What is aseptic technique?

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INTRODUCTION

This report is based on the teachings of aseptic techniques. Aseptic technique have two main purposes and these are:

  • To prevent any contamination of cell cultures in the lab.
  • To prevent contamination from the bacteria to any laboratory workers.

Sterility within the laboratories are essential as it is very easy to contaminate cells and produce very dangerous bacteria that can possibly get out into the environment. ‘All microbial cell cultures should be treated as if they contained potentially harmful organisms.’ (Reed, 2007). The sterility will determine how successful a cell culture will be.

The way in which a sterile environment is contained is by making sure all the equipment is cleaned before and after use, washing of the hands for the correct amount of time and the correct way, any spillages made whilst conducting the experiments cleaned straight away with the correct antimicrobial spray. There are plenty of other ways in which sterility of laboratory can be maintained these will be further looked at in the discussion part.

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Use of a Bunsen burner – this is one of the most effective ways in which to keep equipment in the lab as sterile as possible. The way this works is the Bunsen flame is put on the colour blue (this is the highest heat capacity the flame has). Any equipment such as inoculating bottles and flaming loops is done at the top of the blue triangle. When going to inoculate the bottle the baby finger must be used to remove the lid and keep it in that finger, by doing this there is no transfer of bacteria from the lid of the bottle on to the work surfaces, also there is no contamination from the work surfaces to the bottle. The reason the bottle is inoculated is to remove any bacteria that has managed to find its way to the neck of the bottle.

Cupped – this is where the lid off the agar plate is removed and replaced within the space of a few seconds. This is done to reduce the probability of any airborne bacteria entering the agar plate and contaminating the culture. A way to make sure this is done as effective as possible is by positioning the fingers in a way that will allow the lid to be lifted a small amount and replaced whilst the other hand does whatever is needed on that particular agar plate.

Aseptic technique is method in which medical staff and laboratory workers use to stop the spread of infections and harmful bacteria that can cause infections between people and places. Their objective is to try and gain conditions which reach full asepsis, this is where the environment is completely absent of bacteria, viruses, and other harmful microorganisms.

AIMS

  • To show how many bacteria is carried on the hands and to identify which ones are present.
  • Practise aseptic techniques by successfully inoculating an agar plate.
  • Carry out serial dilutions.
  • Introduce two methods of cell enumeration.

METHODS

This is taken from the DPS1 practical schedule pages 22 – 33. The DPS1 schedule contains the methodology on how to carry out each task needed in this experiment. The only change that was made was to do with the cell counting of the blood and yeast. On page 30 of the schedule the instructions are to use a 1 in 10 dilution for the blood. However, this produced too many cells to count when viewed under the microscope so the dilution factor was changed from that to 1 in 100.

RESULTS

Bacteria on skin:

The agar plate was split in half one side unwashed hand and the other side washed hand. The difference viewed was that on the unwashed side there were very few colonies, only 4. However, on the washed side there was considerably more colonies present these were staphylococci and micrococcus. Both types viewed were very small in size, flat and either had a yellow or white colour to it.

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Streak plate

The Staphylococcus aureus was present on the agar plate and it produced single celled organisms. This will be looked at in more detail and explained in the discussion section.

Figure 1: Results obtained during the experiment from the streak plate.

Cell counting of Staphylococcus aureus (S.aureus)

30 – 300 is the plate that was used.

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

Dilutions

10-2

10-3

10-4

10-5

Number of cells

Too numerous to count (TNTC)

TNTC

TNTC

235

As seen in the table above 3 out of the 4 plates of S.aureus were too numerous to count. The only one that was counted was 10-5 as this appeared to have below 300 cells and more then 30.

A calculation was then taken of the 10-5:

235 in 0.1ml

This was then multiplied by 10 to make the 235 in a solution of 1ml

2350 = 1ml of 10-5

2350×105

2.35×108 Colony Forming Units per ml (I will now refer to it as CFUs)

Gram staining

"" Figure 2a: Gram stain of Esherichia.coli (E.coli)

This E.coli smear produced a negative stain. Its appearance was small rod shaped cells that were pink in colour. This will be looked at further in the discussion section.

""Figure 2b: Gram stain of Saccharomyces cerevisiae (S.cerevisiae)

The S.cerevisiae smear produced neither a positive or negative stain. This will be explained in the discussion section. Its appearance was relatively large rod shaped cells in comparison to E.coli, they were black in colour and looked like yeast cells.

