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An aseptic technique

Introduction

Aseptic means to get rid microorganisms. Aseptic Technique is the procedure that is performed under sterile condition to avoid the development of other microorganisms on the development medium including the Petri dishes comprising the nutritional agar or the real culture. In the event the development medium or the real culture is contaminated with microorganisms from the surroundings, it will leads to misunderstanding and inaccurate data. Hence, it is important to reduce the potential risks of the microorganisms to are exposed to the experimental materials.

In addition, by retaining a clean environment when transferring the culture of microorganism onto the nutritional agar is area of the aseptic technique. This is usually done by disinfecting the desk before and after dealing with microorganism using alcohol. Flaming the experimental materials such as bacteriological loops, bottle or flask necks can assist in sterilizing. It must be done for several seconds in order to raise the temps to wipe out the impurities; however, the bacteriological loop must be cooled for a while before it can be used to get the microorganism as picking right up microorganism with a hot tool will wipe out the skin cells. When getting rid of caps from bottles, it's important to keep the cover in the hand as by placing them on the table, it'll be contaminated. Flaming is also required before exchanging the cover onto the container. It is important to handle available pipes at an perspective so that airborne and other microorganisms will not fall into the tube and cause contamination. During streaking, it is important to keep carefully the lids of the Petri dish over it to avoid contamination. Lastly, stay away from breathing, coughing, sneezing and chatting while transferring the culture so as to reduce the risk on contaminating.

Apart out of this general aseptic technique, there also several other solutions to ensure that damage of living microorganisms in materials and apparatus. One of the methods is to apply dry heat which is sterilising using naked flame or hot air. Sterilising materials or equipment by a naked flame is usually heated up to redness and allowed to cool. They are usually made of steel. Exposure to heat helps to ruin microorganisms in wine glass and porcelain apparatus.

The other method is sterilizing using damp heat which can be found in three different ways which are heating up in water or steam at 100oC, warming in steam under pressure and discontinue heating at low heat. The different ways are used based on the different materials or apparatus used.

The previous method is by using chemical, it could be either in water or gaseous status. They are often used in the disposal of polluted materials and equipment after a laboratory session.

Microorganisms worked with in a laboratory shouldn't be released in to the environment as these strains may contain hereditary markers such as antibiotic resistance. Therefore, they must be discarded properly.

In addition, aseptic approach isn't only applied in lab, it is also applied in professional medical and surgical setting.

Aims

There are two seeks in this test. The first purpose is to show that a sizable amount of microorganisms exist on the surface of our hands. The next aim is perform the aseptic technique properly by transferring 100 % pure culture and inoculating them onto an agar dish.

Materials and Methods

Bacteria on skin

Please make reference to the Lab Manual unless otherwise stated of changes made.

Streak Plate

Please make reference to the Lab Manual unless in any other case explained of changes made. A disposable sterile bacteriological loop is use instead of the metallic sterile bacteriological loop so no heat is necessary.

Discussion

Bacteria on skin

The individual skin's surface do hold a large quantity of microorganism which by cleaning hands, person can reduce the quantity of microorganism noticeably. However, even after having a hand rinse, microorganisms are still present on the surface.

Streak Plate

By utilizing the streaking approach on an agar plate correctly, a single colony can be acquired. Furthermore, it can be used to split up colonies of combined culture. Hence, this natural colony can be found and to be grown up in large quantity. From the result above, it could be observed that solitary colonies of the S. aureus are located. Due to the color and morphology, it can be known that the S. aureus is of a pure culture.

Conclusion

Aseptic approach is a basic laboratory technique that must definitely be hired especially during Microbiology laboratory session so as to prevent any contaminants and influencing the correctness of the effect. Since microorganism can replicated speedily, disposal of pollutants must be achieved properly to be able to protect both equipments and the fitness of individuals.

B- Gram Staining

Introduction

Gram stain is also known as differential stain in which it will split bacterias into two large communities, mainly Gram Positive and Gram Negative. This difference is due to the chemical substance and physical structure of the cell wall called peptidoglycan. During solvent treatment, if the peptidoglycan can wthhold the crystal violet dye, the bacterias will be group as Gram Positive bacterias. However, if it is unable to retain the crystal violet, the bacterias will be group as Gram Negative bacterias and that it will be stained green.

Gram Positive bacteria has a thicker peptidoglycan (50-90% cell wall membrane) as compared to the Gram Negative bacterias (10% cell wall). Furthermore, the Gram Negative bacteria has another part which is constitute 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 counterstaining with safranin.

