This experiment was done to learn proper way of using aseptic technique and sterilization by isolating pure culture of bacterial. Afterwards, the bacterial cells in a sample and their optical density were determined.
First of all, various sterilization methods were introduced. Sterilization is important in a sense that it ensures there is absolutely no contamination in the glassware or apparatus used in the lab. Different sterilization methods are used for different materials. One of them is autoclaving. Autoclaving machine uses high-pressure steam to sterilize and therefore, heat resistant plastics, glass or solutions can be sterilized by autoclaving. As the temperature of the steam is above 100 oC, the organisms cannot survive. Second sterilization method is radiation. As heat sensitive plastics does not have resistance to heat, autoclaving cannot be used and these are often sterilized by using radiation such as UV, gamma-ray or X-ray. The last method is filter sterilization. Some solutions are heat labile, and to sterilize these kind of solutions, filter sterilization can be used. This technique uses the fact that microorganism is around 5micrometer by 1micrometer, and if the filter has a smaller diameter, microorganisms cannot pass through the filter.(1)
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In part B, aseptic technique is learned. This technique prevents any kind of contamination while handling the glassware or transferring. To be more specific, it prevents any contaminant to be introduced in the area of interest. The first step of this technique involves wiping the lab bench with 70% ethanol, which would kill most microorganisms. Then, Bunsen burner is turned on, and the movement of the air goes upwards. Therefore, it minimizes the chance of microorganisms landing on the media of interest. In addition, briefly heating glass tube mouths and minimizing the time of opening lids minimizes contamination.(1)
Using aseptic technique, streaking technique was used to isolate single colonies. To do this, a pure culture of the target microorganism is taken. Then, with an inocular loop, which is flamed with Bunsen burner until red hot, it cooled down. Afterwards, take a bit of pure culture with the loop and streak lines in the medium. The streaking lines should not cross each other to avoid too much diluting.
After streaking, colonies are grown. To count the number of cells, viable cell count method is used. Viable count is only useable with singles colonies and not bacterial lawns. Therefore, in order for cell to have single colonies, appropriate dilution of the bacteria is necessary. The dilution helps for spreading of the cells on the agar. For this, serial dilution, which was introduced last project can be useful. Then, the number of viable cells can be obtained by counting the number of colonies that have developed multiplied by the respective dilution factor. (2)
Material and Methods:
All procedures are performed according to the BIOL 368 lab manual (Concordia Biology Department 2013) except for the following modifications: for the contamination part, we used shoe, finger, E. coli, and E. coli with 70% ethanol.
Results:
Colony isolation by streaking
First of all, the color of the bacteria in all the plates are thick beige colored. In streak 1, extremely small and many colonies were observed. The size of the colonies were very small, they were circular, opaque and smooth. There are 123 colonies. Streak 2 shows chain of bacterial formation, but the number of the colonies is decreased from streak 1. The number of colonies were 60. They were larger than the colonies in streak 1, opaque, circular and smooth as well. In streak 3, single colonies are observed. None of them was huge, but they were larger than the colonies from streak 2. They were opaque, circular and smooth as well. About 9 colonies were observed. In the 4th streak, no single colony was observed. As a result, single colonies of a pure E. coli strain was successfully isolated.
Viable count
Table 1. Raw data of viable count of my group.
Dilution
10-4
10-5
10-6
Number of colony
Too many
Too many
252
Viable count (cfu/ml)
–
–
2.52 x 109
Sample calculation:
Viable count at 10-6 dilution:
Since the plate, -6, has 252 colonies which is in the range of 100-300, I picked the plate to calculate cfu/ml.
