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Introduction

Aseptic agencies to be free from micro-organism. Aseptic Technique is the process that is performed under unfertile status to forestall the growing of other micro-organisms on the growing medium such as the Petri dishes incorporating the alimentary agar or the pure civilization. If the growing medium or the pure civilization is contaminated with micro-organisms from the environment, it will consequences in confusion and inaccurate informations. Hence, it is of import to cut down the hazards of these micro-organisms to come in contact with the experimental stuffs.

In add-on, by keeping a clean environment when reassigning the civilization of micro-organism onto the alimentary agar is portion of the sterile technique. This is normally done by disinfecting the tabular array before and after working with micro-organism utilizing intoxicant. Flaring the experimental stuffs such as bacteriological cringles, bottle or flask cervixs can assist in sterilising. It must be done for several seconds so as to raise the temperature to kill the contaminations ; nevertheless, the bacteriological cringle must be cooled for a piece before it is used to pick up the micro-organism as picking up micro-organism with a hot tool will kill the cells. When taking caps from bottles, it is of import to maintain the cap in the manus as by seting them on the tabular array, it will be contaminated. Flaming is besides required before replacing the cap onto the bottle. It is of import to manage unfastened tubings at an angle so that airborne and other micro-organisms will non fall into the tubing and cause taint. During streaking, it is of import to maintain the palpebras of the Petri dish over it to forestall taint. Last, seek to avoid external respiration, coughing, sneezing and speaking while reassigning the civilization so as to cut down the hazard on contaminating.

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Apart from this general sterile technique, there besides several other methods to guarantee that devastation of populating micro-organisms in stuffs and setup. One of the methods is by utilizing dry heat which is sterilizing utilizing bare fire or hot air. Sterilizing stuffs or setup by a bare fire is normally heated to redness and allowed to chill. They are normally made of metal. Exposure to hot air helps to destruct micro-organisms in glass and porcelain setup.

The other method is sterilising utilizing moist heat which can be used in three different ways which are heating in H2O or steam at 100oC, heating in steam under force per unit area and discontinue warming at low temperature. The different ways are employed harmonizing to the different stuffs or setup used.

The last method is by utilizing chemical, it can be either in liquid or gaseous province. They are frequently used in the disposal of contaminated stuffs and setup after a research lab session.

Microorganisms worked with in a lab should non be released into the environment as these strains may incorporate familial markers such as antibiotic opposition. Therefore, they must be discarded decently.

In add-on, sterile technique is non merely applied in research lab, it is besides applied in clinical and surgical scene.

Purposes

There are two purposes in this experiment. The first purpose is to demo that a big figure of micro-organisms exist on the surface of our custodies. The 2nd purpose is carry out the sterile technique decently by reassigning pure civilization and inoculating them onto an agar home base.

Materials and Methods

Bacterias on tegument

Please refer to the Laboratory Manual unless otherwise stated of alterations made.

Streak Plate

Please refer to the Laboratory Manual unless otherwise stated of alterations made. A disposable unfertile bacteriological cringle is use alternatively of the metal unfertile bacteriological cringle so no warming is required.

Discussion

Bacterias on tegument

The human tegument ‘s surface do transport a big figure of micro-organism and that by rinsing custodies, single can cut down the figure of micro-organism perceptibly. However, even after a manus wash, micro-organisms are still present on the surface.

Streak Plate

By using the streaking technique on an agar home base right, a individual settlement can be obtained. Furthermore, it can be used to separate settlements of assorted civilization. Hence, this pure settlement can be picked up and to be grown in big measure. From the consequence above, it can be observed that individual settlements of the S.aureus are found. Due to the coloring material and morphology, it can be noted that the S.aureus is of a pure civilization.

Decision

Aseptic technique is a basic research lab technique that must be employed particularly during Microbiology research lab session so as to forestall any taint and impacting the truth of the consequence. Since micro-organism can retroflex quickly, disposal of contaminations must be done decently so as to protect both the equipments and the wellness of persons.

B- Gram Staining

Introduction

Gram discoloration is besides known as differential discoloration in which it will split bacteriums into two big groups, chiefly Gram Positive and Gram Negative. This difference is due to the chemical and physical construction of the cell wall called peptidoglycan. During solvent intervention, if the peptidoglycan is able to retain the crystal violet dye, the bacteriums will be group as Gram Positive bacterium. However, if it is non able to retain the crystal violet, the bacteriums will be group as Gram Negative bacterium and that it will be stained pink.

Gram Positive bacterium has a thicker peptidoglycan ( 50-90 % cell wall ) as compared to the Gram Negative bacterium ( 10 % cell wall ) . In add-on, the Gram Negative bacterium has another bed which is make up of liposaccharides and proteins and is separated from the cell wall by the periplasm.

In gm staining, there are four basic stairss which include deluging the heat fixed smear with crystal violet discoloration, following by the add-on of iodine solution to organize complex, adding of intoxicant for decolourisation and counterstaining with saffranine.