After flooding the peptidoglycan with crystal violet stain, the dye will enter into the cells and everything cells will change purple. By adding iodine, a crystal violet-iodine complex will be form such that it will not be able to leave the cells easily. By decolourizing the cell with alcoholic beverages, the peptidoglycan of the Gram Negative bacteria will break down because the alcoholic beverages will dissolves the liposaccharides level and therefore, with removing the covering, the crystal violet-iodine organic will elope which will leads to the loss of the crystal violet stain and the cells turn colourless. Alternatively, the alcohol will dehydrate the Gram Positive bacteria's peptidoglycan, shutting the pores as the peptidoglycan shrinks. As a result, the crystal violet-iodine complex will never be able to run off as the "exits" will be obstructed and they continued to be stained. By counterstaining with safranin, the Gram Negative cell will flip pink and the Gram Positive cells will stay violet.

With gram staining, one is able to differentiate if the culture is a real or a merged, the morphological information on the bacterias and the set up of the bacterias.

Aims

The goal is to get ready smears for staining, observe the morphological details of the bacteria and to have the ability to distinguish between Gram Positive and Gram Negative bacterias.

Materials and Methods

Preparation of Smears for staining

Please refer to the Laboratory Manual unless in any other case mentioned of changes made. A disposable sterile bacteriological loop is use instead of the metal sterile bacteriological loop so no heat is necessary.

Gram Staining Method

Please refer to the Lab Manual unless often stated of changes made.

Discussion

According to the result witnessed, Bacillus subtilis is fishing rod shaped (bacillus). They are stained purple which suggests that they are Gram Positive bacterias. They are organized in singles. Although, endospore cannot be watch in this experiment, they may also be found on Bacillus subtilis. The endospore allows the bacterias to tolerate tough environmental condition such as temperature. Bacillus subtilis can even be known as an individual bacillus bacterium. Escherichia coli is stained pink and thereby, this is a Gram Negative bacterium. The skin cells are also rod shaped but they do not have any particular cell set up. They are located in singles, pairs and even clusters.

Proteus vulgaris is also stained pink and therefore, a Gram Negative bacterium. Its morphology rod shaped which is organized in singles. They may 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 designed, otherwise known cocci and they are usually arranged in grape-like clusters. Therefore, they are simply known as a staphylococci bacterium.

There were no distinctions in the shape and colour observed for each and every of the bacteria, hence, they could be known as a 100 % pure culture.

Conclusion

The Gram staining method is a useful tool used in most laboratories as it helps specific to visualise the bacterias effectively and effectively including the shape, design and even whether the culture is a 100 % pure or combined.

However, it ought to be noted that not all bacteria will give a gram effect as a few of them are gram changing, otherwise known as gram indeterminate. Therefore, they'll give a mix of pink and purple skin cells after gram staining. For a few of the Gram Positive bacteria, their peptidoglycan breaks easily during cell department, hence, after staining, they will give pink cells instead of purple. Furthermore, the length of time of a culture can also have an effect on the gram stain.

C- Cell Counting

Introduction

Cells keeping track of is the accurate and precise counting of cells. They are usually carried out physically or electronically. By keeping track of cells physically, a counting chamber, usually known as the haemocytometer can be used. The counting chamber is utilized to look for the number of skin cells per unit volume of a suspension. Alternatively, a coulter counter is used to count skin cells electronically.

There are two approaches to count the amount of skin cells, mainly total cell counts and the practical counts. Total cell matters are counted directly using the microscope and this both living and lifeless skin cells are counted. That is normally accompanied through the keeping track of chamber or coulter counter. Another approach is the viable counts which only count number the living skin cells. The small level of culture, often known as the dilution of the culture is applied to the surface of the agar dish. After incubating, the colonies are counted, normally colonies between 30-300 are chosen to be used for the computation of attentiveness of the given test. The models given is colony forming devices (CFU) per ml.

The haemocytometer is a altered glass slides with two count chamber of known area. Each chamber grid comprises nine squares which are known as subgrid, each square is 1mm2. Within each large square, there are further sub divisions that help in counting. If the coverslip is put on the grooves of the glide, you will see 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 very small plus they can be observed in a very lot, the suspensions should be diluted enough so that the cells have the ability to disperse uniformly in the counting chamber.

Aims

There are two seeks in this test. Firstly, to have the ability to determine the cell count number in different biological species and subsequently, to have the ability to determine the feasible count of a live bacterias, Staphylococcus aureus.