252 x 10/10-6 =2.52 x 102 x 107 =2.52 x 109 cfu/ml
Table 2. Raw data for viable count for all section
Dilutions
Colony count
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8
Group 9
Group 10
Section 1
10-4
lawn
lawn
lawn
lawn
lawn
lawn
lawn
　
lawn
lawn
10-5
360
lawn
1848
lawn
1028
2168
696
　
3040
1646
10-6
78
287
441
270
234
347
363
　
300
306
Section 2
10-4
920
>300
Lawn
Lawn
Lawn
>300
>300
Lawn
Lawn
Lawn
10-5
249
>300
590
>300
Lawn
>300
>300
406
>300
189
10-6
23
231
189
269
384
222
265
154
180
108
Section 3
10-4
too many
>1000
too many
too many
too many
too many
too many
too many
too many
too many
10-5
too many
>1000
too many
too many
too many
too many
too many
too many
544
too many
10-6
194
420
258
252
295
217
240
197
79
224
Table 3. Viable count for all section data (for 10-6 dilution)
Section
Group
Number of colonies
Cfu/ml
01
1
78
7.8.E+08
2
287
2.87.E+09
3
441
4.41 E+09
4
270
2.70 E+09
5
234
2.34.E+09
6
347
3.47E+09
7
363
3.63E+09
8
–
–
9
300
3.00E+09
10
306
3.06E+09
Max
441
4.41E+09
Min
78
7.8 E+08
Average
291.8
2.92 E+09
Standard Dev.
94.40
2.67E+08
02
1
23
2.3.E+08
2
231
2.31.E+09
3
189
1.89.E+09
4
269
2.69.E+09
5
384
3.84.E+09
6
222
2.22.E+09
7
265
2.65.E+09
8
154
1.54.E+09
9
180
1.80.E+09
10
108
1.08.E+09
Max
384
3.84.E+09
Min
23
2.3.E+08
Average
202.5
2.02.E+09
Standard dev.
93.09
9.31E+08
03
1
194
1.94.E+09
2
420
4.20.E+09
3
258
2.58.E+09
4
252
2.52.E+09
5
295
2.95.E+09
6
217
2.17.E+09
7
240
2.40.E+09
8
197
1.97.E+09
9
79
7.9.E+08
10
224
2.24.E+09
Max
420
4.20.E+09
Min
79
7.9.E+08
Average
237.6
2.38.E+09
Standard Dev.
81.55
8.16.E+08
Max
441
4.41.E+09
Min
23
2.3.E+08
Average
242.3
2.42.E+09
Standard Deviation
96.75
1.15E+09
Sample calculation for STD DEV. (section 1):= 94.40
Bacterial cell count by optical density
Table 4. Cell density for My Group:
OD600 of diluted cultures (Au)
E. Coli Count of diluted Culture (cells/ml)
Original Culture (cells/ml)
Me
0.427
2.18×108
2.18×109
Partner
0.436
2.14×108
2.14×109
E. Coli Count of diluted Culture: 0.202 x (5x 108) = 1.01×108
Original Culture: 1.01×108 x 10 = 1.01×109
Table 5. Raw OD600 values for all sections (unit: Au)
Group
Section 1 (1)
Section 1 (2)
Section 2 (1)
Section 2(2)
Section 3(1)
Section 3 (2)
1
0.389
0.383
0.359
0.371
0.358
0.365
2
0.368
0.369
0.247
0.447
0.345
0.408
3
0.364
0.343
0.344
0.360
0.323
0.335
4
0.374
0.374
0.390
0.338
0.427
0.436
5
0.415
0.430
0.386
0.368
0.320
0.247
6
0.359
0.357
0.302
0.350
0.379
0.352
7
0.347
0.372
0.354
0.369
0.391
0.364
8
–
–
0.362
0.361
0.358
0.328
9
0.370
–
0.35
1.018
0.350
0.341
10
0.737
0.367
0.368
0.353
0.413
0.322
(>1.96 therefore outlier)
Table 6. Diluted Cell Density for all sections (unit: cells/ml)
Group
Section 1 (1)
Section 1 (2)
Section 2 (1)
Section 2(2)
Section 3(1)
Section 3 (2)
1
1.95E+08
1.92E+08
1.80E+08
1.51E+08
1.79E+08
1.90E+08
2
1.84E+08
1.85E+08
1.86E+08
1.75E+08
1.83E+08
1.76E+08
3
1.82E+08
1.72E+08
1.24E+08
1.77E+08
1.73E+08
1.96E+08
4
1.87E+08
1.87E+08
2.24E+08
1.85E+08
2.04E+08
