Introduction to Aseptic techniques
In microbiological and biochemical technology surveies, one about ever trades with a pure civilization or a mixture of known civilizations, except possibly in waste H2O intervention surveies. Unless sterile civilization techniques are followed purely, an originally pure civilization will decidedly go contaminated with other unwanted species. The usage of a contaminated civilization with unknown micro-organisms will merely take to unbelievable consequences that are of small value. Thus, isolation and care of a pure civilization is of extreme importance in many microbiological surveies. It is particularly of import to work with a well-characterised strain if the micro-organism is used for a nutrient readying, every bit good as in antibiotic production where the merchandise is to be taken internally.
The demand for a clean working environment in biological technology surveies is a must and cleanliness is the requirement for any meaningful work. The environment we live in is full of micro-organisms capable of lasting in any status.
When working with micro-organisms it is desirable to work with a pure civilization. A pure civilization is composed of merely one sort of micro-organism. Occasionally a assorted civilization is used. In a assorted civilization there are two or more beings that have distinct features and can be separated easy. In either state of affairs the beings can be identified. When unwanted beings are introduced into the civilization they are known as contaminations.
Aseptic technique is a method that prevents the debut of unwanted beings into an environment. An illustration of utilizing sterile techniques is when turning bacteriums ; sterile techniques are carried out to forestall the taint of the civilization. When working with microbic civilizations sterile technique is used to forestall presenting extra beings into the civilization.
Microorganisms are everyplace in the environment. When covering with microbic civilizations it is necessary to manage them in such a manner that environmental beings do non acquire introduced into the civilization. Microorganisms may be found on surfaces and drifting in air currents. They may fall from objects suspended over a civilization or swim in fluids. Aseptic technique prevents environmental beings from come ining a civilization.
Doors and Windowss are kept closed in the research lab to forestall air currents which may do micro-organisms from surfaces to go airborne.
Once these bugs are airborne they are more likely to acquire into civilizations.
Agar home bases are held in a mode that minimizes the exposure of the surface to the environment. When taking palpebras from tubings, palpebras are held in the manus and non placed on the countertop during the transportation of stuffs from one tubing to another.
Introduction and purposes of this study
This study was based on two lab Sessionss ; the ground for set abouting two lab Sessionss was to let clip for the civilization to turn. In these Sessionss the aim was to larn basic sterile techniques that are required in a lab, and besides to larn how to number cells efficaciously utilizing different methods. Another thing which was seeking to be achieved was to be able to turn a settlement of bacteriums from a individual cell by making streak plating and consecutive dilution.
Overview of patterns and utilizations of sterile techniques
The research lab session involved larning about the sterile techniques. These sterile techniques are of import in a lab because they help maintain the lab sterile, and asepsis is critical in a lab because it allows the scientist to analyze and turn the bacterium they require accurately. Asepsis is besides of import in forestalling bacteriums that are non required from retroflexing and turning on the unfertile growing medium or the agar home base.
There were a few sterile techniques we had to follow while working with bacterium ‘s and unfertile growing medium. To forestall the growing medium from being contaminated by air dullard bacteriums and other free drifting affair, a Bunsen burner was set up near where the growing medium and bacteriums samples were to be used. The Bunsen burner created a convection current that killed and destroyed most of the air borne bacteriums and other free drifting affair near the work station. This reduced the opportunity of the growing medium and bacteriums samples from being contaminated.
The Bunsen burner was besides set up to let the usage of another technique called fire. This technique involves go throughing through the fire of the burner anything that has come in to reach of any bacteriums or anything that is about to come in to reach of the bacterium sample. The points that are flamed are lab equipment such as bacteriological cringles, glass pipette and bottle or flask cervixs. The points must make a temperature of over 100 oC for it to be sterilized.
Another sterile technique is called use. In this technique the smallest finger is used to take the palpebra of the bottle incorporating the bacterium ; this allows the remainder of the fingers to pick up anything else that is required. This technique besides ensures the palpebra of the bottle is non placed down onto the bench where it is apt to taint and therefore polluting the civilization of bacteriums in the bottle.
