Lab 4: Microbial Techniques Flashcards
Name the microorganisms that we worked with in the lab
1) E. coli
2) Bacteriophages
3) Saccharomyces Cerevisiae
4) Neurospora Crassa
5) Sordaria Fimicola
What technique is used to handle microbes?
aseptic technique in order to maintain sterile environment.
Pros and cons of using microorganisms in the lab
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
What are the two basic methods to determine cell density? What are the requirements?
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.
How would you perform a 10 fold serial dilution?
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.
What is the purpose of conducting dilution and plating?
to learn cell # determination techniques
What temp did yeast plates get incubated at?
30 degrees
How are plates of yeast stored? Why?
upside down to prevent condensation falling on the agar surface.
What is the purpose of streak plating?
to isolate pure strains of organism from a mixture. Also aids in miroorganism characterization.
What is a bacteria phage?
viruses that infect bacteria
What’re the two types of bacteriophages
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.
What is a prophage?
an integrated phage genome
What temp were the ecoli plates incubated at?
37 degrees
How a bacteriophage be counted?
plaque assay procedure
When will the pages stop infecting the bacteriophages?
when the bacteria exhaust the nutrients in the medium and stop growing.
How do you know if the phage is working?
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).
How can you tell if there is a mutation in bacteriophage?
mutation of phage genome=altered plaque morphology.
What is conjugation?
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+
What is an episome?
the fertility factor that can exist by part of the main DNA of the donor cell, or exist as a plasmid.
What does it mean when the donor strain if Hfr?
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.
In this lab, what were the characteristics of the Hfr and F- strain?
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.
ECM plate vs NA plate
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.
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?
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.