1.82E+08
5
2.08E+08
2.15E+08
1.72E+08
1.81E+08
1.62E+08
1.79E+08
6
1.80E+08
1.79E+08
1.80E+08
1.81E+08
1.68E+08
1.64E+08
7
1.74E+08
1.86E+08
1.95E+08
1.75E+08
2.14E+08
1.75E+08
8
1.69E+08
5.09E+08
2.18E+08
1.71E+08
9
1.85E+08
1.93E+08
1.84E+08
1.60E+08
2.07E+08
10
1.87E+08
1.84E+08
1.84E+08
1.77E+08
1.24E+08
1.61E+08
Average
1.87E+08
1.95E+08
1.79E+08
Min
1.72E+08
1.24E+08
1.24E+08
Max
2.15E+08
5.09E+08
2.18E+08
Standard Deviation
1.05E+07
7.43E+07
2.13E+07
Sample calculation:
Section 1 group1 student 1: Cell Density = 0.389 x (5x 10^8) = 1.95 x107
Section 1 Average:
Average = ((1.95+1.84+1.82+1.87+2.08+1.80+1.74+1.85+1.87+1.92+1.85+1.72+1.87+2.15+1.79+1.86+1.84) x 108)/ 17 = 1.87 x108
Table 7. Diluted Cell Density for all sections, class analysis
Class (cells/ml)
Average
1.87E+08
Minimum
1.24E+08
Maximum
5.09E+08
Standard Deviation
4.66E+07
Part III. The ubiquity of microorganisms
Table 8. The ubiquity of microorganisms
Place
Observation on TSA plate
Observation on Malt
Shoe
Irregular – orange, red, beige
opaque
Some are large, some are small
Some are smooth some are cracked
10colonies
None
Dirty finger
White and yellow all small colonies (4)
opaque
None
E.coli
Few circular, smooth, flat, beige colonies (lawn)
None
E.coli with 70% ethanol
None
None
Discussion:
The objective of the experiment is to learn aseptic technique, sterilization, and streaking. Part A involved isolating single colonies by streaking, part B involved viable cell count, part C involved bacterial cell count by optical density and lastly part D involved ubiquity of microorganisms.
In part A, a pure E. Coli sample was used to form single colonies by streaking. Four streaks were done in different parts of the plate. As described in the results, 1st streak results in forming smallest and very crowded colonies (123 colonies). The space between the colonies were either very small or even adjacent to each other. The 2nd streak forms a larger and less crowded or less population of colonies (60 colonies). Colonies were found to be further apart from streak 1, but they were adjacent to other colonies, so single colonies were not observed. In the 3rd streak finally, isolated single colonies were observed. This is due to the dilution. As in the streak 1, we have least diluted E. Coli pure sample. Therefore, streak 1 has the biggest cell density, where more colonies would grow. In the streak 2, we streak through the streak 1 once, and so, it is diluted. Then, in the third streak as well, it is even more diluted. This is why we have lesser and lesser colonies in the 2nd and 3rd colonies. Colonies all seem to have same opaque shape with beige color, but they differ in sizes. 1st streak ones have the smallest and 3rd streak ones have the largest. This is due to the fact that as the number of the colonies are bigger and crowded, there are less space to grow, so it tends to be smaller where as in 3rd streak, isolated colonies have more space where they can grow bigger.
