Introduction to Aseptic techniques
In microbiological and biochemical anatomist studies, one almost always deals with a 100 % pure culture or an assortment of known cultures, except perhaps in misuse drinking water treatment studies. Unless aseptic culture techniques are implemented strictly, an actually 100 % pure culture will definitely become contaminated with other unwanted varieties. The use of a contaminated culture with unfamiliar microorganisms will only lead to amazing results that are of little value. Thus, isolation and maintenance of a genuine culture is of utmost importance in many microbiological studies. It is especially important to utilize a well-characterised pressure if the microorganism can be used for a food preparation, as well as in antibiotic production where in fact the product is usually to be considered internally.
The need for a clean working environment in natural engineering studies is a must and sanitation is the prerequisite for any meaningful work. The surroundings we are in is filled with microorganisms capable of surviving in virtually any condition.
When working with microorganisms it is suitable to work with a real culture. A clean culture comprises only 1 kind of microorganism. Once in a while a blended culture can be used. In a blended culture there are several organisms which have particular characteristics and can be segregated easily. In either situation the organisms can be diagnosed. When unwanted microorganisms are introduced in to the culture they are simply known as pollutants.
Aseptic approach is a way that stops the introduction of unwanted microorganisms into a host. A good example of using aseptic techniques is when growing bacteria; aseptic techniques are carried out to prevent the contamination of the culture. Whenever using microbial ethnicities aseptic technique can be used to prevent producing additional organisms in to the culture.
Microorganisms are everywhere in the environment. When coping with microbial civilizations it's important to take care of them in such a way that environmental microorganisms do not get introduced in to the culture. Microorganisms may be found on surfaces and floating in air currents. They could fall from objects suspended more than a culture or swim in essential fluids. Aseptic technique prevents environmental microorganisms from entering a culture.
Doors and house windows are kept finished in the laboratory to avoid air currents which may cause microorganisms from areas to become airborne.
Once these microbes are airborne they are more likely to get into cultures.
Agar plates are held in a manner that minimizes the coverage of the top to the environment. When eliminating lids from pipes, lids are performed in the hand and not put on the countertop during the transfer of materials from one tube to another.
Introduction and goals of this report
This report was based on two lab sessions; the reason behind undertaking two laboratory sessions was to allow time for the culture to increase. In these consultations the objective was to learn basic aseptic techniques that are essential in a laboratory, and to understand how to count cells effectively using different methods. Another thing which was endeavoring to be achieved was to be able to increase a colony of bacterias from an individual cell by doing streak plating and serial dilution.
Overview of procedures and uses of aseptic techniques
The laboratory program involved studying the aseptic techniques. These aseptic techniques are essential in a lab because they help to keep the lab sterile, and sterility is vital in a lab because it allows the scientist to study and increase the bacteria they require accurately. Sterility is also important in avoiding bacteria that are not required from replicating and growing on the sterile progress medium or the agar plate.
There were a few aseptic techniques we had to follow while dealing with bacteria's and sterile expansion medium. To avoid the development medium from being polluted by air bore bacteria and other free floating subject, a Bunsen burner was create near where in fact the progress medium and bacteria samples were to be utilized. The Bunsen burner created a convection current that wiped out and destroyed most of the environment borne bacteria and other free floating matter near the work place. This reduced the chance of the growth medium and bacterias examples from being contaminated.
The Bunsen burner was also setup to allow the use of another strategy called flaming. This technique involves transferring through the flame of the burner whatever has come directly into contact of any bacteria or anything that is going to come directly into contact of the bacterias sample. The things that are flamed are lab equipment such as bacteriological loops, a glass pipette and container or flask necks. The things must reach a temperature of over 100 oC for this to be sterilized.
Another aseptic strategy is named manipulation. In this technique the tiniest finger is utilized to remove the lid of the bottle containing the bacteria; this allows all of those other fingers to get anything else that is required. This technique also ensures the lid of the container is not located down onto the bench where it is likely to contamination and thus contaminating the culture of bacteria in the container.
