Sterilization And Aseptic Technique Biology Essay

Sterilization And Aseptic Technique Biology showThis essay was done to learn proper way of using aseptic technique and sterilization by isolating polished culture of bacteriuml. Afterwards, the bacterial cells in a sample and their optical assiduousness were determined. archetypical of each, various sterilization methods were introduced. Sterilization is important in a sense that it ensures there is absolutely no contamination in the crackpotw be or apparatus arrest in the lab. diametrical sterilization methods atomic number 18 utilize for dissimilar materials. One of them is autoclaving. Autoclaving machine uses high-pressure steam to sterilize and therefore, heat resistant p remnantics, glass or solutions flush toilet be sterilized by autoclaving. As the temperature of the steam is above 100 oC, the organisms cannot survive. fleck sterilization method is radiation. As heat sensitive plastics does not swallow resistance to heat, autoclaving cannot be used and these a re 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 olive-sizeder diameter, microorganisms cannot eradicate through the filter.(1)In part B, aseptic technique is learned. This technique prevents any kind of contamination succession handling the glassware or transferring. To be more specific, it prevents any contaminant to be introduced in the study 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 off 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 supply mouths and minimizing the tim e of opening lids minimizes contamination.(1)Using aseptic technique, salooning 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 prevention lines in the medium. The streaking lines should not cross each other to avoid also much diluting.After streaking, colonies are grown. To count the number of cells, workable cell count method is used. viable count is only useable with singles colonies and not bacterial lawns. Therefore, in order for cell to hurl 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 begin developed figure by the respective dilution factor. (2)Material and MethodsAll procedures are performed according to the BIOL 368 lab manual (Concordia Biology surgical incision 2013) except for the interest modifications for the contamination part, we used shoe, finger, E. coli, and E. coli with 70% ethanol.ResultsColony isolation by streaking graduation of all, the colouring of the bacteria in all the plates are thick beige colored. In streak 1, extremely sharp and many colonies were observed. The size of the colonies were very small, they were orbitual, unintelligible and insipid. 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, muddy, circular and smooth as well. In streak 3, single colonies are observed. no(prenominal) of them was huge, but they were larger than the colonies from streak 2. They were opaque, circular and smooth as well. Ab out 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. feasible count plank 1. birthday suit data of viable count of my group.Dilution10-410-510-6Number of colonyToo manyToo many252Viable count (cfu/ml)2.52 x 109Sample calculationViable count at 10-6 dilutionSince 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/mlTable 2. in the raw data for viable count for all sectionDilutionsColony count separate 1 pigeonholing 2 theme 3Group 4Group 5Group 6Group 7Group 8Group 9Group 10 atom 110-4lawnlawnlawnlawnlawnlawnlawnlawnlawn10-5360lawn1848lawn102821686963040164610-678287441270234347363300306 plane section 210-4920300LawnLawnLawn300300LawnLawnLawn10-5249300590300Lawn30030040630018910-623231189269384222265154180108Section 310-4 to a fault many1000 in like manner many to a fault man y in addition manytoo manytoo manytoo manytoo manytoo many10-5too many1000too manytoo manytoo manytoo manytoo manytoo many544too many10-619442025825229521724019779224Table 3. Viable count for all section data (for 10-6 dilution)SectionGroupNumber of coloniesCfu/ml011787.8.E+0822872.87.E+0934414.41 E+0942702.70 E+0952342.34.E+0963473.47E+0973633.63E+09893003.00E+09103063.06E+09Max4414.41E+09Min787.8 E+08 reasonable291.82.92 E+09 shopworn Dev.94.402.67E+08021232.3.E+0822312.31.E+0931891.89.E+0942692.69.E+0953843.84.E+0962222.22.E+0972652.65.E+0981541.54.E+0991801.80.E+09101081.08.E+09Max3843.84.E+09Min232.3.E+08 second-rate202.52.02.E+09Standard