Project #36565 - microbiology

Bacteriophages can be grown in liquid or solid cultures of bacteria. when solid media are used, the plaque forming method allows bacteriophage to be located. hos bacteria and bacteriophages are mixed together in melted agar, which is then poured into a petri plate containing hardened nutrient agar. each bacteriophage that infects a bacterium multiples, releasing hundred new viruses. the new viruses infect other bacteria, and more new viruses are produced. all the bacteria in the area surrounding the original virus are destroyed, leaving a clear area or plaque against a confluent lawn of bacteria.

in this exercise, we will isolate a bacteriophage from host cells in a natural environment, sewage. because the number of phages in a natural source are low, the desired hos bacteria and additional nutrients are added as an enrichment procedure. after incubation, the bacteriophage can be isolated by centrifugation of the enrichment media and membrane filtration.

Titration of bacteriophage:

label the plates and broth tubes 1 to 6. aseptically add 1 ml of phage suspension to tube 1. mix by carefully aspirating up and down three times with the pipette. using a different pipette, transfer 1 ml to the second tube, mix well, then put 1 ml into the third tube. why is the pipette change? continue until the fifth tube. after mixing this tube, discard 1 ml into a container of disinfectant. there are 6 tubes, what is the purpose of tube 6? with a pipette, add .1 ml of e coli to the soft agar tubes and place them back in the water bath. keep the water bath at 43 degrees to 45 degrees at all times. with the remaining pipette, start with broth tube 6 and aseptically transfer .1 ml from tube 6 to the soft agar tube.

 

Define bacteriophage.

What was the purpose of adding e. coli to the agar plate?

should you make dilutions of E. coli? why or why not?

When making dilutions, why didn’t you put bacteriophage in tube 6?

if you saw plaques in plate 6, what would that indicate?

assuming the first dilution is 1:10 and each dilution after that is a factor of 10, list the dilutions for each tube:

tube 1, tube 2, tube 3, tube 4, tube 5, tube 6

Assuming 0.1 ml sample from each tube to the soft agar/ecoli, write the dilution in each plate:

plate 1, 2, 3, 4, 5, 6

Assume the number of plaques on plate 4 is 55. if the dilutions were done perfectly, how many plaques would there be on each of the other plates?

plate 1, 2, 3, 4, 5, 6

How many PFU/ml would there be in the original suspension? show calculatins.

 

fruit punch problem:

a. add 10 ml of fruit concentrate to 40 ml of 7 up. what dilution of the concentrate is this?

b. the mixture in a tastes too sugary, so you add 50 ml more of 7 up. what dilution of the original concentrate is this?

c. you want to be sure this punch is safe to drink, so you take a 1 ml sample from the mixture you made in b and put it on an agar plate. what dilution f the organisms in the concentrate is this?

d. to be thorough, you now put a .1 sample of b on an agar plate. what dilution of organisms in the concentrate is this?

e. on the plate you made in d, 93 colonies grow, how many CFU/ml were in the original concentrate?

f. if you have done your experiment perfectly, how many colonies will grow on the plate you make in C?

g. if there were 300 colonies on the plate you made in d, how would you determine the cfu/ml in the original concentrate?

h. what does CFU stand for? how does Cfu differ from the total number of organisms?

i. do you think the original concentrate has been pasteurized? if yes, do you think the pasteurization was effective? if no, would you recommend pasteurization of future batches of concentrate?

j. what general types of organisms will pasteurization kill?

k. if your punch is left out of the refrigerator on a warm day and ferments, name one organism that can cause this.

you are given a flask containing a 1:10 dilution of chicken broth, without making any further dilutions, how much of this chicken broth must be plated in order to achieve each of the following plated dilutions of organisms in the broth?

1:2

1:10

1:100

devise a scheme to prepare 1:20, 1:40, and 1:80 dilutions of disinfectant draw and completely label your scheme.

 

 

 

Subject Science
Due By (Pacific Time) 07/28/2014 03:00 am
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