An aseptic technique

An aseptic techniqueIntroductionAseptic means to be free from microorganisms. Aseptic Technique is the procedure that is performed under sterile condition to pr counterbalancet the growth of divergently microorganisms on the growth medium such as the Petri dishes containing the nutrient agar or the pure nicety. If the growth medium or the pure culture is contaminated with microorganisms from the environment, it volition results in confusion and in unblemished data. Hence, it is measurable to reduce the risks of these microorganisms to come in contact with the experimental materials.In addition, by maintaining a clean environment when transferring the culture of microorganism onto the nutrient agar is ploughsh atomic issuance 18 of the aseptic technique. This is usu whollyy d i by disinfecting the table before and after working with microorganism utilize alcohol. Flaming the experimental materials such as bacteriologic loops, bottle or flask necks screwing help in sterili zing. It must be d bingle for several seconds so as to raise the temperature to kill the contaminants however, the bacteriologic loop must be cooled for a while before it is used to pick up the microorganism as picking up microorganism with a hot marionette will kill the stalls. When removing caps from bottles, it is important to keep the cap in the hand as by putting them on the table, it will be contaminated. Flaming is also required before replacing the cap onto the bottle. It is important to handle open tubes at an angle so that airborne and an an other(a)(prenominal)(prenominal) microorganisms will not fall into the tube and cause contamination. During streaking, it is important to keep the lids of the Petri dish over it to prevent contamination. Lastly, try to avoid breathing, coughing, sneezing and talking while transferring the culture so as to reduce the risk on contaminating.Apart from this general aseptic technique, there also several other methods to ensure that dest ruction of breathing microorganisms in materials and apparatus. One of the methods is by using dry heat which is sterilising using in the buff flame or hot air. Sterilising materials or apparatus by a naked flame is usually heated to redness and allowed to cool. They ar usually made of metal. Exposure to hot air helps to destroy microorganisms in glass and porcelain apparatus.The other method is sterilizing using moist heat which sack be used in three different ways which are heat up in water supply or steam at 100oC, heating in steam under pressure and discontinue heating at low temperature. The different ways are employed according to the different materials or apparatus used.The last method is by using chemical, it can be either in liquid or gaseous state. They are often used in the disposal of contaminated materials and apparatus after a laboratory session.Microorganisms worked with in a lab should not be released into the environment as these strains may contain genetic markers such as antibiotic resistance. Therefore, they must be discarded properly.In addition, aseptic technique is not only applied in laboratory, it is also applied in clinical and surgical setting.AimsThere are two rails in this experiment. The first aim is to show that a large(p) keep down of microorganisms exist on the appear of our hands. The second aim is carry out the aseptic technique properly by transferring pure culture and inoculating them onto an agar plate.Materials and MethodsBacteria on skinPlease come to to the Laboratory Manual unless otherwise stated of changes made.Streak scurfPlease refer to the Laboratory Manual unless otherwise stated of changes made.A disposable sterile bacteriological loop is use instead of the metal sterile bacteriological loop so no heating is required.DiscussionBacteria on skinThe human skins surface do carry a large number of microorganism and that by washing hands, individual can reduce the number of microorganism noticeably. Howev er, even after a hand wash, microorganisms are salve present on the surface.Streak PlateBy employing the streaking technique on an agar plate correctly, a single colony can be obtained. Furthermore, it can be used to separate colonies of mixed culture. Hence, this pure colony can be picked up and to be grown in large quantity. From the result above, it can be observed that single colonies of the S.aureus are found. Due to the comment and morphology, it can be noted that the S.aureus is of a pure culture.ConclusionAseptic technique is a basic laboratory technique that must be employed especially during Microbiology laboratory session so as to prevent any contamination and bear upon the accuracy of the result. Since microorganism can replicated rapidly, disposal of contaminants must be done properly so as to protect both the equipments and the health of individuals.B- gee catchingIntroduction thou stain is also cognize as differential stain in which it will divide bacteria into two large classifys, chiefly Gram Positive and Gram blackball. This difference is due to the chemical and physical structure of the electric electric cell wall called peptidoglycan. During solvent treatment, if the peptidoglycan is able to retain the crystal majestic dye, the bacteria will be group as Gram Positive bacteria. However, if it is not able to retain the crystal violet, the bacteria will be group as Gram Negative bacteria and that it will be stained pink.Gram Positive bacteria has a thicker peptidoglycan (50-90% cell wall) as compared to the Gram Negative bacteria (10% cell wall). In addition, the Gram Negative bacteria has another layer which is make up of liposaccharides and proteins and is