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In part B, viable count was used to estimate the number of bacterial cells in the sample. Firstly, we prepared 4-fold, 5-fold and 6-fold diluted solutions of E. Coli and they were incubated at 37 degrees Celsius. As we can see in the table 2, 4-fold dilution and 5-folded dilution are too concentrated that bacterial lawn is observed where we cannot apply viable count: they have small viable count to work with and would result in high uncertainty (1). 6-folded dilution appears to be fine to apply viable count and therefore, we used 6-folded dilution to analyze. Looking at the all section data, most of them have the viable counts ranged between 30 and 300. In addition, the average viable count of our section is very close to the class average: 2.38 x 109 to 2.42 x 109 cfu/ml. Also, my group value is very close to the class average as well: 2.52 x 109 cfu/ml. This means that our result is pretty accurate compared to the class result. Speaking of the minimum and the maximum value, there is a high chance that the errors come from these as these values are furthest from the average. Section 1 has the maximum value which is 4.41 x 109 cfu/ml and section 2 has the minimum value which is 2.3 x 108 cfu/ml. The minimum value seem to be “okay” but maximum value seem to lie over 300 colonies, and therefore, the biggest error comes from that value. However, none of these biggest error comes from our section, and therefore, we can say that our section value has the least error.
Part C was done to take cell density by optical density. CAG12033 was taken and was diluted with LB broth. When analyzing, the group 9 student2’s value was 1.018, which appeared to be as an outlier. Grubb’s test was done, and it was eventually an outlier, so we excluded it from further analysis. Speaking of the cell density, as there are more and more of cell in the solution, the density increases. As well, the result shows that as absorbance increases, the cell density increases. Comparing the class average to our section average, it is fairly close: 1.79x 108 and 1.87 x 108cells/ml. However, we have the class minimum value which is 1.24 x 108 cells/ml so we have one of the largest errors. But this is not very far from the average value, which is 1.87 x 108 cells/ml it is not the biggest error. The class maximum value however is very far from the class average value: 5.09×108 cells/ml. This value is in the section 2 data. Section 1 has the best result over the class with closest average value to the class average having no minimum nor maximum values; 1.87 x 108cells/ml which is the same as class average. Comparing my cell density value to the section value, I had 2.18x109cells/ml, whereas the class average was 1.87 x 108 cells/ml. I have a fairly close value and it can be considered that CAG12033 is diluted fairly correctly.
Now comparing viable count method to the cell density measured by spectrophotometer, they can be considered the same. The class average value for the cell density was1.87 x 108 cells/ml and the class average result for the viable count method was 2.42 x 109 cfu/ml. They can be considered the same with the following reasons. First of all, for the optical density method, there is an assumption that there are 5 x 108 cells/ml when the absorbance is 1 Au. This is an assumption and is not an accurate value. Secondly, there are experimental errors such as when diluting, the dilution was not done perfectly, where the error would increase as serial dilution was done in viable count part. As a result, factor of 10 difference is quiet big, but within these assumptions and errors, they can be considered as similar.
Part D was done to see what contamination looks like and how it is. TSA and malt medium were used to contaminate. Different samples were taken with a sterilized rod and were streaked different parts of the plates on both TSA and malt. They were then incubated at 37 degrees Celsius if it is from internal body or incubated at 30 degrees otherwise. TSA is usually considered the best under neutral to slightly basic conditions and required high N for bacteria to grow. On the other hand, malt is best under acidic condition and high in C and N. Malt is best for fungi. First of all, the shoe was rubbed, and streaked on both TSA and Malt plates. A week later, all different kinds of bacteria were grown. Various colored and various sizes were observed: orange, red and beige. Some were really huge and flat, some were small, opaque and smooth. 10 colonies were observed. On malt, nothing grew. Due to the fact that nothing grew on malt, the colonies have to be bacteria. Another possibilities is that malt plate was put in the 37 degrees Celsius which is inappropriate. In quarter of the plate, dirty finger was used to contaminate. 4 colonies of white and yellow were observed. They were all opaque. Nothing grew on malt. In another part of the plate, we put E. Coli sample. Circular, smooth, flat colonies were observed. There were a lot of colonies (bacterial lawn) grown. Again nothing grew on malt. Lastly, we put E. coli with 70% ethanol. Absolutely nothing grew on both malt and TSA. Overall, nothing grew on malt. It is maybe because there was no fungi, or the plates were incubated in the wrong temperature (37 degrees Celsius instead of 30 degrees Celsius). Also, we can say that 70% ethanol kills most of the bacteria or at least enough to prevent them to grow.
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