After deluging the peptidoglycan with crystal violet discoloration, the dye will come in the cells and all cells will turn violet. With the add-on of I, a crystal violet-iodine composite will be organize such that it will non be able to go out the cells easy. By bleaching the cell with intoxicant, the peptidoglycan of the Gram Negative bacteriums will interrupt down because the intoxicant will fade out the liposaccharides bed and hence, with the remotion of the bed, the crystal violet-iodine composite will run off which will consequences in the loss of the crystal violet discoloration and the cells turn colourless. On the other manus, the intoxicant will desiccate the Gram Positive bacterium ‘s peptidoglycan, shuting the pores as the peptidoglycan psychiatrists. As a consequence, the crystal violet-iodine composite will non be able to run off as the “ issues ” will be blocked and they remained stained. By counterstaining with saffranine, the Gram Negative cell will turn pink and the Gram Positive cells will stay violet.

With gram staining, one is able to distinguish if the civilization is a pure or a assorted, the morphological inside informations of the bacteriums and the agreement of the bacteriums.

Purposes

The purpose is to fix vilifications for staining, observe the morphological inside informations of the bacteriums and to be able to distinguish between Gram Positive and Gram Negative bacterium.

Materials and Methods

Preparation of Smears for staining

Please refer to the Laboratory Manual unless otherwise stated of alterations made. A disposable unfertile bacteriological cringle is use alternatively of the metal unfertile bacteriological cringle so no warming is required.

Gram Staining Method

Please refer to the Laboratory Manual unless otherwise stated of alterations made.

Discussion

Harmonizing to the consequence observed, Bacillus subtilis is rod shaped ( B ) . They are stained violet which suggests that they are Gram Positive bacteriums. They are arranged in singles. Although, endospore can non be observe in this experiment, they can besides be found on Bacillus subtilis. The endospore enables the bacterium to digest rough environmental status such as high temperature. Bacillus subtilis can besides be known as a individual B bacteria. Escherichia coli is stained pink and thereby, it is a Gram Negative bacteria. The cells are besides rod shaped but they do non hold any peculiar cell agreement. They are found in singles, braces and even bunchs.

Proteus vulgaris is besides stained pink and hence, a Gram Negative bacteria. Its morphology rod shaped and is arranged in singles. They can besides be known as a individual B bacteria.

Staphylococcus aureus is a Gram Positive bacteria as it is stained dark purple after gm staining. It has a spherical shaped, otherwise known coccus and they are normally arranged in grape-like bunchs. Therefore, they are known as a staphylococci bacteria.

There were no differences in the form and coloring material observed for each of the bacteriums, hence, they can be known as a pure civilization.

Decision

The Gram staining method is a utile tool used in most research labs as it helps single to visualize the bacteriums accurately and efficaciously such as the form, agreement and even whether the civilization is a pure or assorted.

However, it should be noted that non all bacteriums will give a gram reaction as some of them are gram variable, otherwise known as gm indeterminate. Therefore, they will give a mix of pink and purple cells after gm staining. For some of the Gram Positive bacteriums, their peptidoglycan interruptions easy during cell division, hence, after staining, they will give tap cells alternatively of purple. In add-on, the continuance of a civilization can besides impact the gm discoloration.

C- Cell Counting

Introduction

Cells numeration is the accurate and precise numeration of cells. They are normally carried out manually or electronically. By numbering cells manually, a numeration chamber, otherwise known as the haemocytometer is used. The counting chamber is used to find the figure of cells per unit volume of a suspension. On the other manus, a colter counter is used to number cells electronically.

There are two attacks to number the figure of cells, chiefly entire cell counts and the feasible counts. Total cell counts are counted straight utilizing the microscope and that both life and dead cells are counted. This is usually accompanied by the usage of the numbering chamber or colter counter. Another attack is the feasible counts which merely count the life cells. The little volume of civilization, otherwise known as the dilution of the civilization is applied to the surface of an agar home base. After incubating, the settlements are counted, usually settlements between 30-300 are chosen to be used for the computation of concentration of the given sample. The units given is colony organizing units ( CFU ) per milliliter.

The haemocytometer is a modified glass slides with two count chamber of known country. Each chamber grid is composed of nine squares which are known as subgrid, each square is 1mm2. Within each big square, there are farther bomber divisions that help in numeration. When the coverslip is placed over the channels of the slide, there will be a thickness of 0.1mm. Hence, the volume is 0.1mm3 or 1 ten 10-4ml. Therefore, the cell concentration will be calculated as the figure of cells multiply by 1 tens 104ml and once more, multiplying the dilution factor.

Since cells are really little and they can be observed in a really high figure, the suspensions should be diluted plenty so that the cells are able to administer uniformly in the numeration chamber.

Purposes

There are two purposes in this experiment. First, to be able to find the cell count in different biological species and secondly, to be able to find the feasible count of a unrecorded bacterium, Staphylococcus aureus.