The previous but the most crucial aseptic approach is who someone prevents bacterias from themselves contaminating the laboratory and the gear. Every person carries a sizable amount of bacteria inside and the outside of your body. Whenever using bacterias in a laboratory, we had to wearing a lab coating, this prevent bacteria from our clothes and systems spreading away in the laboratory. Also we'd to be careful that people don't cough or sneeze on the progress medium, as this would lead to the progress of the bacterias released by your body. Also after performing the experiment it was vital that hands were washed with antibacterial soap to help prevent cross contaminants. If hands are not washed correctly and if bacteria remain still left on hands they could multiply at an exponential rate and can cause bacterial infections.
The first part of the experiment was to see different deviation and amount of bacteria on hands pre rinse and after wash. This is done by placing the hands in a Petri dish with nutrient agar. Nutrient agar is a microbiological progress medium commonly used for the boring cultivation of bacteria. The dish was segregated in two and was labelled with one part of dish having prints from pre washed hands and the other part after rinse. The dish was then positioned into incubation at 37 diplomas as it is the optimum temps where bacteria have the ability to multiply at an exponential rate depending on some factors an example being the quantity of food available or space.
The next part of the experiment contains doing a streak plate. This was done using the bacteria Staphylococcus aureus. Small test of the bacteria SA was used and put on a on the sterile loop and streak an agar medium. An example of the streak dish which was carried out is shown on the diagram below:
Diagram showing method of streak plating
1. Flame the loop and line and streak a loopful of broth as at A in the diagram.
2. Reflame the loop and cool it.
3. Streak as at B to propagate the original bacteria over more of the agar.
4. Reflame the loop and cool it.
5. Streak as at C, D E and F pursuing same procedure after each streak as quoted above.
6. Label the plate and incubate it inverted.
The next part of the first period was to do serial dilution. This enables you to look for the number of skin cells in a bacterial culture. Since bacterial cell statistics are usually high in the initial test, plating out this test in an undiluted fashion would just lead to the creation of the bacterial backyard (a smear of several, many individual bacteria colonies that are growing next to or on top of each other).
Bacterial cell statistics have to be reduced, which is performed by consistently diluting the amount of bacterias in the test. A small amount of bacteria sample is mixed with a diluent solution (such sterile broth), and then successive dilutions are made. A small amount of each of the diluted bacteria samples is then multiply onto an agar plate. The amounts of bacterias colonies that expand on each plate are counted. By working backwards using multiplication with the "dilution factor" (the number of times that you have diluted the bacteria test with the diluent solution), we were able to make a dedication of the numbers of bacteria in the initial sample. Following the dilutions were created 100 l of each dilution was transferred to an agar plate using a pipette, it was then pass on across the agar plate with a spreader. These six agar plates were then put into incubation at 37 C every day and night. When distributing the bacterial backyard the plate with the dilution level 10-5 was done first and then the others 10-4, 10-3, 10-2. it is because the spreader that was used was plastic so the lower focused bacterium was pass on first as the clear plastic spreader could not be flamed to destroy the bacteria. If this aseptic strategy was not used and the best concentration of bacterias was used first it would have meant that the bacterial meals could have become polluted and also one colonies of bacterias would not be gained. If a cup spreader was used then it might did in ascending order as the a glass could be flamed by putting ethanol on the surface killing the bacterias on the glass spreader before doing another part of the serial dilution.
The final area of the first lab classes was to get ready smears of bacterias for gram staining. Gram staining is a common technique used to differentiate two large groups of bacteria predicated on their different cell wall structure constituents. The Gram stain process distinguishes between Gram positive and Gram negative groups by colouring these skin cells pink or purple. Gram positive bacterias stain purple because of the presence of any thick coating of peptidoglycan in their cell wall surfaces, which retains the crystal violet these cells are stained with. Additionally, Gram negative bacterias stain pink, which is attributed to a leaner peptidoglycan wall structure, which will not wthhold the crystal violet through the decolouring process.
Gram staining involves three techniques: staining with a water-soluble dye called crystal violet, decolourisation, and counterstaining, usually with safanin. Due to distinctions in the width of an peptidoglycan covering in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacterias (with a thicker peptidoglycan level) preserve crystal violet stain through the decolourisation process, while Gram negative bacterias lose the crystal violet stain and are instead stained by the safranin in the ultimate staining process. The process involves three steps:
1. Cells are stained with crystal violet dye. Next, a Gram's iodine solution (iodine and potassium iodide) is added to form a organic between your crystal violet and iodine. This complex is a larger molecule than the original crystal violet stain and iodine and is insoluble in water.