The last but the most of import sterile technique is who person prevents bacteria from themselves polluting the lab and the equipment. Every individual carries a big sum of bacteriums inside and the exterior of the organic structure. When working with bacteriums in a lab, we had to have oning a lab coat, this prevent bacterium from our apparels and organic structures distributing out in the lab. Besides we had to be careful that we do n’t cough or sneeze on the growing medium, as this would take to the growing of the bacteriums released by the organic structure. Besides after executing the experiment it was critical that custodies were washed with antibacterial soap to assist forestall transverse taint. If custodies are non washed right and if bacteriums are still left on custodies they are able to multiply at an exponential rate and can do bacterial infections.
The first portion of the experiment was to see the different fluctuation and sum of bacteriums on custodies pre wash and after wash. This was done by puting the fingers in a Petri dish with alimentary agar. Nutrient agar is a microbiological growing medium normally used for the everyday cultivation of bacteriums. The dish was separated in two and was labelled with one side of dish holding prints from pre washed fingers and the other side after wash. The dish was so placed into incubation at 37 grades as it is the optimal temperature where bacteriums are able to multiply at an exponential rate depending on some factors an illustration being the sum of nutrient available or infinite.
The following portion of the experiment consisted of making a streak home base. This was done utilizing the bacterium Staphylococcus aureus. Small sample of the bacteriums SA was taken and put on a on a unfertile cringle and streak an agar medium. An illustration of the run home base which was carried out is shown on the diagram below:
Diagram to demo process of run plating
A 1.A Flame the cringle and wire and streak a loopful of stock as at A in the diagram.
A 2.A Reflame the cringle and cool it.
A 3.A Streak as at B to distribute the original bacterium over more of the agar.
A 4.A Reflame the cringle and cool it.
A 5.A Streak as at C, D E and F following same process after each run as quoted above.
A 6.A Label the home base and incubate it upside-down.
The following portion of the first session was to make consecutive dilution. This allows you to find the figure of cells in a bacterial civilization. Since bacterial cell Numberss are normally really high in the original sample, plating out this sample in an undiluted manner would merely take to the creative activity of a bacterial lawn ( a vilification of many, many single bacteriums settlements that are all turning following to or on top of one another ) .
Bacterial cell Numberss need to be reduced, which is done by repeatedly thining the sum of bacteriums in the sample. A little sum of bacteriums sample is assorted with a diluent solution ( such unfertile stock ) , and so consecutive dilutions are made. A little sum of each of the diluted bacterium samples is so dispersed onto an agar home base. The Numberss of bacterium settlements that grow on each home base are counted. By working backwards utilizing generation with the “ dilution factor ” ( the figure of times that you have diluted the bacteriums sample with the diluent solution ) , we were able to do a finding of the Numberss of bacteriums in the original sample. After the dilutions were created 100 Aµl of each dilution was transferred to an agar home base utilizing a pipette, it was so spread around the agar home base with a spreader. These six agar home bases were so put into incubation at 37 A°C for 24 hours. When distributing the bacterial lawn the home base with the dilution degree 10-5 was done foremost and so the others 10-4, 10-3, 10-2.this is because the spreader which was used was fictile so the lower concentrated bacteria was spread foremost as the plastic spreader could non be flamed to kill the bacteriums. If this sterile technique was non used and the highest concentration of bacterium was used first it would hold meant that the bacterial dishes would hold become contaminated and besides individual settlements of bacteriums would non be gained. If a glass spreader was used so it could hold done in go uping order as the glass could be flamed by puting ethyl alcohol on the surface killing the bacterium on the glass spreader before making the following portion of the consecutive dilution.
The concluding portion of the first lab Sessionss was to fix vilifications of bacteriums for gm staining. Gram staining is a common technique used to distinguish two big groups of bacteriums based on their different cell wall components. The Gram discoloration process distinguishes between Gram positive and Gram negative groups by coloring these cells pink or violet. Gram positive bacteriums stain violet due to the presence of a thick bed of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteriums stain pink, which is attributed to a dilutant peptidoglycan wall, which does non retain the crystal violet during the decolouring procedure.
Gram staining involves three procedures: staining with a water-soluble dye called crystal violet, decolourisation, and counterstaining, normally with safanin. Due to differences in the thickness of a peptidoglycan bed in the cell membrane between Gram positive and Gram negative bacteriums, Gram positive bacteriums ( with a thicker peptidoglycan bed ) retain crystal violet discoloration during the decolourisation procedure, while Gram negative bacteriums lose the crystal violet discoloration and are alternatively stained by the saffranine in the concluding staining procedure. The procedure involves three stairss:
1. Cells are stained with crystal violet dye. Next, a Gram ‘s iodine solution ( iodine and potassium iodide ) is added to organize a composite between the crystal violet and I. This composite is a larger molecule than the original crystal violet discoloration and I and is indissoluble in H2O.
