Bacterias are single-cell micro-organisms that are able to populate in life beings or inanimate things. They are procaryotic so they do non hold any membrane-bound cell organs and karyon. Some types of bacteriums can photosynthesize and bring forth its ain nutrient. However, there are types of bacteriums which are non able to put to death that map.
Bacterias are the type of most normally used micro-organisms in research labs because they reproduce so fast. [ 1 ] The bacteriums Escherichia coli were named for the Austrian physician, Theodor von Escherich ( 1857-1911 ) . E.coli was foremost isolated the genus of bacteriums belonging to the household Enterobacteriaceae, folk Eschericheae by him. [ 2 ] E.coli is a big group of bacteriums. Some strains of E.coli are harmless, nevertheless, some of them may do diarrhoea. When working with cell civilizations, one can work with a ‘pure civilization ‘ which is a good stray settlement or a civilization that has been studied in the research lab before and go known how the cells appear and react, it is called strain.
There are some microbiological techniques in order to transport out the research decently. Microbiological techniques are used in cell civilization and for placing micro-organisms. While working with micro-organisms, there is ever a hazard of taint. To forestall and cut down the hazard of taint, sterile techniques must be used. One of the of import techniques is disinfection which helps to cut down the entree of any life beings. Largely, fire is used for disinfection. Other technique which is used to forestall taint is sterilisation. It is required to destruct micro-organisms that can pollute civilizations. There are 3 types of sterilisation: Heating, utilizing chemicals and irradiation. Using heat for sterilisation in an autoclave causes the devastation of micro-organisms by denaturation of enzymes and proteins. The recommendations for sterilisation in an sterilizer are 15 proceedingss at 121-124 & A ; deg ; C. [ 3 ]
Bacterias can turn on any nutrient that contains C and N. Solid or liquid food that bacteriums turn on is called medium. 2 types of medium were used in research lab: LB broth which is liquid and LB agar which is solid.
Petri dishes incorporating LB agar
1.5 milliliter Eppendorf tubes incorporating LB stock
15 ml Falcon tubing
Plastic vaccination cringle
Glass vaccination cringle
First, to forestall any taint, baseball mitts were worn. First measure of the experiment was vaccination. Alcohol burner was burned and fictile vaccination cringle was heated for a 2nd. Then the palpebra of falcon tubing which is incorporating E.coli was opened and oral cavity of falcon tubing was passed through the fire. After that, plastic cringle was inserted in falcon tubing and picked up a bead of liquid. Then palpebra of petri dish which is incorporating LB agar was opened and E.coli was spread over the agar. Finally, fictile cringle was burned in order to kill bacteriums.
Second measure of the experiment was streaking. Plastic incubation cringle was besides used in this measure. It was heated and cooled a small in order non to kill E.coli. Lid of petri dish was opened and a line was drawn to the Centre by utilizing cringle. Then, the same cringle was moved in a zigzag form until ? of the home base was covered. After that, petri was rotated about 45 grades and cringle was moved from the terminal of first zigzag to untasted country with the same form. Same technique was applied until all countries in petri dish were done. Finally, palpebra was closed and swathed with paraffin. Plastic cringle was burned, once more.
The 3rd measure was consecutive dilution. Five empty petri dishes were labeled as the dilution that will be used: 10-1, 10-2, 10-3, 10-4 and 10-5. In add-on, 100 µl E.coli was added to 1.5 milliliters eppendorf tubing which is incorporating 900 µl LB stock. Then, tip was attached to pipettor and 100 µl of liquid from eppendorf was taken. For about 20 times, liquid was transferred between new eppendorf tubing and pipettor in order to disperse bacteriums every bit. Consequently, 10-1 dilution of E.coli was obtained and transferred to eppendorf tubing. After that, same method was applied for eppendorf which contains 10-1 E.coli. In this manner, 10-2 E.coli was obtained and transferred to new eppendorf tubing. This process was carried out until five eppendorf tubings which are incorporating 10-1, 10-2, 10-3, 10-4 and 10-5 dilution of E.coli were obtained. Later on, distributing method was applied. Petri dish which is labeled as 10-1 was taken and E.coli was transferred to petri from pipettor, so tips were burned and discarded. After that, glass vaccination cringle was heated to sterilise and cooled non to kill bacteriums in the petri dish. By the near of fire, E.coli was spread all over the agar by utilizing glass cringle at one clip. Petri was rotated easy while distributing. Same process was applied all the dilutions. Besides, all process was applied by the near of fire.
