Week 3 Microbiology Ch 9-10

ISOLATION METHODS • QUADRANT STREAK PLATE METHOD Preparing initial inoculum of quadrant streak plating

1 Label agar plate with marker 2 Turn on Bunsen burner 3 Sterilize loop by placing into flame of Bunsen burner 4 Pick up bacterial culture tube. Remove cap. Briefly heat mouth of tube in Bunsen burner flame to reduce contamination 5 Insert loop into tube to acquire bacterial sample 6 Briefly heat mouth of tube, replace cap, and return tube to rack 7 Use loop to streak bacterial sample onto one quadrant of plate 8 Sterilize loop using Bunsen burner

Three steps of the PCR technique

1. Heating target DNA to 94 degrees Celsius to separate it into two strands denaturation 2. Cooling to approximately 60 degrees Celsius to allow the oligonucleotide primers to base-pair with specific sites priming 3. Raising the temperature to 72 degrees Celsius and adding DNA polymerase and nucleotides extension

True statements regarding transcription and translation in prokaryotes

1. Prokaryotic mRNAs often contain information from several genes in series. 2. Transcription and translation can occur at the same time in prokaryotes. 3. Translation occurs in the cytoplasm of prokaryotes.

Plasmids (bacteria)

Extra pieces of DNA found in bacteria are called __________.

1. genetic alteration of the frost gene in Pseudomonas syringae 2. genetic alteration of Pseudomonas syringae that would prevent the formation of ice crystals on plant surfaces 3. insertion of an insecticide gene from Bacillus thuringiensis into Pseudomonas fluorescens 4. the alteration of oncolytic adenoviruses to attack cancer cells

Four examples of GMOs

adenine cytosine guanine thymine

Four nitrogen base found in DNA

1. immune treatments 2. hormones 3. enzymes 4. vaccines

Four types of protein product obtained through recombinant DNA technology

ISOLATION METHODS • SUBCULTURING OF BACTERIA Lab data and questions 1. How many types of bacteria appear on a successful subculture to a slant? 2. If you are attempting to obtain a pure culture, what must you avoid when subculturing bacteria from a mixed plate to a sterile agar plate?

Answer: 1. One 2. Contamination of the transfer tool or media containers.

72oC

At which of the following temperatures would you predict maximum activity from a heat-stable polymerase such as Taq polymerase?

template

Components of a PCR reaction with the function they serve (1/5) source of DNA to be amplified...

primer

Components of a PCR reaction with the function they serve (2/5) specifies the region of DNA to be amplified...

dNTPs

Components of a PCR reaction with the function they serve (3/5) The components of DNA that are joined together by DNA polymerase...

polymerase

Components of a PCR reaction with the function they serve (4/5) Reads the template and assembles a new copy of DNA...

thermal cycler

Components of a PCR reaction with the function they serve (5/5) Alters the reaction temperature to make the template available for DNA polymerase and to facilitate DNA synthesis via primer annealing...

gene therapy

Correction or repair of a faulty gene in humans suffering from a fatal or debilitating disease is a process known as __________.

Agar slant

Bacterial culture medium containing agar in a test tube that is allowed to solidify at an angle and forms a solid, slanted surface. Several regions of the tube, known as the "slant" (top) and the "butt" (bottom) can be identified in a properly created agar slant.

Agar plate

Bacterial culture medium containing agar poured into a petri dish.

Agar media

Bacterial culture medium that contains agar as a solidifying agent and is used for growing microorganisms.

ISOLATION METHODS • QUANTITATIVE DILUTION OF BACTERIA After completing steps to create serial dilutions, each bottle now contains what proportion of cells from the initial culture sample?

Bottle A: 1:100 Bottle B: 1:10,000 Bottle C: 1:1,000,000

Smaller fragments move through the gel faster

How does the size of a DNA fragment relate to its speed of passage through the agarose gel?

True

In the laboratory, the chemistry of DNA allows varying temperatures to be used to separate the two strands and replicate the DNA.

No

Is uracil a nitrogen base found in DNA? yes or no

Central Dogma of Biology Steps and Image

It involves copying a gene's DNA sequence to make an RNA molecule. Transcription is performed by enzymes called RNA polymerases, which link nucleotides to form an RNA strand (using a DNA strand as a template). Transcription has three stages: initiation, elongation, and termination.

Utilizing insulin made from recombinant DNA technology is beneficial because __________.

It will not cause an allergic reaction

ISOLATION METHODS • QUADRANT STREAK PLATE METHOD During the steps, the bacterial culture is...

Only added directly to the algar plate one time

polymerase

PCR is dependent on a heat-stable ________.

Which method often results in colonies developing down throughout the agar and some colonies on the surface?

Pour plate

Restriction enzymes are used to cut DNA at specific locations.

Role of restriction enzymes in genetic engineering.

BACTERIAL GENETICS • DNA PROFILING Gel electrophoresis What would be the result of running the gel too long?

The DNA sample will move through the entire gel and exit at the bottom

amplify

The Polymerase Chain Reaction is used to ________ a specific sequence of DNA.

Genome

The __________ is the sum total of genetic material in a cell.

Transformation

The acceptance by a bacterial cell of small DNA fragments from the surrounding environment is termed __________.

Inoculum

The correct microbiological term for the tiny sample of specimen that is put into a nutrient medium in order to produce a culture is the _____.

hydrogen

The denaturing and annealing steps of PCR involve the breaking or formation of ________ bonds.

Substitution mutation

Types of mutation (3/4) Changing of single base in the DNA code that may result in the placement of a different amino acid?

spontaneous mutation

Types of mutations (1/4) Random change in the DNA arising from errors in replication that occur without a known cause?