separated from the cell wall by the periplasm.In gram staining, there are four basic steps which include flooding the heat fixed smear with crystal violet stain, following by the addition of iodine solution to form complex, adding of alcohol for decolourisation and counterstainin g with safranin.After flooding the peptidoglycan with crystal violet stain, the dye will enter the cells and all cells will deal purple. With the addition of iodine, a crystal violet-iodine complex will be form such that it will not be able to exit the cells easily. By decolourizing the cell with alcohol, the peptidoglycan of the Gram Negative bacteria will break down because the alcohol will dissolves the liposaccharides layer and hence, with the removal of the layer, the crystal violet-iodine complex will run take away which will results in the loss of the crystal violet stain and the cells turn colourless. On the other hand, the alcohol will dehydrate the Gram Positive bacterias peptidoglycan, close the pores as the peptidoglycan shrinks. As a result, the crystal violet-iodine complex will not be able to run off as the exits will be block and they remained stained. By counterstaining with safranin, the Gram Negative cell will turn pink and the Gram Positive cells will remain violet. With gram staining, one is able to signalize if the culture is a pure or a mixed, the morphological details of the bacteria and the arrangement of the bacteria.AimsThe aim is to prepare smears for staining, observe the morphological details of the bacteria and to be able to differentiate between Gram Positive and Gram Negative bacteria.Materials and MethodsPreparation of Smears for stainingPlease refer to the Laboratory Manual unless otherwise stated of changes made.A disposable sterile bacteriological loop is use instead of the metal sterile bacteriological loop so no heating is required.Gram Staining MethodPlease refer to the Laboratory Manual unless otherwise stated of changes made.DiscussionAccording to the result observed, Bacillus subtilis is rod regulate ( atomic number 5). They are stained purple which suggests that they are Gram Positive bacteria. They are arranged in singles. Although, endospore cannot be observe in this experiment, they can also be found on Baci llus subtilis. The endospore enables the bacteria to tolerate harsh environmental condition such as high temperature. Bacillus subtilis can also be known as a single bacillus bacterium.Escherichia coli is stained pink and thereby, it is a Gram Negative bacterium. The cells are also rod shaped and they do not have any particular cell arrangement. They are found in singles, pairs and even clusters.Proteus vulgaris is also stained pink and hence, a Gram Negative bacterium. Its morphology rod shaped and is arranged in singles. They can also be known as a single bacillus bacterium.Staphylococcus aureus is a Gram Positive bacterium as it is stained dark purple after gram staining. It has a spherical shaped, otherwise known cocci and they are usually arranged in grape-like clusters. Therefore, they are known as a staphylococci bacterium. There were no differences in the shape and colour observed for for each one of the bacteria, hence, they can be known as a pure culture.ConclusionThe Gr am staining method is a useful tool used in most laboratories as it helps individual to visualise the bacteria accurately and effectively such as the shape, arrangement and even whether the culture is a pure or mixed.However, it should be noted that not all bacteria will give a gram reaction as some of them are gram variable, otherwise known as gram indeterminate. Therefore, they will give a mix of pink and purple cells after gram staining. For some of the Gram Positive bacteria, their peptidoglycan breaks easily during cell division, hence, after staining, they will give pink cells instead of purple. In addition, the duration of a culture can also call for the gram stain.C- Cell numberIntroductionCells enumerate is the accurate and precise counting of cells. They are usually carried out manually or electronically. By counting cells manually, a counting house, otherwise known as the haemocytometer is used. The counting chamber is used to determine the number of cells per unit vo lume of a suspension. On the other hand, a coulter counter is used to count cells electronically.There are two approaches to count the number of cells, mainly total cell counts and the viable counts. congeries cell counts are counted directly using the microscope and that both living and dead cells are counted. This is normally accompanied by the use of the counting chamber or coulter counter. Another approach is the viable counts which only count the living cells. The small volume of culture, otherwise known as the dilution of the culture is applied to the surface of an agar plate. After incubating, the colonies are counted, normally colonies between 30-300 are chosen to be used for the calculation of concentration of the given sample. The units given is colony forming units (CFU) per ml. The haemocytometer is a modified glass slides with two count chamber of known area. Each chamber grid is composed of nine squares which are known as subgrid, each square is 1mm2. Within each larg e square, there are further sub divisions that help in counting. When the coverslip is placed over the grooves of the slide, there will be a thickness of 0.1mm. Hence, the volume is 0.1mm3 or 1 x 10-4ml. Therefore, the cell concentration will be faced as the number of cells multiply by 1 x 104ml and again, multiplying the dilution factor.Since cells are very small and they can be observed in a very high number, the suspensions should