Lab 4: Microbial Techniques

Question 1

Name the microorganisms that we worked with in the lab

Answer 1

1) E. coli
2) Bacteriophages
3) Saccharomyces Cerevisiae
4) Neurospora Crassa
5) Sordaria Fimicola

Question 2

What technique is used to handle microbes?

Answer 2

aseptic technique in order to maintain sterile environment.

Question 3

Pros and cons of using microorganisms in the lab

Answer 3

Pros:

• reproduce quickly
• easy to culture and simple nutritional needs
• haploid life cycles allow for easy analysis because phenotype matches genotype.

Cons:

• many are pathenogenic
• difficult to count
• needs aseptic technique to avoid contamination

Question 4

What are the two basic methods to determine cell density? What are the requirements?

Answer 4

1) viable cell counts: counting living cells.
- in order for the technique to work , plates can only have 30-300 colonies. In order to accomplish the 30-300 colony requirement, dilutions and plating must happen in aseptic conditions.

2) total cell counts: counts living and dead cells.

Question 5

How would you perform a 10 fold serial dilution?

Answer 5

take 9ml of sterile water and 1 ml stock solution and vortex it to create a solution that 1/10 as concentrated than the original.

Question 6

What is the purpose of conducting dilution and plating?

Answer 6

to learn cell # determination techniques

Question 7

What temp did yeast plates get incubated at?

Answer 7

30 degrees

Question 8

How are plates of yeast stored? Why?

Answer 8

upside down to prevent condensation falling on the agar surface.

Question 9

What is the purpose of streak plating?

Answer 9

to isolate pure strains of organism from a mixture. Also aids in miroorganism characterization.

Question 10

What is a bacteria phage?

Answer 10

viruses that infect bacteria

Question 11

What’re the two types of bacteriophages

Answer 11

1) lysogenic: a phage that can integrate into the genome of a host bacteria and enter into a metastable association. the phage dna gets replicated each time bacterial DNA gets replicated. When the host cell gets damaged, the prophage gets excised and the host cell lysis, releasing progeny phage.
2) lytic: a phage attaches to host cell and injects its DNA, it uses the host cell organelles to replicate and form it protein components. The components self assemble to from mature phages, and the host cell lysis.

Question 12

What is a prophage?

Answer 12

an integrated phage genome

Question 13

What temp were the ecoli plates incubated at?

Answer 13

37 degrees

Question 14

How a bacteriophage be counted?

Answer 14

plaque assay procedure

Question 15

When will the pages stop infecting the bacteriophages?

Answer 15

when the bacteria exhaust the nutrients in the medium and stop growing.

Question 16

How do you know if the phage is working?

Answer 16

plaques and holes show where infection has occurred. Ecoli plates look hazy, and when they lyse and stop growing/get killed off, the plate surface looks clear (hole).

Question 17

How can you tell if there is a mutation in bacteriophage?

Answer 17

mutation of phage genome=altered plaque morphology.

Question 18

What is conjugation?

Answer 18

the process of a donor cell giving its fertility facto (Hfr, F+) to the recipient cell, who has no fertility factor, F-, through a conjugation tube. after conjugation, the recipient cell converts from F- to F+

Question 19

What is an episome?

Answer 19

the fertility factor that can exist by part of the main DNA of the donor cell, or exist as a plasmid.

Question 20

What does it mean when the donor strain if Hfr?

Answer 20

its fertility factor is integrated into the main bacterial chromosome, it has a high frequency of recombination.

When the integrated F+ conjugates with the F- strain, pushing some replicated fertility factor through the conjugation tube, the recipient can undergo recombination with homologous sequences, resulting in the production of genetically recombinant cells. The resulting cells are a combination of donor and recipient DNA.

Question 21

In this lab, what were the characteristics of the Hfr and F- strain?

Answer 21

Hfr was prototrophic thr+leu+thie+lac+str s (protein+) and antibiotic sensitive.
F- was auxotrophic thr-leu-thi-lac- str r and antibiotic resistant.

Question 22

ECM plate vs NA plate

Answer 22

The ecm plate has thiamine and streptomycin and glucose on it. because it is laced with antibiotics, only an antibiotic resistant strain can grow. it also needs to be able to make its own amino acids.

an Na plate is nutritionally complete, and has no streptomycin. All strains, even the F- strain that cannot make its own nutrients, can grow on it because it has a supply.

Question 23

If the resulting recombinant cells from conjugation show specific traits ,what can be said about these traits in terms of location on the bacterial chromosome?

Answer 23

If you see a certain trait in the progeny upon combining an Hfr and F- strain, it is likely the the gene for that trait is close to the integrated fertility factor and was replicated with it and transferred to the F- along with the factor during conjugation.