Materials and Methods

Cell Counting using Counting Chamber

Please make reference to the Lab Manual unless often mentioned of changes made. Serial Dilution is carried out before the test is loaded in to the Neubauer Manual Keeping track of Chamber. Normal saline (0. 9% NaCl) is used to dilute the blood and broth medium can be used to dilute the brewer's yeast ( Saccharomyces cervisiae). Both blood vessels and brewer's yeast are dilute in the proportion of 1 1:10 and 1:100. The 1:10 dilution is prepared by diluting the 10L of complete blood or yeast with 90L normal saline or broth medium respectively. The 1:100 dilution is made by extracting 10L from the individual test from 1:10 and adding 90L of normal saline and fungus into the respective sample.

Cell Counting of Live Bacterias, S. aureus (after Serial Dilution)

Please make reference to the Laboratory Manual unless usually mentioned of changes made. Two changes were made in Step one 1 and Step 12 respectively. Only three nutritional agar plates, 10-3, 10-4 and 10-5 were labelled and culture in these dilutions were distributed on the individual agar plates. A new spreader and pipette idea were used everything a different dilution culture was spread on the agar plate. Count the amount of colonies on the three different agar plates. Choose the agar dish with colonies between 30-300 to analyze the awareness of the initial sample.

Discussion

Using the counter chamber, person can provide a quick assessment on the amount of cells considering that all the task on planning and loading the sample about it. One of it is the fact that suspension system/sample is not combined before loading. That is because of the fact that cells tend to settle at the bottom of the pipe and therefore, while pipetting the sample right out of the tube, individual do not have the real or accurate variety of cells. Therefore, to get a uniform suspension for a far more accurate result, blending the tube before pipetting is recommended. In addition, it can also help in minimizing the clumping of skin cells.

Furthermore, improper filling up of chambers can lead to inaccurate volume of suspension system in the chamber and leading to inaccurate cell focus. Improper filling of chamber includes overfilling or under filling of sample.

Moreover, there must be a persistence in counting skin cells which is in touch with the boundary lines (ie. the three lines just outside the grid) or when the skin cells are clump collectively. Individual must determine which cells to rely and which never to count especially a cell which is situated on a boundary such as though a cell has half its area beyond your border, individual do not count up those cells.

The other method of cell counting is the practical count in which a single cell will give rise to a colony which is noticeable to the naked sight on the agar dish. Therefore, by counting the amount of colonies on the agar dish, individual is ready calculate the cell attention. However, only plates which have 30 to 300 colonies are used to determine the cell amount.

In the result for viable count of S. aureus, the dish 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 amount of colonies (328) surpass the amount of colonies of 300 that we were supposed to chose, this 10-5 dilution plate has the closest amount to 300. However, we should dilute even more because a single colony can have clumps or chain of skin cells in it and hence, resulting in inaccurate amount of colonies/cells in which the actual range of skin cells should actually be more than the calculated number of skin cells.

The benefit of viable cell counting would be that the organism counted is a positive one (ie. S. aureus) instead of any organism as though there exists contaminant, the morphology or color will be different.

Another downside of viable cell counting, other than cells that clump together or have chains which will form a single colony, is that organism will only increase in condition which is ideal for their progress on the agar plate.

Cell counting usually is combined with serial dilution as it is impossible to depend the number of skin cells if the amount is too much as it will lead to an extremely high number of skin cells.

Conclusion

There are several other methods, other than using counting chamber and feasible cell matter, to count skin cells in a suspension system. However, they will be the least expensive and is able to give accurate lead to a very short time of their time.

References

  • Abcam, 2009. Cell matters utilizing a haemocytometer. Abcam plc. Source: http://www. abcam. com/index. html?pageconfig=resource&rid=11454 Accessed: 1 Oct 2009
  • George Xu, 2007. Background of the Gram Stain and How it operates. University of Pennsylvania. Source: http://www. uphs. upenn. edu/bugdrug/antibiotic_manual/Gram1. htm Accessed: 1 Oct 2009
  • H. Kayser, A. Bienz, Johannes Eckert. M. Zinkernagel. 2005. Medical Microbiology. Thieme Stuttgart. , New York. p. 264-270.
  • Kenneth Todar, 2008. The Progress of Bacterial Society. Todar's Online Textbook of Bacteriology. Source: http://textbookofbacteriology. net/growth_2. html Accessed: 6 October 2009
  • Linda B, Mary R, 2007. Aseptic Copy. Austin Community College or university. Source: http://www. austincc. edu/microbugz/aseptic_technique. php Accessed: 3 Oct 2009
  • Steve Hogg, 2008. The Gram Stain. Newcastle University or college. Source: http://www. ncl. ac. uk/dental/oralbiol/oralenv/tutorials/gramstain. htm Accessed: 6 October 2009
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