be diluted full so that the cells are able to distribute uniformly in the counting chamber.AimsThere are two aims in this experiment. Firstly, to be able to determine the cell count in different biological species and secondly, to be able to determine the viable count of a live bacteria, Staphylococcus aureus.Materials and MethodsCell Counting using Counting ChamberPlease refer to the Laboratory Manual unless otherwise stated of changes made.Serial Dilution is carried out before the sample is loaded into the Neubauer Manual Counting Chamber. Normal sa line (0.9% NaCl) is used to dilute the blood and broth medium is used to dilute the brewers yeast ( Saccharomyces cervisiae). Both blood and brewers yeast are dilute in the ratio of 110 and 1100. The 110 dilution is prepared by diluting the 10L of whole blood or yeast with 90L normal saline or broth medium several(prenominal)ly. The 1100 dilution is prepared by extracting 10L from the respective sample from 110 and adding 90L of normal saline and yeast into the respective sample. Cell Counting of Live Bacteria, S. aureus (after Serial Dilution)Please refer to the Laboratory Manual unless otherwise stated of changes made.Two changes were made in Step 1 and Step 12 respectively.Only three nutrient agar plates , 10-3, 10-4 and 10-5 were labelled and culture in these dilutions were spread on the respective agar plates. A new spreader and pipette tip were used everything a different dilution culture was spread on the agar plate. Count the number of colonies on the three different agar p lates. Choose the agar plate with colonies between 30-300 to calculate the concentration of the original sample.DiscussionUsing the counter chamber, individual is able to give a quick assessment on the number of cells given that all the procedure on preparing and loading the sample onto it. One of it is that suspension/sample is not mixed before loading. This is due to the fact that cells tend to settle at the bottom of the tube and hence, while pipetting the sample out from the tube, individual do not have the actual or accurate number of cells. Therefore, to get a uniform suspension for a more accurate result, mixing the tube before pipetting is recommended. In addition, it can also help in reducing the clumping of cells.Furthermore, out-of-the-way filling of chambers can lead to inaccurate volume of suspension in the chamber and leading to inaccurate cell concentration. Improper filling of chamber includes overfilling or under filling of sample.Moreover, there must be a consiste ncy in counting cells which is in contact with the boundary lines (ie. the three lines just outside the grid) or when the cells are clump together. Individual will have to determine which cells to count and which not to count especially a cell which is locate on a border such as if a cell has half of its area outside the border, individual do not count those cells.The other method of cell counting is the viable count where a single cell will give rise to a colony which is visible to the naked eyes on the agar plate. Therefore, by counting the number of colonies on the agar plate, individual is able calculate the cell concentration. However, only plates which have 30 to 300 colonies are used to calculate the cell concentration.In the result for viable count of S.aureus, the plate chosen was 10-5 because there was 82 colonies in one quadrant which is equivalent to 328 (82 x 4) colonies on the agar plate. Although, the number of colonies (328) exceed the number of colonies of 300 tha t we were supposed to chose, this 10-5 dilution plate has the closest number to 300. However, we should dilute even further because a single colony can have clumps or chain of cells in it and hence, resulting in inaccurate number of colonies/cells in which the actual number of cells should actually be more than the calculated number of cells.The advantage of viable cell counting is that the organism counted will be a positive one (ie. S.aureus) instead of any other organism as if there is contaminant, the morphology or colour will be different.Another disadvantage of viable cell counting, other than cells that clump together or have chains which will form a single colony, is that organism will only grow in condition which is suitable for their growth on the agar plate.Cell counting usually is accompanied by serial dilution as it is impossible to count the number of cells if the concentration is too high as it will lead to a very high number of cells.ConclusionThere are several other methods, other than using counting chamber and viable cell count, to count cells in a suspension. However, they are the least expensive and is able to give accurate result in a very short period of time.ReferencesAbcam, 2009. Cell counts using a haemocytometer. Abcam plc. consultation http//www.abcam.com/index.html?pageconfig=resourcerid=11454Accessed 1 October 2009George Xu, 2007. History of the Gram Stain and How it Works. University of Pennsylvania.Source http//www.uphs.upenn.edu/bugdrug/antibiotic_manual/Gram1.htmAccessed 1 October 2009H. Kayser, A. Bienz, Johannes Eckert. M. Zinkernagel. 2005. Medical Microbiology. Thieme Stuttgart., New York. p. 264-270.Kenneth Todar, 2008. The Growth of Bacterial Population. Todars Online Textbook of Bacteriology.Source http//textbookofbacteriology.net/growth_2.htmlAccessed 6 October 2009Linda B, Mary R, 2007. Aseptic Transfer. Austin Community College.Source http//www.austincc.edu/microbugz/aseptic_technique.phpAccessed 3 October 2009Steve Hogg, 2008. The Gram Stain. Newcastle University.Source http//www.ncl.ac.uk/dental/oralbiol/oralenv/tutorials/gramstain.htmAccessed 6 October 2009