive to erythromycin to give it a scientif ic
meaning
Comment 41
Why would you think this?? Did you notice any bacterial growth? Also the cells would
probably dead af ter 48hours if they would have been cotaminated
Comment 42
If you have a 100% conf luency your cells have been happily growing and the erythromycin
had not an ef f ect on them. That is you result and that does not mean that your experiment
went wrong!!
Comment 43
This could be the only reason of all the ones mentioned above.
Comment 44
Phrasing
Comment 45
You were not looking at bacterial growth you were looking if erythromycin had a toxic ef f ect
on cells!!!
Comment 46
Here you get you are looking at the toxic ef f ect on cells
Comment 47
Conf irmed, scientif ic writing
Comment 48
Cell morphology has changed
Comment 49
Any ref erences f or this statement??
PAGE 11
Comment 50
Phrasing also other cell lines could be used
Comment 51
Phrasing
Comment 52
Why do you think so, either they are not as sensitive to erythromycin or you did not add it
Comment 53
No it showed a cytotoxic ef f ect on LTK cells, no bacteria involved in the whole project!!
PAGE 12
Comment 1
And many more.
Comment 2
I would put this paragraph in later
Comment 3
Good
Comment 4
You should refer to the article you have looked at not to NCBI
Comment 5
Third person
Comment 6
Third person
The prediction
Comment 7
We are not looking at bacterial growth!!
Comment 8
You don’t have to mention all of it in the introduction a short general hypothesis is ok.
The rest can be described in more detail I the results and discussion
Comment 9
This could go into the methods and the outcome in the results
Comment 10
Methods
Comment 11
Comment 12
Results
Comment 13
Results
Comment 14
The concentration of erythromycin used in the dif f erent wells
Comment 15
Term
Comment 16
TO DETAILED JUST MENTION THAT ALL THE DIF F ERENT ERYTHROMYCIN CONCENTRATIONS WERE DONE IN
DUPLICATES
Comment 18
You have to mention that the cells were f ixed with 4% f omaldehyde
Comment 19
Crystal violet was extracted f rom the wells using 200 ul of Ethanol
Put the tables that show the results next to the text
What do you mean with def orming describe the morphology
No morphology change could be observed
Of viable cells that take up the stain
Conf luency is suff icient
Comment 31
Objective used?
Comment 32
This interpretation goes not into the f ig legend
Again that is not a scientif ic value lots of cells etc.
Suf f icient to mention conf luency
See comments above conf luency tell a cell biologist everything
Image of LTK cells exposed to 2uM erythromycin af ter crystal violet stain using a 40x
objective
Add the objective that has been used
Axis label
What are the numbers? Well number mention erythromycin concentrations or explain the
concentration in the f igure legend
Comment 39
Axis lable
You could argue that CHO cell are less sensitive to erythromycin to give it a scientif ic
meaning
Why would you think this?? Did you notice any bacterial growth? Also the cells would
probably dead af ter 48hours if they would have been cotaminated
If you have a 100% conf luency your cells have been happily growing and the erythromycin
had not an ef f ect on them. That is you result and that does not mean that your experiment
went wrong!!
This could be the only reason of all the ones mentioned above.
Phrasing
You were not looking at bacterial growth you were looking if erythromycin had a toxic ef f ect
on cells!!!
Here you get you are looking at the toxic ef f ect on cells
Conf irmed, scientif ic writing
Cell morphology has changed
Any ref erences f or this statement??
Phrasing also other cell lines could be used
Why do you think so, either they are not as sensitive to erythromycin or you did not add it
No it showed a cytotoxic ef f ect on LTK cells, no bacteria involved in the whole project!!
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