Lab Practical 2 Flashcards
What is transformation?
Transformation is the uptake of naked DNA by a bacterial cell
What promoter controls the GFP gene?
Transcription of the GFP gene is controlled by the promoer for arabinose metabolism.
What factors effect transformation of bacteria?
Methods of transformation
- The method of preparation of competent cells
- the length of time of heat shock
- temperature of heat shock
- incubation time after heat shock
- growth medium used and various additives
- Bacterial colonies’ phase of growth
- Size and state of the foreign DNA
What is meant by competency?
Competency is the relative ability of a bacterial cell to incorporate naked DNA (plasmid) and undergo transformation.
Type of medium that contains reagant that will change color or react due to the growth of metabolism of bacteria.
Differential Media
What type of medium is this?
Ex: The reagants in MaConkey Agar (MAC) will turn bacterial colonies pink if they are able to ferment lactose.
Differential Media
What is the purpose of a selective marker?
Selectable markers show the success of a transfection or other procedure meant to introduce foreign DNA into a cell. It is a technique in gene targeting and gene knockout.
Ex: Beta-lactamase which confers ampicillin resistance to bacterial hosts.
What is the purpose of the heat shock step?
Heat shock forms transient pores in the bacteria’s cell wall, providing a path for the plasmid (foreign DNA) to approach the bacterial cell more readily.
Why are cells held on ice prior to heat shock?
What structure is commonly used by bacteria for motility?
Flagella are a common physiological feature of motile bacteria
Are all bacteria motile?
No but whether a bacterium is motile or non-motile can be a useful characteristic for differentiation
You have a suspected gram-positve culture. Would it be more appropriate to grow it on MacConkey agar or PEA agar to confirm these results?
PEA agar, because PEA agar is selective for Gram- positive bacteria. Because the growth of Gram-negative bacteria is suppressed by the interaction of the alcohol with the outer membrane, suppressing growth.
MAC is selective for Gram negative bacteria
In addition to selecting for halophilic bacteria, MSA can detect the fermentation of mannitol. What color will the plate turn if mannitol is fermented?
Yellow
If the halotolerant organism is able to ferment mannitol, the acids produced will cause the phenol red in the medium to turn yellow.
You disover that an unidentified bacterium you have been given is Gram-negative. Would you use MAC, PEA, or MSA to determine if the bacterium ferments lactose?
MAC, because MacConkey Aga is selective for Gram-negative bacteria and differential in lactose fermentation.
- Not:
- PEA is selective only for Gram- postive bacteria
- MSA is selective for Gram-negative but differential for mannitol fermentation
If an organism does not grow on MacConkey, what conclusions can you draw regarding its ability to ferment lactose.
No conclusions on the bacteria’s ability to ferment lactose can be drawn, since MAC is a selective and differential test.
- If an organism does not grow on the MAC agar, no conclusions can be drawn regarding lactose fermentation because selective assay takes precedence over the differential assay.
When using selective media, why must a culture also be grown on TSA?
The TSA plate acts as a positve control in order to compare and differentiate among the enzymatic processes of bacteria, different media (such as selective and differentail assays) to detect metabolic and physiological differences
What are the selective reagants in PEA?
Phenylethyl Alcohol
- The Gram-negative bacteria is suppressed by the interaction of alcohol with the outer membrane
- The Gram-positive bacteria are typically unaffected by the phenylethyl alcohol
What are the selective reagants in MAC?
Bile salts and crystal violet
- The bile salts and crystal violet inhibit the growth of Gram-positive bacteria when compared to the TSA control plate
What are the selective reagants in MSA?
7.5% NaCl
- MSA selects for halotolerant organisms, bacteria that can survive in a relatively high salt environment
What are the differential reagents in MAC?
Sugar and neutral red (a pH indicator)
- If a Gram-negative organism is able to ferment lactose, the colonies will turn pink and may have a haze around them
- Gram-negative organisms unable to ferment lactose will be colorless
What are the differential reagents in MSA?
Sugar mannitol and phenol red (a pH indicator)
- If a halotolerant organism is able to ferment mannitol, the acids produced will cause the phenol red in the medium to turn yellow
- Bacterial growth with no color change indicates halotolerance, but no ability to ferment mannitol
What type of media contains reagents that will change color or react due to the growth or metabolism of bacteria?
Differential Media
What is this an example of?
The reagants in MacConkey Agar (MAC) will turn bacterial colonies pink if they are able to ferment lactose?
Differential media
__________ ingredients do not suppress growth, they react in response to growth.
Differential