2. A decolouriser such as ethyl alcoholic beverages or acetone is put into the test, which dehydrates the peptidoglycan coating, shrinking and tensing it. The large crystal violet-iodine organic is not able to permeate this tightened peptidoglycan covering, and it is thus captured in the cell in Gram positive bacteria. Conversely, the outer membrane of Gram negative bacterias is degraded and the thinner peptidoglycan layer of Gram negative cells struggles to wthhold the crystal violet-iodine complex and the color is lost.
3. A counter stain, like the weakly normal water soluble safranin, is added to the test, staining it green. Since the safranin is lighter than crystal violet, it does not disrupt the crimson coloration in Gram positive skin cells. However, the decolourised Gram negative cells are stained red.
(The descriptive methods are shown in the handbook for all experiments. )
Results for the gram staining
After following method as stated in the handbook we examined the slides under a microscope using the petrol immersion goal of 100x. We then observed the condition of the bacteria that may be seen and the color being crimson (Gram positive) or green (Gram negative). Here are the traces of the bacterias which could be observed under the microscope.
Figure 1 - Staph aureus - gram positive (purple)
Description of what could be seen:
- Cocci molded bacteria
- Irregular clusters of bacterial cells
Figure 2 - Bacillus cereus - gram positive (purple)
Description of what could be seen:
- Rod designed bacterial cell
- Singular bacteria
Figure 3 - Saccharomyces cerevisiae - gram positive (purple)
Description of what could be observed:
- Cocci formed bacteria
- Clusters of bacteria closely packed
Figure 4 - E-coli - gram negative (green)
Description of what could be seen:
- Rod shaped bacteria cells
- Associated (string like, filamentous)
Results of the experiments
After 24 hours the agar plates with the bacterias were ready to be viewed. Firstly the agar dishes with the hands prints were looked at. Below is a diagram of the agar dish and the bacteria which was present:
There will vary bacteria that have been present while watching the dish, these were the following
A - The first bacteria which were seen and labelled being a were the largest of the three visible colonies which are circle in shape and yellow in color with smooth corners, they could be seen to have a just a little humped surface.
B - These are slightly smaller in size than those described above and are also group in form but are white in coloring, again the edges are even and the surface is humped.
C- These bacterium were only seen after cleansing hands they had no specific condition and were a lighter color which was not very clear. They were flat with harsh edges.
The next dish that was seen was the streak plating dish, this had been remaining to incubate for 24 hours also. The results are shown on the picture below:
A test of Staphylococcus aureus was inoculated onto an agar dish using the streak dish method.
From this diagram it could be seen that part 1 shows an increased concentration of bacteria. Parts 2, 3 have fewer bacteria but nonetheless there are extremely few sole colonies. Part 4 shows many different sole colonies of Staphylococcus aeurus and are often visible.
The last dish which was viewed after a day of incubation was the dish made up of Staphylococcus aureus where it turned out diluted to 10-5. These results is seen below where there is diagram of the Petri dish like the bacterium.
From keeping track of the colonies on the dish the amount which was computed was 486. The amount of colonies calculated was still quite high as the most well-liked number of colonies would have been from 30-300. This might have been achieved if the serial dilution was transported further.
To calculate the quantity of cells in this agar dish first the following was done:
0. 1ml of solution = 4. 86x10-2 (486)
1ml of solution = 4. 86x10-3
so as it was the serial dilution of 10-5 the calculation was then multiplied by 5 to give the ultimate answer amount of bacterial skin cells = 4. 86x10-8
The last part of the lab treatment was to count up cells using a better Neubauer Keeping track of Chamber also sometimes known as a haemocytometer. The primary objective of this treatment was to be able to calculate the full total skin cells in the given test. Below is a diagram of any haemocytometer with the glide located over it:
The haemocyometer is made up of 9 large squares under the microscope at the 40X lens. The area of the square can be assessed at 1mm2. The best way to differentiate these squares from one another is by the tripe thick lines. Within each large square there are smaller grids that can be used to help during counting. Also when the counting the bacteria skin cells there is a arranged way to do this as shown below in the diagram:-
So as can be seen in the diagram if the bacterias cells are at positioned on the edge of the tiny squares they will not be documented. The orange lines representing cells will never be recorded because they are the finish of the grid.
