Sterilization And Aseptic Technique Biology Essay

Sterilization And Aseptic Technique Biology EssayThis experiment was done to learn proper way of using infertile technique and sterilization by isolating pure culture of bacterial. after(prenominal)wards, the bacterial cells in a strain and their optical density were determined.First of alone, various sterilization methods were introduced. Sterilization is important in a sense that it ensures there is absolutely no contamination in the glassw atomic number 18 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 kindle be sterilized by autoclaving. As the temperature of the steam is above 100 oC, the organisms thronenot survive. Second sterilization method is radiation. As heat sensitive plastics does not go resistance to heat, autoclaving send wordnot be used and these are often sterilize d by using radiation such as UV, gamma-ray or X-ray. The last method is sieve sterilization. Some solutions are heat labile, and to sterilize these kind of solutions, filter out 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)In part B, antiseptic technique is learned. This technique prevents any kind of contamination while handling the glassware or transferring. To be more(prenominal) 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 violent death 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 s pring lids minimizes contamination.(1)Using aseptic technique, raveling technique was used to isolate atomic number 53 colonies. To do this, a pure culture of the target microorganism is administern. 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 excessively much diluting.After streaking, colonies are bafflen. To librate the number of cells, practicable 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 workable cells can be obtained by counting the number of colonies that have devel oped multiplied by the respective dilution factor. (2)Material and MethodsAll 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.ResultsColony isolation by streakingFirst of all, the color of the bacteria in all the plates are thick beige washed-out. In streak 1, extremely small and many another(prenominal) colonies were observed. The size of the colonies were very small, they were circular, opaque and smooth. There are 123 colonies. grade insignia 2 shows chain of bacterial formation, and the number of the colonies is decreased from streak 1. The number of colonies were 60. They were bigger 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 s mooth as well. About 9 colonies were observed. In the 4th streak, no single colony was observed. As a way out, single colonies of a pure E. coli strain was successfully isolated.Viable countTable 1. Raw data of possible count of my group.Dilution10-410-510-6Number of colonyToo manyToo many252Viable count (cfu/ml)2.52 x 109 specimen 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. Raw data for viable count for all segmentationDilutionsColony countGroup 1Group 2Group 3Group 4Group 5Group 6Group 7Group 8Group 9Group 10 slit 110-4lawnlawnlawnlawnlawnlawnlawnlawnlawn10-5360lawn1848lawn102821686963040164610-678287441270234347363300306 theatrical role 210-4920300LawnLawnLawn300300LawnLawnLawn10-5249300590300Lawn30030040630018910-623231189269384222265154180108Section 310-4too many1000too manytoo manytoo manytoo manytoo manytoo ma nytoo manytoo many10-5too many1000too manytoo manytoo manytoo manytoo manytoo many544too many10-619442025825229521724019779224Table 3. Viable count for all member 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+09 max4414.41E+09Min787.8 E+08Average291.82.92 E+09Standard 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+08Average202.52.02.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 densityT able 4. carrel density for My GroupOD600 of diluted cultures (Au)E. Coli Count of diluted burnish (cells/ml)Original Culture (cells/ml)Me0.4272.181082.18109Partner0.4362.141082.14109E. Coli Count of diluted Culture 0.202 x (5x 108) = 1.01108Original Culture 1.01108 x 10 = 1.01109Table 5. Raw OD600 cling tos 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 outlier)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+081.75E+081.83E+081.76E+0831.82E+081.72E+08 1.24E+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, discipline analysisClass (cells/ml)Average1.87E+08Minimum1.24E+08Maximum5.09E+08Standard Deviation4.66E+07Part III. The omnipresence of microorganismsTable 8. The ubiquity of microorganismsPlaceObservation on TSA plateObservation on MaltShoeIrr egular 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 move 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.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 shed light on average 2.38 x 109 to 2.42 x 109 cfu/ml. Also, my group nurture 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 utmost value, there is a high chance that th e errors come from these as these values are furthest from the average. Section 1 has the maximal 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 all 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 experiment 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 se ction 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 5.09108 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 premise 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 usuall y considered the best under neutral to meagrely 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 uncontaminating 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 colon ies (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 ill-treat 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.