The concluding measure was incubation. After the spreading, petri dishes incorporating E.coli were put into a machine called brooder which provides equal temperature for bacteriums. After 24 hours incubation, E.coli had been reproduced adequate to see with bare oculus.
Figure 1: Streaking Method Figure 2: Petri serve that we took place
Figure 3: Spreading 10-1 Figure 4: Spreading 10-2
Figure 5: Spreading 10-3 Figure 6: Spreading 10-4
Figure 7: Spreading 10-5
To cipher the entire figure of E.coli through distributing method, 10-5 dilution of E.coli was counted. Then, it is calculated that how many E.coli would be counted in 100 dilutions.
If 10-5 dilution has 8 E.coli, than 100 dilution has 8*105 E.coli.
? = 8*105 E.coli in 100 µl
Subsequently, it should be calculated that how many E.coli would be counted in 100 milliliter. This measure is necessary because distributing method had been applied in 100 µl. ( 1 milliliter = 1000 µl )
If 100 µl have 8*105 E.coli, than in 100 milliliter, figure of E.coli is 8*108.
100 µl 8*105
? = 8*108 E.Coli
As a consequence, streaking method was non applied accurately. However, distributing method was applied successfully because bacteriums settlements were easy denumerable. In add-on, assorted beings were seen in petri dish that was taken place.
In this experiment, how microbiological techniques which are sterile techniques, vaccination, streaking, consecutive dilution and incubation are applied has been learned. First of wholly, to forestall any taint, sterilisation has to be provided. For case, tabular array was cleaned with % 70 ethyl alcohol before and after the experiment. Besides, filtered tips were used for the pipettors and baseball mitts were worn to forestall any taint. First process of this experiment was vaccination. Most of import thing while transporting out vaccination was working by the near of fire. It is of import because air watercourse which is produced by fire helps to maintain any micro-organisms or peculiar off. Therefore, taint hazard was reduced. Besides, it is necessary to fire the cringle after work with it. It is because bacteriums on that cringle may reassign and pollute the experiment. Streaking method is used for acquiring stray settlements of bacteriums, look intoing whether civilization is pure or contaminated and able to turn. [ 4 ] Unfortunately, the streaking method was non accomplished accurately. It is understood from that settlements were non individually denumerable. The ground for this error may be the incorrect application of fictile cringle or non to get down streaking where it is ended. Other ground for that error can be non to revolve petri plenty to streak all over the surface. Following stairss which are consecutive dilution and spreading were done successfully. Aim of consecutive dilution is to find the figure of bacteriums in the original civilization. [ 5 ] As ever, consecutive dilution was applied by the near of fire in order to forestall any taint. Using pipettor decently was important since incorrect use of pipettor such as non to halt pressing the speculator after first point may do pull the liquid into the pipettor so taint. It is besides of import to fire tips after utilizing because of the bacteriums taint. The important portion of distributing method was sterilising glass cringle and utilizing it accurately. After keeping glass cringle through the fire, it is of import to wait a piece until it cooled because high temperature may kill bacteriums. Furthermore, distributing must be applied while revolving petri dish in order to distribute bacteriums every bit. Best consequence was taken with 10-5 dilution in distributing so computation was made harmonizing to that dilution. In decision, it is identified that assorted micro-organisms reproduced in petri dish and formed settlements.