3 dense lines separating each large square
The way the cells were counted was to recognize which squares were heading to be utilized to observe and calculate the amount of bacteria present. The way this is done was there have been 9 squares in support of 5 squares were chosen as shown in the diagram below:
Squares 1, 3, 5, 7 and 9 were the squares that have been used to count the bacterias.
Once the haemocytometer was arranged and the proposed samples were put into the counting chambers and then put under a microscope to see (long method is defined in the module handbook). The cell count was done for two different cell suspensions whole blood vessels (ovine) and brewer's candida (Saccharomyces cervisiae) the samples weren't diluted. These were then counted and the results are shown in the stand below.
Table showing cell count up from haemocytometer for your blood
Number Of Cells Present
To calculate the full total cell count in the cool solution a calculation was needed. First of all the average variety of cells was would have to be worked out. The amount was 237/5=47. 4. To work through the cellular number it was multiplied by 1x10-4=4. 74x10-5 So then finally to work through the cell yield the number of skin cells/ml was used that was 4. 7x10-5 and was multiplied by the full total level of 10ml and so the yield computed was 4. 74x10-6.
Table to show cell count from haemocytometer for the Brewer's yeast
Number Of Skin cells Present
The same steps were taken to workout the cellular number and produce for the Brewer's yeast.
Cell quantity = 1. 62x10-5
Cell Yield = 1. 62x10-6
Table to show cellular number and cell yield of both samples
Discussion of results
In this area of the report I'll make reference to the results obtained and determine if they were correct or not to the study of aseptic techniques. Firstly the practical which engaged looking at bacterias on your skin exhibited that after cleaning there was fewer bacterias but another form of bacterial cells started to expand. The explanation for this may have been that when closing the taps I may have used my hands. The taps in the laboratory are created o be closed down by the wrist therefore the bacteria is unable to come in contact with the top of your hands. The reason behind it is because if you wash your hands and then close the taps with your hands again then you are just collecting the bacteria off of the taps again. In some cases the amount of microbacterium on your skin can increase after cleansing, this is because by covering the skin with normal water you are making conditions for microrganisms more favourable and thus more will increase. Hence, it is understandable that the development of microorganisms depends upon the chemical composition of the skin, for example if it is dried or whether it has a minimal pH. Most microorganisms that are present on the skin can be found near hair follicles or sweating glands it is because they provide the nutrients and the right environment for there progress.
Also another reason behind the bacterias still being there after cleansing hands on the agar dish was as a result of process of cleansing hands. The procedure of cleansing hands should be done surgically as there isn't such thing as part sterile. So to make certain that all germs and bacteria are washed away from skin the procedure of hand cleaning should be adopted correctly.
There are over 100 different kinds of bacteria on hands. The most frequent types of bacteria found on hands are familiar household labels: Propionobacterium (the bacteria responsible for acne), strep, and staph (which the infamous methicillin resistant staph aureus, MRSA is a subtype). Not absolutely all these bacteria are hazardous as skin attacks do not come up because you have bacteria on your skin layer. Rather, they come up because the kind of bacteria on contaminated skin is not healthy bacteria but hostile pathogenic bacterias.
Streak plating discussion
From looking at the results extracted from the streak plating it could be seen that the streak dish had not been very accurate as the supposed result was not achieved. The purpose of this test was to try and gain one colonies but the problem with the streak plate which I acquired completed was that there is not enough room for the solitary colonies to boost. This was because the initial inoculation streaks were too heavy and so used too much space thus departing little space in the middle of the plate for single colonies. Hence, it is required that the original streaks are created thinner and cover, as a abrasive estimate, the outside 2cm of the agar plate thus, leaving abundant space at the centre of the dish for sole colonies to expand. The situation with this process is that each colony may well not signify the progeny from one cell, as two or more cells which are extremely close mutually could appear as you colony. Another problem which might have induced difficulty achieving single colonies may have been the awareness of the bacteria. If the bacterias were diluted it may have helped to accomplish solitary colonies.