dev.93.099.31E+080311941.94.E+0924204.20.E+0932582.58.E+0942522.52.E+0952952.95.E+0962172.17.E+0972402.40.E+0981971.97.E+099797.9.E+08102242.24.E+09Max4204.20.E+09Min797.9.E+08Average237.62.38.E+09Standard Dev.81.558.16.E+08Max4414.41.E+09Min232.3.E+08Average242.32.42.E+09Standard Deviation96.751.15E+09Sample calculation for STD DEV. (section 1)= 94.40Bacterial cell count by optical stringencyTable 4. Cell density for My GroupOD600 of cut cultures (Au)E. Coli Count of cut shade (cells/ml)Original Culture (cells/ml)Me0.4272.181082.18109Partner0.4362.141082.14109E. Coli Count of cut Culture 0.202 x (5x 108) = 1.01108Original Culture 1.01108 x 10 = 1.01109Table 5. Raw OD600 values for all sections (unit Au)GroupSection 1 (1)Section 1 (2)Section 2 (1)Section 2(2)Section 3(1)Section 3 (2)10.3890.3830.3590.3710.3580.36520.3680.3690.2470.4470.3450.40830.3640.3430.3440.3600.3230.33540.3740.3740.3900.3380.4270.43650.4150.4300.3860.3680.3200.24760.3590.3570.3020.3500.3790.35270.3470.3720.3540.3690.3910.36480.3620.3610.3580.32890.3700.351.0180.3500.341100.7370.3670.3680.3530.4130.322(1.96 therefore out duplicityr)Table 6. Diluted Cell Density for all sections (unit cells/ml)GroupSection 1 (1)Section 1 (2)Section 2 (1)Section 2(2)Section 3(1)Section 3 (2)11.95E+081.92E+081.80E+081.51E+081.79E+081.90E+0821.84E+081.85E+081.86E+08 1.75E+081.83E+081.76E+0831.82E+081.72E+081.24E+081.77E+081.73E+081.96E+0841.87E+081.87E+082.24E+081.85E+082.04E+081.82E+0852.08E+082.15E+081.72E+081.81E+081.62E+081.79E+0861.80E+081.79E+081.80E+081.81E+081.68E+081.64E+0871.74E+081.86E+081.95E+081.75E+082.14E+081.75E+0881.69E+085.09E+082.18E+081.71E+0891.85E+081.93E+081.84E+081.60E+082.07E+08101.87E+081.84E+081.84E+081.77E+081.24E+081.61E+08Average1.87E+081.95E+081.79E+08Min1.72E+081.24E+081.24E+08Max2.15E+085.09E+082.18E+08Standard Deviation1.05E+077.43E+072.13E+07Sample calculationSection 1 group1 student 1 Cell Density = 0.389 x (5x 108) = 1.95 x107Section 1 AverageAverage = ((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 x108Table 7. Diluted Cell Density for all sections, course of study analysis crystalise (cells/ml)Average1.87E+08Minimum1.24E+08Maximum5.09E+08Standard Deviation4.66E+07Part III. The ubiquity of microorganismsTable 8. The ubiquity of microorganisms outObs ervation on TSA plateObservation on MaltShoeIrregular orange, red, beigeopaqueSome are large, some are smallSome are smooth some are cracked10coloniesNoneDirty fingerWhite and yellow all small colonies (4)opaqueNoneE.coliFew circular, smooth, flat, beige colonies (lawn)NoneE.coli with 70% ethanolNoneNoneDiscussionThe 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 plaza 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 moreover apart from streak 1, but they were adjacent to other colonies, so single colonies were not observed. In the third streak finally, isolated single colonies were observed. This is due to the dilution. As in the streak 1, we have least(prenominal) 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 equal opaque formula 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 lieu to grow, so it tends to be smaller where as in 3rd streak, isolated colonies have m ore station where they can grow bigger.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 confuse 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 fair(a) viable count of our section is very close to the break up median(a) 2.38 x 109 to 2.42 x 109 cfu/ml. Also, my group value is very close to the ramify average as well 2.52 x 109 cfu/ml. This means that our result is pretty accurate compared to the class result. Sp eaking 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 student2s value was 1.018, which appeared to be as an outlier. Grubbs test was done, and it was eventually an outlier, so we excluded it from barely 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, th e cell density increases. Comparing the class average to our section average, it is somewhat 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 5.09108 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 malted milk 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 and so incubated at 37 degrees Celsius i f it is from internal body or incubated at 30 degrees otherwise. TSA is usually considered the best at a lower place 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(prenominal) 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 pu t 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. short 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.