Induced mutation

Types of mutations (2/4) Result from exposure to known mutagens, which are primarily physical or chemical agents that damage DNA?

frameshift mutation

Types of mutations (4/4) Addition or deletion of bases that changes the reading of mRNA codons?

recombinant plasmid

When a specific gene from one organism is introduced into a plasmid, the plasmid is referred to as a __________________.

gel electrophoresis

Which of the following is used to detect the products of PCR?

Patterns of DNA fragments in a gel that are unique to an individual organism.

Which statement best describes the purpose of DNA profiling?

agar plate

You have a nutrient broth, an agar slant, and an agar plate with bacterial growth in or on each. If you wanted to evaluate the macroscopic appearance of the bacteria growing in or on the medium, which of the cultures would be the best choice?

gel electrophoresis

________ can be used to separate nucleic acids based on size.

Plasmids

__________ are excellent cloning vectors because they are small, well characterized, easy to manipulate, and can be transferred into appropriate host cells through transformation.

A mutation that causes a change in a single nucleotide in DNA...

changes the corresponding nucleotide in mRNA, resulting in a different codon

BACTERIAL GENETICS • DNA PROFILING What does the EcoRi/Pstl enzyme mixture do?

cuts DNA at specific locations, creating various-sized fragments

solid medium

essential for development of discrete, isolated colonies

When preparing pure cultures, dilution is necessary for?

reducing the number of inoculated organisms so that isolated colonies can develop.

Choose the statement that best describes the role of restriction enzymes in DNA profiling.

used to cut DNA at specific locations

Colony

visible mass of bacteria growing together on an agar plate or slant. Colonies are derived from a single microorganism. Colonies may form on the top surface of the agar or within the agar (referred to as "subsurface colonies").

Culture

many colonies growing on a plate, on a slant, or in broth. A culture is referred to as a "pure culture" when there is only one type (species) of organism in it.

Vegetative cell

metabolically active and growing

A pure culture contains only

one species of microorganism

Select ALL of the possible applications of DNA profiling (also known as DNA fingerprinting).

- Identifying the source of blood or tissue left at crime scene - Tracking pathogens during investigation of an epidemic - Identification of disaster victims - Determining parentage.

Steps of the Pour Plate Method

- Liquefy nutrient agar tubes and maintain at 50 degrees -Remove loopful of organisms from culture using aspetic technique -Add loopful of organisms to an agar tube -Transfer one loopful from one agar tube into another agar tube, and repeat this again with an third agar tube -Pour liquified agar tubes into empty Petri dishes -Incubate for 24-48 hours.

Place the steps of the Polymerase Chain Reaction in the correct sequence.

- denaturing: heating target DNA to 954 degrees C to separate it into two strands - Annealing (priming) cooling to 60 degrees C to allow t he oligonucleotide primers to base-pair with their complementary sites - extending (replication) raising temperature to 72 degrees C and adding DNA polymerase and extension

BACTERIAL GENETICS • POLYMERASE CHAIN REACTION (PCR) What does the master mix solution contain?

- heat stable polymerase - free deoxynucleotides

BACTERIAL GENETICS • DNA PROFILING Gel electrophoresis What is the purpose for adding loading dye to the samples before they are loaded into the gel?

- it contains a colored indicator, allowing migration of the samples to be monitored during electrophoresis - it contains glycerol, which allows the samples to sink into the wells

ISOLATION METHODS • POUR PLATING METHOD Use inoculated agar to create pour plates for isolation

- pour plates show isolated colonies, both surface and subsurface, from E. coli sample and dilutions - Precautions to take are... 1. plates should be placed in the incubator upside-down to prevent contamination from condensation. 2. plates should be accurately labeled, with writing on the agar side of the petri dish

ISOLATION METHODS • QUADRANT STREAK PLATE METHOD If your completed quadrant streak plate showed two different and distinct colony appearances, what could you conclude?

-Initial bacterial inoculum contained two unique bacterium species -your plate was inoculated with a single species but break in aseptic technique allowed containment bacteria to also grow

Please select all of the statements which are TRUE regarding isolated colonies.

-The colony results from a single cell or a cluster of cells multiplying into a visible mass. -The cells within the colony are all the same species. -Isolated colonies form on solid nutrient media.

ISOLATION METHODS • QUADRANT STREAK PLATE METHOD If you stopped the procedure here and analyzed your quadrant streak plate after incubation, which of the following would correctly describe the outcome?

-heavy growth visible -appears in only one quadrant -no isolated colonies present

ISOLATION METHODS • POUR PLATING METHOD Performing dilution preps for pour plating

1Turn on Bunsen burner 2Sterilize loop by placing into flame of Bunsen burner 3Pick up E. coli tube from rack and tube I from water bath in same hand 4Remove caps from tubes. Heat mouths of tubes 5Use loop to transfer sample from E. coli tube into tube I 6Heat mouths of tubes. Replace caps. Heat loop 7Click to shake tubes. Return E. coli tube to rack 8While holding tube I, pick up tube II from water bath 9Remove caps from tubes. Heat mouths of tubes 10Use loop to transfer sample from tube I into tube II 11Heat mouths of tubes. Replace caps. Heat loop 12Shake tubes. Return tube I to water bath 13While holding tube II, pick up tube III from water bath 14Remove caps from tubes. Heat mouths of tubes 15Use loop to transfer sample from tube II into tube III 16Heat mouths of tubes. Replace caps. Heat loop. Return loop to holder 17Shake tubes. Return tubes II and III to water bath

plasmid

A(n) ________ is an extrachromosomal DNA molecule in a bacterial cell that replicates independent of the bacterial chromosome.

Select the statement that is true about DNA profiling.

DNA profiling is the analysis of the collection of fragments produced by cutting DNA with specific restriction endonucleases.