The materials that were used in this lab included a 100/1000 μL micropipetter, 4 agar plates with pre-poured LB broth , ice bath, hot water bath, micro centrifuge tubes, sterile loops, UV light, as well as pGLO plasmids and E.coli. The first step of the experiment that occurred is to take the two microcentrifuge tubes and label the first +pGLO and the second –pGLO. Next the micropipetter was set to 250 microliters and 250 microliters of calcium chloride was added to each microcentrifuge tube, using a fresh tip if it came into contact with the solution, then the tubes were put on ice until the bacterial was obtained. Next our group placed an E.coli colony into each of the tubes using a sterile loop and spinning the loop so that the bacteria was all in the solution, be sure to use a different sterile loop for each tube. Then we took a third clean sterile loop and placed it into the solution of the desired …show more content…
plasmid, pGLO in this case, and use the sterile loop to transfer it to the tube marked +pGLO, make sure there is no pGLO plasmid in the –pGLO tube and that there is a film across the sterile loop, place the tubes back into the ice bath.
Next our group iced the tubes for ten minutes and labeled the plates with the materials that are inside of them, in this case +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, -pGLO LB. Following the ten minutes on ice, the tubes were placed in water at 42 degrees Celsius for 50 seconds and then immediately moved back to the ice bath. After two more minutes on ice they were removed and, using the micropipetter with a new tip each time, 250 microliters of nutrient broth was added to each tube, they were then let to sit for ten minutes. When the ten minutes was up the tubes were gently flicked to agitate the solution and 100 microliters of the +pGLO tube was pipetted to each of the agar plates labeled
+pGLO and 100 microliters of the –pGLO tubes was added to each of the plates labeled –pGLO. Then we took a sterile loop and spread the solution around the surface of the agar being sure to use a different sterile loop for each plate. The final step was to put all the plates together in a stack and store them at 37 degrees Celsius for 24 hours, and refrigerated after that (Weedman 2015).
I identified the genus and species of an unknown bacterial culture, #16, and I applied the following knowledge of morphologic, cultural and metabolic characteristics of the unknown microorganism according to the laboratory manual as well as my class notes and power point print outs. I was given an incubated agar slant labeled #16 and a rack of different tests to either examine or perform myself; the tests are as follows: Gram Stain; Nutrient Gelatin Test; Carbohydrate Fermentation; Dextrose, Lactose and Sucrose; IMVIC tests; Citrate, Indole, Mythel-Red and Vogues Proskauer test; as well as a Urease and TSI Test. Materials and Methods/Results Upon receiving the Microorganism (M.O.) #16, I prepared a slide by cleaning and drying it. Then, using a bottle of water I placed a sterile drop of water on the slide and used an inoculating loop, flame sterilized, I took a small sample of the unknown growth in my agar slant and smeared it onto the slide in a dime sized circle and then heat fixed it for ten minutes.
The unknown bacterium that was handed out by the professor labeled “E19” was an irregular and raised shaped bacteria with a smooth texture and it had a white creamy color. The slant growth pattern was filiform and there was a turbid growth in the broth. After all the tests were complete and the results were compared the unknown bacterium was defined as Shigella sonnei. The results that narrowed it down the most were the gram stain, the lactose fermentation test, the citrate utilization test and the indole test. The results for each of the tests performed are listed in Table 1.1 below.
We used the pipette filler and filled the glucose rinsed pipette to add 10ml of 10% of glucose in test tube 0.
...et light. If the LAA plate glows green under exposure to ultraviolet light, then we can conclude that our unknown insert piece of DNA would be the kan gene. If it does not glow green under exposure to ultraviolet light, then then we streak the colony from our LAA plate onto the LAC plate using a sterile glass spreader. When the LAC plate is dray, we place it upside down in the microfuge rack so that it can be incubated at 37 ºC. Incubation at 37 ºC will allow the transformed bacterial cells to grow. If we see bacterial growth on the LA plate containing chloramphenicol, we can conclude that our unknown insert piece of DNA would be the cat gene, since the cat gene is resistant to chloramphenicol. Afterwards, we then grab the microfuge tube labeled NP and repeat the aforementioned steps shown above pertaining to the LA plates. This would be considered our control.
In this experiment the heat shock method will be used to deliver a vector (plasmid) of GFP to transform and grow E. coli bacteria. Four plates containing Luria Bertani (LB) broth and either –pGLO and +pGLO will have E. coli bacteria added to it. The plate containing –pGLO (no pGLO) and LB will show growth as ampicillin will be present killing bacteria but no glowing because no arabinose will be present for glowing to be activated, the same result will be seen in the plate containing +pGLO, LB and ampicillin. The plate with –pGLO, LB and ampicillin will show no growth and no glowing as no arabinose is present for glowing to be activated
Equipment list: Test tubes were used to hold the milk, the lipase and the milk and lipase solutions. Test tube racks were used to hold the test tubes
After the incubation period, the tube will be centrifuge using table top centrifuge machine for 10 minutes at 2500 rpm. Centrifuging process should causing the bacteria and other cell debris to form a pellet at the bottom of the tube. The supernatant produce will be taken up about 3 mL using a syringe. A 0.22 micron filter will be attach to the base of the syringe and the supernatant will be filter into a sterile tube. Any remaining bacteria are prevented from passing through the filter except for phage. The filtrate will be label as ‘Enrich Phage’ and store it in refrigerator. By the way, one more overnight culture of Salmonella will be set up.
Add 350 µL Buffer N3, mix by inverting the tube four to five times, and incubate the tube on ice for 5 minutes.
After a 2-day incubation, the Micro-ID unit was placed flat on the bench and opened; 0.1 ml of 20% KOH was added to the inoculation well of the VP test only. The tray was then closed and held upright until the KOH flowed into the VP test solution.
There were five test solutions used in this experiment, water being the control, which were mixed with a yeast solution to cause fermentation. A 1ml pipetman was used to measure 1 ml of each of the test solutions and placed them in separated test tubes. The 1 ml pipetman was then used to take 1ml of the yeast solution, and placed 1ml of yeast into the five test tubes all containing 1 ml of the test solutions. A 1ml graduated pipette was placed separately in each of the test tubes and extracted 1ml of the solutions into it. Once the mixture was in the pipette, someone from the group placed a piece of parafilm securely on the open end of the pipette and upon completion removed the top part of the graduated pipette.
Stout, M.A, et al. "Microbiology Lab Notebook". Lab handbook. University of Texas. Arlington. 2014. Print.
2. In the large beaker, put water and boil it completely. After that, remove the beaker from heat. 3. Sample tubes (A-D) should be labeled and capped tightly.
== § Test tubes X 11 § 0.10 molar dm -3 Copper (II) Sulphate solution § distilled water § egg albumen from 3 eggs. § Syringe X 12 § colorimeter § tripod § 100ml beaker § Bunsen burner § test tube holder § safety glasses § gloves § test tube pen § test tube method = == = =
Leboffe, M. J., & Pierce, B. E. (2010). Microbiology: Laboratory Theory and Application, Third Edition 3rd Edition (3rd Ed.). Morton Publishing
In experiment three, there were a lot of different kinds of organisms persent on the agar. On the plate that contained nasal micro-organisms, large yellow colonies appeared on the Mannitol Salt Agar, indicating Staphylococcus aureus. On the TSA side, small creamy white colonies were also present. These could possibly be yeast. On the plate that contained the skin swab, there was a large amount of large, fuzzy white and black growth, which is mold. There was a single light-orange colony on the TSA side, possibly a growth of Flavobacterium.