Materials and Methods

Cell Counting utilizing Counting Chamber

Please refer to the Laboratory Manual unless otherwise stated of alterations made. Consecutive Dilution is carried out before the sample is loaded into the Neubauer Manual Counting Chamber. Normal saline ( 0.9 % NaCl ) is used to thin the blood and broth medium is used to thin the beer maker ‘s barm ( Saccharomyces cervisiae ) . Both blood and beer maker ‘s barm are dilute in the ratio of 1:10 and 1:100. The 1:10 dilution is prepared by thining the 10AµL of whole blood or barm with 90AµL normal saline or broth medium severally. The 1:100 dilution is prepared by pull outing 10AµL from the several sample from 1:10 and adding 90AµL of normal saline and barm into the several sample.

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

Please refer to the Laboratory Manual unless otherwise stated of alterations made. Two alterations were made in Step 1 and Step 12 severally. Merely three alimentary agar home bases, 10-3, 10-4 and 10-5 were labelled and civilization in these dilutions were spread on the several agar home bases. A new spreader and pipette tip were used everything a different dilution civilization was spread on the agar home base. Count the figure of settlements on the three different agar home bases. Choose the agar home base with settlements between 30-300 to cipher the concentration of the original sample.

Discussion

Using the counter chamber, single is able to give a speedy appraisal on the figure of cells given that all the process on preparing and lading the sample onto it. One of it is that suspension/sample is non assorted before lading. This is due to the fact that cells tend to settle at the underside of the tubing and hence, while pipetting the sample out from the tubing, single do non hold the existent or accurate figure of cells. Therefore, to acquire a unvarying suspension for a more accurate consequence, blending the tubing before pipetting is recommended. In add-on, it can besides assist in cut downing the clip-clop of cells.

Furthermore, improper filling of Chamberss can take to inaccurate volume of suspension in the chamber and taking to inaccurate cell concentration. Improper filling of chamber includes overfilling or under filling of sample.

Furthermore, there must be a consistence in numbering cells which is in contact with the boundary lines ( Internet Explorer. the three lines merely outside the grid ) or when the cells are clump together. Individual will hold to find which cells to count and which non to number particularly a cell which is situated on a boundary line such as if a cell has half of its country outside the boundary line, single do non number those cells.

The other method of cell numeration is the feasible count where a individual cell will give rise to a settlement which is seeable to the bare eyes on the agar home base. Therefore, by numbering the figure of settlements on the agar home base, single is able calculate the cell concentration. However, merely plates which have 30 to 300 settlements are used to cipher the cell concentration.

In the consequence for feasible count of S.aureus, the home base chosen was 10-5 because there was 82 settlements in one quarter-circle which is tantamount to 328 ( 82 x 4 ) settlements on the agar home base. Although, the figure of settlements ( 328 ) exceed the figure of settlements of 300 that we were supposed to take, this 10-5 dilution home base has the closest figure to 300. However, we should thin even further because a individual settlement can hold bunchs or concatenation of cells in it and therefore, ensuing in inaccurate figure of colonies/cells in which the existent figure of cells should really be more than the deliberate figure of cells.

The advantage of feasible cell numeration is that the being counted will be a positive 1 ( Internet Explorer. S.aureus ) alternatively of any other being as if there is contaminant, the morphology or coloring material will be different.

Another disadvantage of feasible cell numeration, other than cells that clump together or have ironss which will organize a individual settlement, is that being will merely turn in status which is suited for their growing on the agar home base.

Cell numbering normally is accompanied by consecutive dilution as it is impossible to number the figure of cells if the concentration is excessively high as it will take to a really high figure of cells.

Decision

There are several other methods, other than utilizing numbering chamber and feasible cell count, to number cells in a suspension. However, they are the least expensive and is able to give accurate consequence in a really short period of clip.

Mentions

  • Abcam, 2009. Cell counts utilizing a haemocytometer. Abcam plc. Beginning: hypertext transfer protocol: //www.abcam.com/index.html? pageconfig=resource & A ; rid=11454 Accessed: 1 October 2009
  • George Xu, 2007. History of the Gram Stain and How it Works. University of Pennsylvania. Beginning: hypertext transfer protocol: //www.uphs.upenn.edu/bugdrug/antibiotic_manual/Gram1.htm Accessed: 1 October 2009
  • H. 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. Todar ‘s Online Textbook of Bacteriology. Beginning: hypertext transfer protocol: //textbookofbacteriology.net/growth_2.html Accessed: 6 October 2009
  • Linda B, Mary R, 2007. Aseptic Transfer. Austin Community College. Beginning: hypertext transfer protocol: //www.austincc.edu/microbugz/aseptic_technique.php Accessed: 3 October 2009
  • Steve Hogg, 2008. The Gram Stain. Newcastle University. Beginning: hypertext transfer protocol: //www.ncl.ac.uk/dental/oralbiol/oralenv/tutorials/gramstain.htm Accessed: 6 October 2009

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