2. A decolouriser such as ethyl intoxicant or propanone is added to the sample, which dehydrates the peptidoglycan bed, shriveling and fastening it. The big crystal violet-iodine composite is non able to perforate this tightened peptidoglycan bed, and is therefore trapped in the cell in Gram positive bacteriums. Conversely, the outer membrane of Gram negative bacterium is degraded and the dilutant peptidoglycan bed of Gram negative cells is unable to retain the crystal violet-iodine composite and the coloring material is lost.
3. A counter discoloration, such as the decrepit H2O soluble saffranine, is added to the sample, staining it pink. Since the saffranine is lighter than crystal violet, it does non interrupt the violet colour in Gram positive cells. However, the decolourised Gram negative cells are stained pink.
( The descriptive methods are shown in the enchiridion for all experiments. )
Consequences for the gm staining
After following the method as stated in the enchiridion we examined the slides under a microscope utilizing the oil submergence aim of 100x. We so noted the form of the bacteriums that could be seen and the coloring material being violet ( Gram positive ) or tap ( Gram negative ) . Below are the hints of the bacteriums which could be seen under the microscope.
Figure 1 – Staphylococcus aureus – gm positive ( purple )
Description of what could be seen:
– Coccus shaped bacteriums
– Guerrilla bunchs of bacterial cells
Figure 2 – Bacillus cereus – gm positive ( purple )
Description of what could be seen:
– Rod shaped bacterial cell
– Singular bacteriums
Figure 3 – Saccharomyces cerevisiae – gm positive ( purple )
Description of what could be seen:
– Coccus shaped bacteriums
– Bunchs of bacteriums closely packed
Figure 4 – E-coli – gm negative ( pink )
Description of what could be seen:
– Rod shaped bacteriums cells
– Linked ( threading like, filiform )
Consequences of the experiments
After 24 hours the agar plates with the bacteriums were ready to be viewed. First the agar dishes with the manus prints were viewed. Below is a diagram of the agar dish and the bacterium which was present:
There are different bacteriums which were present while detecting the dish, they were the following
A – The first bacteriums which were seen and labelled as A were the largest of the three seeable settlements which are circle in form and yellow in coloring material with smooth borders, they can be seen to hold a somewhat crookback surface.
B – These are somewhat smaller in size than the 1s described above and are besides circle in form but are white in coloring material, once more the borders are smooth and the surface is humped.
C- These bacteria were merely seen after rinsing custodies they had no specific form and were a lighter coloring material which was non really clear. They were level with unsmooth borders.
The following dish which was observed was the run plating dish, this had been left to incubate for 24 hours besides. The consequences are shown on the image below:
A sample of Staphylococcus aureus was inoculated onto an agar home base utilizing the run home base method.
From this diagram it can be seen that portion 1 shows a higher concentration of bacteriums. Partss 2, 3 have fewer bacteriums but still there are really few individual settlements. Separate 4 shows many different individual settlements of Staphylococcus aeurus and are easy seeable.
The concluding dish which was viewed after 24 hours of incubation was the dish containing Staphylococcus aureus where it had been diluted to 10-5. These consequences can be seen below where there is diagram of the Petri dish including the bacteria.
From numbering the settlements on the dish the figure which was calculated was 486. The sum of settlements calculated was still rather high as the preferable figure of settlements would hold been from 30-300. This may hold been achieved if the consecutive dilution was carried farther.
To cipher the sum of cells in this agar home base foremost the followers was done:
0.1ml of solution = 4.86×10-2 ( 486 )
1ml of solution = 4.86×10-3
so as it was the consecutive dilution of 10-5 the computation was so multiplied by 5 to give the concluding reply figure of bacterial cells = 4.86×10-8
The last portion of the lab session was to number cells utilizing an Improved Neubauer Counting Chamber besides sometimes known as a haemocytometer. The chief aim of this session was to be able to cipher the entire cells in the given sample. Below is a diagram of a haemocytometer with the slide placed over it:
The haemocyometer contains 9 big squares under the microscope at the 40X lens. The country of the square can be measured at 1mm2. The manner to separate these squares from one another is by the tripe dense lines. Within each big square there are smaller grids which can be used to assist during numeration. Besides when the numbering the bacterium cells at that place was a fit manner to make this as shown below in the diagram: –
So as can be seen in the diagram if the bacterium cells are at placed on the border of the little squares so they will non be recorded. The orange lines stand foring cells will non be recorded as they are the terminal of the grid.