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Figure 2c: Gram stain of Bacillus cereus (B.cereus)

The B.cereus smear produced a positive stain. These cells were purple in colour and singly short chain rods which had some branched and others single celled.

""Figure 2d: Gram stain of S.aureus

The S.aureus smear produced a positive stain. These cells were purple in colour similar to E.coli however, the arrangement of them were grape like clusters.

Cell counting of blood and yeast

Table 2: Number of blood cells 1 in 100 dilution counted in 5 squares of the counting chamber with the average 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 too many cells to count on the magnification ×10 so 1 in 100 was used. The way this experiment was conducted was to mix 990 micrometers of phosphate buffer (PBS) with 10 micrometers of blood then mix that solution. It was placed on the counting chamber 0.1mm deep, and looked at under a microscope.

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The average cells counted for the blood were 1776. A simple calculation was then done in order to determine the cell yield of blood. A multiplication 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 = 1.07×109 cells per ml

Table 3: Number of yeast cells 1 in 10 dilution counted in 5 squares of the counting chamber with the average 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 too many. The same this as in the blood experiment was done however, instead of using blood we used yeast with the PBS and also it was only done to the first stage. As we didn’t need to do the second stage because the dilution needed was only 1 in 10. Therefore 900 micrometers of PBS and 100 micrometers of yeast.

The average of cells counted for the yeast were 670

A simple calculation is then done to find out the cell yield however, unlike the blood a multiplication of 10 is taken as the yeast was only diluted 1 in 10.

129+152+169+104+116 = 670

670 ÷ 5 = 134

134×10 = 1340×104

1.34×107 CFUs per ml

DISCUSSION

Bacteria on skin

The results from the experiment showed that initially there wasn’t many bacteria on the skin not even the resident bacteria that live on the skin. However, after the hands were washed there were many resident bacteria present on the skin. This could have been due to many factors such as; once the hands have been washed the transfer between the fingers and the agar plate is more ideal because it works better when the fingers are a little wet. Another reason for the increase in bacteria was soap strips any dead cells off the skin and exposes bacteria on the surface of the skin. Although bacteria is present on the skin even after being washed this is a good thing as these bacteria Staphylococci and micrococcus protect our skin, stimulate the immune system and also prevent colonisation from more dangerous bacteria.

The results could also indicate that the ethnic group of this individual was African as there wasn’t many bacteria present on the agar plate. The reason behind this deduction was that the melanin in the skin is antimicrobial which is more protective.

Something that was not present on the skin before washing was any transient cells as these don’t grow on the skin however, are picked up from the environment. The reason they don’t occur on the skin after washing is because of its transient nature.

Errors that could have occurred were; when individuals went to wash their hands they need to touch the taps with their fingers to turn it on and even though they have just washed they hands they then proceed to close this with their same clean fingers. This has now just transferred all the bacteria left on the tap when they opened it back onto their clean hands. Ways to overcome this is by either having sensory taps that don’t need to be touched to turn on or off or even simply closing the taps after use with the elbow. Another error that could have occurred was the individual didn’t wash their hands properly and for a long enough period of time. The way to overcome this is by having posters of how the NHS wash their hands near all the taps so people can view this as they are washing their hands. Also as the NHS teach the individual can sing happy birthday in their head twice to make sure they have thoroughly cleaned their hands for the correct amount of time.

Streak plate

The results from the experiment shown in figure 1 produced single celled organisms as expected. Other members that didn’t receive the correct results could have contaminated their experiment by either not flaming their loop for a long enough amount of time or not cooling their loop on the side of the agar plate by the edges before going to spread the bacteria. Some individuals could have misunderstood the way in which they were meant to spread the bacteria on the agar plate. Another error could have been in the initial step of taking the bacteria from the test tube and drawing it on the plate – if too much bacteria was taken out then the agar plate would be too concentrated and the individual would run out of space trying to spread the bacteria in the streak like manner to produce the single celled colonies.

Cell counting of S.aureus

As explained in the result section 3 out of the 4 tests resulted in 235 cells being counted. When conducting the experiment the dilution of 10-5 was used to spread on the agar plate first as it was the least concentrated and would have no effect on the concentrations above this.

