What is aseptic technique? Essay

Introduction

This study is based on the instructions of sterile techniques. Aseptic technique have two chief intents and these are:

  • To forestall any taint of cell civilizations in the lab.
  • To forestall taint from the bacteriums to any laboratory workers.

Asepsis within the research labs are indispensable as it is really easy to pollute cells and bring forth really unsafe bacteriums that can perchance acquire out into the environment. ‘All microbic cell civilizations should be treated as if they contained potentially harmful organisms.’ ( Reed, 2007 ) . The asepsis will find how successful a cell civilization will be.

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The manner in which a sterile environment is contained is by doing certain all the equipment is cleaned before and after usage, lavation of the custodies for the right sum of clip and the right manner, any spillages made whilst carry oning the experiments cleaned directly off with the right antimicrobic spray. There are plentifulness of other ways in which asepsis of research lab can be maintained these will be farther looked at in the treatment portion.

Use of a Bunsen burner – this is one of the most effectual ways in which to maintain equipment in the lab every bit unfertile as possible. The manner this works is the Bunsen fire is put on the coloring material blue ( this is the highest heat capacity the fire has ) . Any equipment such as inoculating bottles and flaring cringles is done at the top of the bluish trigon. When traveling to inoculate the bottle the babe finger must be used to take the palpebra and maintain it in that finger, by making this there is no transportation of bacteriums from the palpebra of the bottle on to the work surfaces, besides there is no taint from the work surfaces to the bottle. The ground the bottle is inoculated is to take any bacteriums that has managed to happen its manner to the cervix of the bottle.

Cupped – this is where the palpebra off the agar home base is removed and replaced within the infinite of a few seconds. This is done to cut down the chance of any airborne bacteriums come ining the agar home base and polluting the civilization. A manner to do certain this is done every bit effectual as possible is by positioning the fingers in a manner that will let the palpebra to be lifted a little sum and replaced whilst the other manus does whatever is needed on that peculiar agar home base.

Aseptic technique is method in which medical staff and research lab workers use to halt the spread of infections and harmful bacteriums that can do infections between people and topographic points. Their aim is to seek and derive conditions which reach full antisepsis, this is where the environment is wholly absent of bacteriums, viruses, and other harmful micro-organisms.

Purposes

  • To demo how many bacteriums is carried on the custodies and to place which 1s are present.
  • Practise sterile techniques by successfully inoculating an agar home base.
  • Carry out consecutive dilutions.
  • Introduce two methods of cell numbering.

Method

This is taken from the DPS1 practical agenda pages 22 – 33. The DPS1 agenda contains the methodological analysis on how to transport out each undertaking needed in this experiment. The lone alteration that was made was to make with the cell numeration of the blood and barm. On page 30 of the agenda the instructions are to utilize a 1 in 10 dilution for the blood. However, this produced excessively many cells to number when viewed under the microscope so the dilution factor was changed from that to 1 in 100.

Consequence

Bacterias on tegument:

The agar home base was split in half one side common manus and the other side washed manus. The difference viewed was that on the common side there were really few settlements, merely 4. However, on the washed side there was well more settlements present these werestaphylococcusandMicrococcus. Both types viewed were really little in size, level and either had a xanthous or white coloring material to it.

Streak home base

TheStaphylococcus aureuswas present on the agar home base and it produced individual celled beings. This will be looked at in more item and explained in the treatment subdivision.

Figure 1: Consequences obtained during the experiment from the run home base.

Cell numeration ofStaphylococcus aureus ( S.aureus )

30 – 300 is the home base that was used.

Table 1: Cells that were counted on 5 different agar home bases.

Dilutions

10-2

10-3

10-4

10-5

Number of cells

Excessively legion to number ( TNTC )

TNTC

TNTC

235

As seen in the tabular array above 3 out of the 4 home bases ofS.aureuswere excessively legion to number. The lone 1 that was counted was 10-5as this appeared to hold below 300 cells and more so 30.

A computation was so taken of the 10-5:

235 in 0.1ml

This was so multiplied by 10 to do the 235 in a solution of 1ml

2350 = 1ml of 10-5

2350?105

2.35?108Colony Forming Unit of measurements per milliliter ( I will now mention to it as CFUs )

Gram staining

Figure 2a: Gram discoloration ofEsherichia.coli ( E.coli )

ThisE.colivilification produced a negative discoloration. Its visual aspect was little rod shaped cells that were pink in coloring material. This will be looked at further in the treatment subdivision.

Figure 2b: Gram discoloration ofSaccharomyces cerevisiae ( S.cerevisiae )

TheS.cerevisiaevilification produced neither a positive or negative discoloration. This will be explained in the treatment subdivision. Its visual aspect was comparatively big rod shaped cells in comparing toE.coli, they were black in coloring material and looked like barm cells.

Figure 2c: Gram discoloration ofBacillus Cereus ( B.cereus )

TheB.cereusvilification produced a positive discoloration. These cells were violet in coloring material and singly short concatenation rods which had some branched and others individual celled.