3 heavy line dividing each big square
The manner the cells were counted was to place which squares were traveling to be used to detect and cipher the figure of bacteria nowadays. The manner this was done was there were 9 squares and merely 5 squares were chosen as shown in the diagram below:
Squares 1, 3, 5, 7 and 9 were the squares which were used to number the bacteriums.
Once the haemocytometer was set and the proposed samples were placed into the numeration Chamberss and so placed under a microscope to position ( extended method is described in the faculty enchiridion ) . The cell count was done for two different cell suspensions whole blood ( ovine ) and beer maker ‘s barm ( Saccharomyces cervisiae ) the samples were non diluted. They were so counted and the consequences are shown in the tabular array below.
Table to demo cell count from haemocytometer for the whole blood
Number Of Cells Present
To cipher the sum cell count in the orderly solution a computation was needed. First the mean figure of cells was needed to be worked out. The amount was 237/5=47.4. To work out the cell figure it was multiplied by 1×10-4=4.74×10-5 So so eventually to work out the cell yield the figure of cells/ml was used which was 4.7×10-5 and was multiplied by the entire volume of 10ml and therefore the output calculated was 4.74×10-6.
Table to demo cell count from haemocytometer for the Brewer ‘s barm
Number Of Cells Present
The same stairss were taken to work out the cell figure and output for the Brewer ‘s barm.
Cell figure = 1.62×10-5
Cell Yield = 1.62×10-6
Table to demo cell figure and cell output of both samples
Brewer ‘s barm
Discussion of consequences
In this portion of the study I will mention to the consequences obtained and assess if they were accurate or non to the survey of sterile techniques. First the practical which involved looking at bacteriums on the tegument showed that after rinsing at that place was fewer bacteriums but another signifier of bacterial cells started to turn. The ground for this could hold been that when shuting the lights-outs I may hold used my custodies. The lights-outs in the lab are made o be closed by the carpus so the bacterium is unable to come in contact with the surface of your custodies. The ground for this is because if you wash your custodies and so shut the lights-outs with your custodies once more so you are merely roll uping the bacteriums off the lights-outs once more. In some instances the sum of microbacterium on the tegument can increase after rinsing, this is because by covering the tegument with H2O you are doing conditions for microrganisms more favorable and therefore more will turn. It is hence apprehensible that the growing of micro-organisms will depend upon the chemical composing of the tegument, for illustration if it is dry or whether it has a low pH. Most micro-organisms that are present on the tegument are located near hair follicles or sudate secretory organs this is because they provide the foods and the right environment for there growing.
Besides another ground for the bacteriums still being at that place after rinsing custodies on the agar dish was because of the procedure of rinsing custodies. The procedure of rinsing custodies should be done surgically as there is non such thing as portion sterile. So to do certain that all sources and bacteriums are washed off from clambering the process of manus lavation should be followed right.
There are over 100 different types of bacteriums on custodies. The most common types of bacteriums found on custodies are familiar family names: Propionobacterium ( the bacterium responsible for acne ) , strep, and staph ( of which the ill-famed methicillin immune staph aureus, MRSA is a subtype ) . Not all these bacteriums are harmful as skin infections do non originate because you have bacteria on your tegument. Rather, they arise because the type of bacteriums on septic tegument is non healthy bacteriums but aggressive infective bacteriums.
Streak plating treatment
From looking at the consequences obtained from the run plating it can be seen that the run home base was non really accurate as the intended consequence was non achieved. The purpose of this experiment was to seek and derive individual settlements but the job with the run home base which I had carried out was that there was non adequate room for the individual settlements to progress. This was because the initial vaccination runs were excessively thick and so took up excessively much infinite therefore go forthing small infinite in the center of the home base for individual settlements. It is hence needed that the initial runs are made dilutant and screen, as a unsmooth estimation, the outer 2cm of the agar home base therefore, go forthing plentiful infinite at the Centre of the home base for individual settlements to turn. The job with this process is that each settlement may non stand for the offspring from one cell, as two or more cells which are really near together could look as one settlement. Another job which may hold caused trouble accomplishing individual settlements may hold been the concentration of the bacteriums. If the bacteriums were diluted it may hold helped to accomplish individual settlements.
