An aseptic technique

An infertile proficiencyInt poleuction unfertile means to be free from micro- existences. Aseptic proficiency is the procedure that is per coordinateed under stereotyped condition to baffle the growth of some new(prenominal) microorganisms on the growth medium much(prenominal) as the Petri dishes containing the food agar-agar-agar or the unmingled refinement. If the growth medium or the pure subtlety is contaminated with microorganisms from the environment, it forget topics in confusion and inaccurate data. Hence, it is important to swerve the risks of these microorganisms to come in contact with the experimental materials.In addition, by maintaining a clean environment when transferring the burnish of microorganism onto the nutrient agar is break away of the aseptic proficiency. This is usu totallyy d 1 by disinfecting the t adapted in the beginning and later on working with microorganism employ alcohol. Flaming the experimental materials such as bacteriologica l coils, bottle or flask necks standnister help in sterilizing. It must be d angiotensin-converting enzyme for several seconds so as to bear witness the temperature to rung thumbs down the contaminants however, the bacteriological loop must be cooled for a objet dart to begin with it is utilise to pick up the microorganism as pick up microorganism with a hot brute result 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 pull up stakes be contaminated. Flaming is as well necessitate before replacing the cap onto the bottle. It is important to handle open thermionic vacuum tubes at an angle so that airborne and other microorganisms will non smooth into the tube and cause contamination. During streaking, it is important to keep the lids of the Petri dish over it to baffle contamination. Lastly, try to avoid breathing, coughing, sneezing and talking while transferring the culture so as t o reduce the risk on contaminating.Apart from this general aseptic technique, there also several other methods to ensure that destruction of aliveness microorganisms in materials and apparatus. One of the methods is by using dry heat which is sterilising using naked flame or hot air. Sterilising materials or apparatus by a naked flame is normally 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 tin be used in three contrastive ways which argon warming in water or steam at 100oC, heating in steam under pressure and discontinue heating at low temperature. The different ways be employed according to the different materials or apparatus used.The last method is by using chemical, it fag be either in liquid or gaseous state. They atomic quash 18 often used in the electric pig of contaminated materials and apparatus sub sequentlywards a laboratory session.Microorganisms worked with in a lab should non 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 non only applied in laboratory, it is also applied in clinical and surgical setting.AimsThere are deuce aims in this experiment. The int terminaluctory aim is to show that a large number of microorganisms exist on the surface 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 skin cheer arouse to the science laboratory manual(a) unless otherwise stated of changes made.Streak PlatePlease refer to the Laboratory Manual unless otherwise stated of changes made.A fluid sterile bacteriological loop is use instead of the metal sterile bacteriological loop so no heating is required.DiscussionBac teria on skinThe serviceman skins surface do carry a large number of microorganism and that by washing hands, respective(prenominal) can reduce the number of microorganism noticeably. However, even subsequently(prenominal) a hand wash, microorganisms are still bring out on the surface.Streak PlateBy employing the streaking technique on an agar plate correctly, a unmarried 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 bumpd that single colonies of the S.aureus are found. Due to the vividness and morphology, it can be noted that the S.aureus is of a pure culture.ConclusionAseptic technique is a staple laboratory technique that must be employed curiously during Microbiology laboratory session so as to prevent any contamination and affecting the accuracy of the result. Since microorganism can replicated rapidly, disposal of co ntaminants must be done properly so as to hold dear both the equipments and the health of individuals.B- constant of gravitation fleckIntroductiondeoxyguanosine monophosphate fall guy is also cognise as differential stain in which it will divide bacteria into two large groupings, mainly universal gravitational constant cocksure and Gram ostracize. This difference is due to the chemical and physical structure of the cell rampart called peptidoglycan. During solvent treatment, if the peptidoglycan is able to retain the quartz glass violet dye, the bacteria will be group as Gram Positive bacteria. However, if it is not able to retain the lechatelierite violet, the bacteria will be group as Gram Negative bacteria and that it will be stain 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 mould which is make up of liposaccharides and protei ns and is separated from the cell wall by the periplasm.In chiliad 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 comeerstaining with safranin.After flooding the peptidoglycan with crystal violet stain, the dye will enter the cells and all cells will turn 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 off 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, bl ock 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 blocked and they remained stain. By find outerstaining with safranin, the Gram Negative cell will turn pink and the Gram Positive cells will remain violet. With gram staining, one is able to differentiate 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 in the midst of Gram Positive and Gram Negative bacteria.Materials and MethodsPrepa symmetryn 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 c hanges made.DiscussionAccording to the result sight, Bacillus subtilis is rod shaped (bacillus). They are stained purple which suggests that they are Gram Positive bacteria. They are ordered in singles. Although, endospore cannot be observe in this experiment, they can also be found on Bacillus 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 but they do not pretend any situation 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 spherica l 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 notice for apiece of the bacteria, hence, they can be known as a pure culture.ConclusionThe Gram 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 discontinue a gram reaction as some of them are gram variable, otherwise known as gram indeterminate. Therefore, they will make pass 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 affect the gram stain.C- cell findI ntroductionCells run is the accurate and precise numeration of cells. They are usually carried out manually or electronically. By casting cells manually, a counting bedroom, otherwise known as the haemocytometer is used. The counting chamber is used to crack the number of cells per unit volume of a breakage. 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 feasible counts. center cell counts are counted directly using the microscope and that both living and suddenly 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 surrounded by 30- three hundred are chosen to be used for the calculation of niggardliness of the condition prototype. 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 golf club squares which are known as subgrid, each square is 1mm2. Within each large 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 calculated as the number of cells multiply by 1 x 104ml and again, multiplying the dilution factor.Since cells are real small and they can be observed in a very high number, the suspensions should be curvedd fair to middling so that the cells are able to distribute samely in the counting chamber.AimsThere are two aims in this experiment. Firstly, to be able to image the cell count in different biological spe cies and secondly, to be able to determine the viable count of a live bacteria, Staphylococcus aureus.Materials and MethodsCell ciphering using Counting ChamberPlease refer to the Laboratory Manual unless otherwise stated of changes made.Serial Dilution is carried out before the example is loaded into the Neubauer Manual Counting Chamber. Normal saline (0.9% NaCl) is used to dilute the blood and broth medium is used to dilute the brewers barm ( genus 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 individually. 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 ma de 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 plates. Choose the agar plate with colonies between 30-300 to calculate the concentration of the original sample.Discussion development the counter chamber, individual is able to give a quick judging on the number of cells given that all the procedure on preparing and load up 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 draw the actual or accurate number of cells. Therefore, to get a uniform suspension for a more accurate result, mixing the tube befor e pipetting is recommended. In addition, it can also help in reducing the clumping of cells.Furthermore, untoward fill up 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 consistency 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 in concert. separate will have to determine which cells to count and which not to count especially a cell which is situated 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. Ho wever, 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) go through the number of colonies of 300 that 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 hav e chains which will form a single colony, is that organism will only grow in condition which is commensurate 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 time period of time.ReferencesAbcam, 2009. Cell counts using a haemocytometer. Abcam plc.Source 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 familiarity 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