Figure 2d: Gram discoloration ofS.aureus

TheS.aureusvilification produced a positive discoloration. These cells were violet in coloring material similar toE.colinevertheless, the agreement of them were grape like bunchs.

Cell numeration of blood and barm

Table 2: Number of blood cells 1 in 100 dilution counted in 5 squares of the numbering chamber with the norm taken.

Squares on the grid

1

2

3

4

5

Number of blood cells

393

345

327

402

309

The dilution 1 in 10 produced excessively many cells to number on the magnification ?10 so 1 in 100 was used. The manner this experiment was conducted was to blend 990 microns of phosphate buffer ( PBS ) with 10 microns of blood so mix that solution. It was placed on the numbering chamber 0.1mm deep, and looked at under a microscope.

The mean cells counted for the blood were 1776. A simple computation was so done in order to find the cell output of blood. A generation of 100 was taken as this solution was diluted as a 1 in 100.

393+345+327+402+309 = 1776

1776 ? 5 = 355.2

355.2?100 = 35520?104

3.55?108?3 = 1.07?109cells per milliliter

Table 3: Number of yeast cells 1 in 10 dilution counted in 5 squares of the numbering chamber with the norm taken.

Squares on the grid

1

2

3

4

5

Number of yeast cells

129

152

169

104

116

The dilution 1 in 10 was used as under the magnification of ?10 the cells were present and there weren’t excessively many. The same this as in the blood experiment was done nevertheless, alternatively of utilizing blood we used yeast with the PBS and besides it was merely done to the first phase. As we didn’t need to make the 2nd phase because the dilution needed was merely 1 in 10. Therefore 900 microns of PBS and 100 microns of barm.

The norm of cells counted for the barm were 670

A simple computation is so done to happen out the cell output nevertheless, unlike the blood a generation of 10 is taken as the barm was merely diluted 1 in 10.

129+152+169+104+116 = 670

670 ? 5 = 134

134?10 = 1340?104

1.34?107CFUs per milliliter

Discussion

Bacterias on tegument

The consequences from the experiment showed that ab initio there wasn’t many bacteriums on the tegument non even the occupant bacteriums that live on the tegument. However, after the custodies were washed there were many resident bacteriums nowadays on the tegument. This could hold been due to many factors such as ; once the custodies have been washed the transportation between the fingers and the agar home base is more ideal because it works better when the fingers are a small moisture. Another ground for the addition in bacteriums was soap strips any dead cells off the tegument and exposes bacteriums on the surface of the tegument. Although bacterium is present on the tegument even after being washed this is a good thing as these bacteriumsStaphylococcusandMicrococcusprotect our tegument, excite the immune system and besides prevent colonization from more unsafe bacteriums.

The consequences could besides bespeak that the cultural group of this person was African as there wasn’t many bacteriums present on the agar home base. The ground behind this tax write-off was that the melanin in the tegument is antimicrobic which is more protective.

Something that was non present on the tegument before rinsing was any transeunt cells as these don’t grow on the tegument nevertheless, are picked up from the environment. The ground they don’t occur on the tegument after rinsing is because of its transeunt nature.

Mistakes that could hold occurred were ; when persons went to rinse their custodies they need to touch the lights-outs with their fingers to turn it on and even though they have merely washed they hands they so proceed to shut this with their same clean fingers. This has now merely transferred all the bacterium left on the pat when they opened it back onto their clean custodies. Wayss to get the better of this is by either holding sensory lights-outs that don’t necessitate to be touched to turn on or off or even merely shuting the lights-outs after usage with the cubitus. Another mistake that could hold occurred was the single didn’t wash their custodies decently and for a long adequate period of clip. The manner to get the better of this is by holding postings of how the NHS wash their custodies near all the lights-outs so people can see this as they are rinsing their custodies. Besides as the NHS learn the person can sing happy birthday in their caput twice to do certain they have exhaustively cleaned their custodies for the right sum of clip.

Streak home base

The consequences from the experiment shown in figure 1 produced individual celled beings as expected. Other members that didn’t receive the right consequences could hold contaminated their experiment by either non flaring their cringle for a long adequate sum of clip or non chilling their cringle on the side of the agar home base by the borders before traveling to distribute the bacterium. Some persons could hold misunderstood the manner in which they were meant to distribute the bacteriums on the agar home base. Another mistake could hold been in the initial measure of taking the bacterium from the trial tubing and pulling it on the home base – if excessively much bacterium was taken out so the agar home base would be excessively concentrated and the person would run out of infinite seeking to distribute the bacteriums in the run like mode to bring forth the individual celled settlements.

Cell numeration ofS.aureus

As explained in the consequence subdivision 3 out of the 4 trials resulted in 235 cells being counted. When carry oning the experiment the dilution of 10-5was used to distribute on the agar home base foremost as it was the least concentrated and would hold no consequence on the concentrations above this.

Mistakes that could hold been incurred whilst carry oning this experiment were ; the dilutions could hold been excessively strong which would hold resulted in each agar home base holding excessively many cells to number. Another error could hold been that whilst doing the dilutions the barm in the bottle could non hold been shaken before being added to the PBS. This would hold meant that the precipitate of the barm would hold been left at the underside of the beaker, hence ensuing in a less dilute concentration of barm.