Errors that could have been incurred whilst conducting this experiment were; the dilutions could have been too strong which would have resulted in each agar plate having too many cells to count. Another mistake could have been that whilst making the dilutions the yeast in the bottle could not have been shaken before being added to the PBS. This would have meant that the precipitate of the yeast would have been left at the bottom of the beaker, therefore resulting in a less dilute concentration of yeast.

Does this method count all the cells in S.aureus culture?

No, as this method only counts all the viable cells within this culture. The only way to see all the cells is by looking under a microscope.

Why is it only plates between 30 – 300 colonies chosen?

Any colonies with over 300 cells is too numerous to count with the naked eye and get an accurate answer. The cells in this colony are all competing for nutrients and space. This means that they are all clustered together so you may over count or under count. They also interact with each other and in doing so the cells might inhibit or stimulate each other. This is another factor that affects how an individual will count the cells in a colony. Each of these reasons is why when conducting an experiment over 300 is not counted and used.

Scientist have proven that by using colonies with under 30 cells results show a random statistical error. This is due to the fact that because of random variation it could be any number between 0 – 30.

If a colony produces at least 30 cells this decreases the random statistical error.

Gram staining

What is gram staining?

Gram staining is used to differentiate different types of bacteria. The way in which this is done is by looking at its structural differences. The cell wall is what determines whether it will be pink in colour or purple. The pink colour produces a negative gram stain, and the purple colour produces a positive gram stain. This will be further looked at when discussing each set of results obtained by the gram staining.

Gram stain for E.coli

The results obtained from the E.coli was that it produced a negative smear. The layer of the cell wall peptidoglycan is relatively thin in comparison to the positive gram stains. Any bacteria with a relatively thin cell wall will produce a negative staining as the cell wall does not hold the affinity to retain the purple colour from the crystal violet and Grams iodide when washed with alcohol, as the outer layer of the bacteria cell wall breaks down due to the alcohol. When this doesn’t produce a result the bacteria is then further stained with the Safranine which gives it its pink colour.

Gram stain of B.cereus and Gram stain of S.aureus

The results obtained from the B.cereus and S.aureus showed a positive gram staining. This is due to the thickness of the cell wall. As previously explained the gram negative staining had a relatively thin wall with comparison to the gram positive. When any bacteria is washed with alcohol if it is positive the cell wall will shrink and trap the stain from the crystal violet and gram iodide producing a purple stain around the bacteria molecules.

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

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

Gram stain of S.cerevisiae

(Figure 3b: Structure of S.cerevisiae bacteria)

This result proved to be neither positive nor negative. The reason for this is because this is a eukaryotic yeast cell. The cell wall in eukaryotes are different then in prokaryotes as they do not contain peptidoglycan. Peptidoglycan is what is responsible for the staining therefore the absence of this means that the staining method doesn’t work on any eukaryotic cells.

Errors that could have occurred whilst conducting this experiment were:

Individuals could have accidently left the staining on the slides for too long resulting in incorrect visual images of the colour of the molecule when looking under a microscope. The way in which to overcome this is simply by trying to follow the instructions as closely as possible however, mistakes like this can’t be avoided as it is natural human error.

Another error could have been use of old gram staining or an old bacteria or yeast molecule being used, which could result in the bacteria and yeast not reacting with the gram staining correctly. The way to overcome this is by simply making sure that the staining is changed on a regular basis.

Cell counting of blood and yeast

This experiment looked at the amount of cells present in the blood and yeast. The conclusion drawn from the results show that blood has a higher cell concentration then yeast. There was a vast difference in the averages, with bloods average being almost double the amount of yeast. The blood and yeast cells were diluted as explained in the results section. The reason for this is because had they not been diluted there would have been too many cells to count which was first witnessed when the experiment for blood was conducted at a 1 in 10 dilution (there was too many cells to count).

Errors that could have incurred whilst conducting this experiment could have been: too much light coming through the microscope, when counting the cells in each grid there could have either been an over estimate or an under estimate. These are all human errors and are unavoidable the only action that can be taken against these is to take extreme care whilst conducting the experiment. However, this answer doesn’t accurately tell us how many cells are present in the blood and yeast as we are taking an average of five squares.

References

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

http://www.healthline.com/health/aseptic-technique#Overview1

http://www.academia.edu/3555414/Biology_Escheria_coli_Gram_staining_-_Practical_report

Reed, R. (2007). Practical skills in biomolecular sciences. Harlow: Prentice Hall.

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