The solitary colonies that have been achieved were all similar to one another this demonstrates the bacterium that was present in them colonies was the same bacterias. This was achieved as the inoculating loop was sterilised each time so only the bacterium that was being used grew on the agar dish.
In this part of the practical there have been four different bacterias which were analyzed utilizing the gram staining process to see if indeed they were gram positive or gram negative. The first bacterium was Staphylococcus aureus, Bacillus cereus and Saccharomyces cerevisiae which were gram positive bacterium as after examining the bacteria under the microscope it revealed that it was stained purple. The SA under the microscope was seen just like a bunch of grapes as its names suggest as Staphyle in Greek conditions designed grapes. Staphylococcus aureus is a bacterium, frequently living on the skin or in the nostril of a healthy person that can cause illnesses ranging from small skin attacks and abscesses, to life-threatening diseases such as pneumonia, meningitis, endocarditis and septicemia.
The Bacillus cereus is a facultative anaerobic bacterium associated with food poisoning in humans. The food poisoning is because ingesting toxins made by the bacterias. B. cereus is popular in the garden soil and the food industry in such foods as natural remedies, spices, dairy, and vegetables. Transmitting of the disease results not only from contaminated foods, but also from inappropriate food handling/storage space and improper air conditioning of cooked food. The bacterias seen under the microscope so that as is seen in the diagram (figure 2) shows the bacterias as rod formed bacterias which do not cluster jointly and are separated around the slip, in different guidelines and aren't in any particular order as they all in different sides.
The Saccharomyces cerevisiae is also known better as candida which could be used for cooking or used while making alcoholic beverages. These skin cells where seen under the microscope as solo cells that have been rounded shaped cells and were directly packed jointly in communities.
The last bacterium was the E. coli which was stained green as this is a gram negative bacterium. This bacterium is situated in animals and wild birds in the lower intestines it helps with the digestion of food. If E. coli is ingested it'll cause the small intestine to be inflamed. People can written agreement an E. coli infection by drinking contaminated water, eating berries or vegetables that have been watered with polluted water, sipping unpasteurised dairy, or eating undercooked earth meat.
In Gram-positive skin cells, peptidoglycan makes up around 90% of the dense cell wall; more than 20 levels of this polymer stacked jointly. These peptidoglycan tiers are the outermost cell wall framework of Gram positive skin cells, whereas in Gram negative cells, the thinner peptidoglycan aspect is covered by an external lipopolysaccharide (LPS) membrane.
Serial dilution - agar dish 10-5
This practical was done to see if individual colonies could actually be produced therefore the skin cells could be counted. The main aim was to attain from 30 -300 individual colonies. The amount of colonies which I stated in my agar dish was determined and counted at 486. The method used was to try and calculate the amount of skin cells in 1 ml solution of SA. This may only be achieved by serial dilution as it might be too difficult to depend the skin cells if the bacterium solution was not diluted. The other food had way too many colonies to count up just by using the naked eye because it looked like a bacteria grass. To achieve an improved result and fewer colonies the test could have eliminated further and rather than having a attentiveness of 10-5 the solution might have been diluted further. By diluting down the perfect solution is it also allows the bacterium to develop in ideal conditions as they do not have problems such as less space or food.
Cell counting utilizing a haemocytometer
In this area of the sensible two solution were offered and cells were counted through a haemocytometer and a microscope. While counting the bacteria it could have been misjudged as some bacterias may well not have been keeping track of this is one reason why the number saved were quite low as the solution had not been diluted as well. Also when counting the bacteria it's a complete cell count so that it is the living and useless bacteria so the results are not as accurate if only performing a live cell count. A better idea is always to execute a serial dilution when doing a live cell count number as only the living skin cells will increase into individual colonies. For the whole blood the quantity of cells which were computed was 237 and in the brewer's candida there were 81 cells. Grounds for the brewers yeast having less amount of skin cells maybe that the skin cells form flocks of skin cells so it may be hard to aesthetically see separate cells so when keeping track of them a few cells may be counted as one cell. Also when keeping track of these skin cells the same person was used to matter the skin cells in both solution the explanation for this being as different people have different judgments and utilizing the same person it can help gain fair and even more appropriate results.
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