The individual settlements which were achieved were all similar to one another this shows that the bacteria which was present in them settlements was the same bacterium. This was achieved as the vaccinating cringle was sterilised each clip so merely the bacteria which was being used grew on the agar dish.
In this portion of the practical there were four different bacteriums which were tested by utilizing the gm staining procedure to see if they were gram positive or gram negative. The first bacteria was Staphylococcus aureus, Bacillus Cereus and Saccharomyces cerevisiae which were gms positive bacteria as after proving the bacterium under the microscope it showed that it was stained purple. The SA under the microscope was seen like a clump of grapes as its names suggest as Staphyle in Greek footings meant grapes. Staphylococcus aureus is a bacteria, often populating on the tegument or in the olfactory organ of a healthy individual that can do unwellnesss runing from minor tegument infections and abscesses, to dangerous diseases such as pneumonia, meningitis, endocarditis and blood poisoning.
The Bacillus Cereus is a facultative anaerobiotic bacteria associated with nutrient toxic condition in worlds. The nutrient toxic condition is a consequence of consuming toxins produced by the bacteriums. B. Cereus is widespread in the dirt and the nutrient industry in such nutrients as herbs, spices, milk, and veggies. Transmission of this disease consequences non merely from contaminated nutrients, but besides from improper nutrient handling/storage and improper chilling of cooked nutrient. The bacterium seen under the microscope and as can be seen in the diagram ( figure 2 ) shows the bacterium as rod shaped bacteriums which do non constellate together and are separated around the slide, in different waies and are non in any peculiar order as they all in different angles.
The Saccharomyces cerevisiae is besides known better as barm which could be used for baking or used while doing intoxicant. These cells where seen under the microscope as individual cells which were rounded molded cells and were closely packed together in groups.
The last bacteria was the E. coli which was stained pink as this was a gram negative bacteria. This bacteria is found in animate beings and birds in the lower bowels it helps with the digestion of nutrient. If E. coli is ingested it will do the little bowel to go inflamed. Peoples can contract an E. coli infection by imbibing contaminated H2O, eating fruit or veggies that have been watered with contaminated H2O, imbibing unpasteurized milk, or eating undercooked land meat.
In Gram-positive cells, peptidoglycan makes up every bit much as 90 % of the midst cell wall ; more than 20 beds of this polymer stacked together. These peptidoglycan beds are the outermost cell wall construction of Gram positive cells, whereas in Gram negative cells, the dilutant peptidoglycan constituent is covered by an external lipopolysaccharide ( LPS ) membrane.
Consecutive dilution – agar dish 10-5
This practical was done to see if single settlements were able to be produced so the cells could be counted. The chief purpose was to make from 30 -300 single settlements. The sum of settlements which I produced in my agar dish was calculated and counted at 486. The method used was to seek and cipher the figure of cells in 1 ml solution of SA. This could merely be done by consecutive dilution as it would be excessively hard to number the cells if the bacteria solution was non diluted. The other dishes had excessively many settlements to number merely by utilizing the bare oculus because it looked like a bacterium lawn. To seek and accomplish a better consequence and fewer settlements the experiment could hold gone farther and alternatively of holding a concentration of 10-5 the solution could hold been diluted further. By thining down the solution it besides allows the bacteria to turn in optimal conditions as they do non hold jobs such as less infinite or nutrient.
Cell numbering utilizing a haemocytometer
In this portion of the practical two solution were supplied and cells were counted by the usage of a haemocytometer and a microscope. While numbering the bacteriums it may hold been misjudged as some bacteriums may non hold been numbering this is one ground why the figure recorded were rather low as the solution was non diluted every bit good. Besides when numbering the bacterium it ‘s a entire cell count so it is the life and dead bacteriums so the consequences are non as accurate if merely making a unrecorded cell count. A better thought would be to make a consecutive dilution when making a unrecorded cell count as merely the life cells will turn into single settlements. For the whole blood the sum of cells which were calculated was 237 and in the beer maker ‘s barm there were 81 cells. A ground for the beer makers yeast holding less sum of cells possibly that the cells form flocks of cells so it may be difficult to visually see separate cells so when numbering them a few cells may be counted as one cell. Besides when numbering these cells the same individual was used to number the cells in both solution the ground for this being as different people have different judgements and by utilizing the same individual it will assist derive just and more accurate consequences.