Does this method count all the cells inS.aureuscivilization?

No, as this method merely counts all the feasible cells within this civilization. The lone manner to see all the cells is by looking under a microscope.

Why is it merely plates between 30 – 300 settlements chosen?

Any settlements with over 300 cells is excessively legion to number with the bare oculus and acquire an accurate reply. The cells in this settlement are all viing for foods and infinite. This means that they are all clustered together so you may over count or under count. They besides interact with each other and in making so the cells might suppress or excite each other. This is another factor that affects how an person will number the cells in a settlement. Each of these grounds is why when carry oning an experiment over 300 is non counted and used.

Scientist have proven that by utilizing settlements with under 30 cells consequences show a random statistical mistake. This is due to the fact that because of random fluctuation it could be any figure between 0 – 30.

If a settlement produces at least 30 cells this decreases the random statistical mistake.

Gram staining

What is gram staining?

Gram staining is used to distinguish different types of bacterium. The manner in which this is done is by looking at its structural differences. The cell wall is what determines whether it will be pink in coloring material or purple. The pink coloring material produces a negative gm discoloration, and the violet coloring material produces a positive gm discoloration. This will be farther looked at when discoursing each set of consequences obtained by the gm staining.

Gram discoloration forE.coli

The consequences obtained from theE.coliwas that it produced a negative vilification. The bed of the cell wall peptidoglycan is comparatively thin in comparing to the positive gm discolorations. Any bacteriums with a comparatively thin cell wall will bring forth a negative staining as the cell wall does non keep the affinity to retain the violet coloring material from the crystal violet and Grams iodide when washed with intoxicant, as the outer bed of the bacteriums cell wall interruptions down due to the intoxicant. When this doesn’t produce a consequence the bacterium is so further stained with the Safranine which gives it its pink coloring material.

Gram discoloration ofB.cereusandGram discoloration ofS.aureus

The consequences obtained from theB.cereusandS.aureusshowed a positive gm staining. This is due to the thickness of the cell wall. As antecedently explained the gm negative staining had a comparatively thin wall with comparing to the gm positive. When any bacterium is washed with intoxicant if it is positive the cell wall will shrivel and pin down the discoloration from the crystal violet and gram iodide bring forthing a violet discoloration around the bacterium molecules.

As shown in figure 3 the differences between the cell wall for negative and positive differ drastically. The peptidoglycan in the gm positive is much thicker than the peptidoglycan in the gm negative.

http://water.me.vccs.edu/courses/env108/clipart/cellwall.gif

Figure 3: Structure of a gm positive and gram negative cell wall

Gram discoloration ofS.cerevisiae

( Figure 3b: Structure ofS.cerevisiaebacteriums )

This consequence proved to be neither positive nor negative. The ground for this is because this is a eucaryotic barm cell. The cell wall in eucaryotes are different so in procaryotes as they do non incorporate peptidoglycan. Peptidoglycan is what is responsible for the staining therefore the absence of this means that the staining method doesn’t work on any eucaryotic cells.

Mistakes that could hold occurred whilst conducting this experiment were:

Persons could hold accidently left the staining on the slides for excessively long ensuing in wrong ocular images of the coloring material of the molecule when looking under a microscope. The manner in which to get the better of this is merely by seeking to follow the instructions every bit closely as possible nevertheless, errors like this can’t be avoided as it is natural human mistake.

Another mistake could hold been usage of old gm staining or an old bacterium or barm molecule being used, which could ensue in the bacteriums and yeast non responding with the gm staining right. The manner to get the better of this is by merely doing certain that the staining is changed on a regular footing.

Cell numeration of blood and barm

This experiment looked at the sum of cells present in the blood and barm. The decision drawn from the consequences show that blood has a higher cell concentration so yeast. There was a huge difference in the norms, with bloods mean being about double the sum of barm. The blood and barm cells were diluted as explained in the consequences subdivision. The ground for this is because had they non been diluted there would hold been excessively many cells to number which was foremost witnessed when the experiment for blood was conducted at a 1 in 10 dilution ( there was excessively many cells to number ) .

Mistakes that could hold incurred whilst carry oning this experiment could hold been: excessively much visible radiation coming through the microscope, when numbering the cells in each grid at that place could hold either been an complete estimation or an under estimation. These are all human mistakes and are ineluctable the lone action that can be taken against these is to take utmost attention whilst carry oning the experiment. However, this reply doesn’t accurately state us how many cells are present in the blood and barm as we are taking an norm of five squares.

Mentions

hypertext transfer protocol: //www.lifetechnologies.com/uk/en/home/references/gibco-cell-culture-basics/aseptic-technique.html

hypertext transfer protocol: //www.healthline.com/health/aseptic-technique # Overview1

hypertext transfer protocol: //www.academia.edu/3555414/Biology_Escheria_coli_Gram_staining_-_Practical_report

Reed, R. ( 2007 ) . Practical accomplishments in biomolecular scientific disciplines. Harlean carpenter: Prentice Hall.

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