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
__________ contain ingredients that suppress the growth of some type of organism and allow a different type of organism to grow.
Selective Media
Selective media are interpreted with regard to the ___________ or _________ of growth
presence or absence
Differential media are interpreted by the _______ or ___________of the growth
color or lack of color
What is this an example of?
The ingredients of the MacConkey agar (MAC) suppress the growth of Gram-positve cells, while selecting the growth of Gram-negative cells.
Selective Media
If an organism does not grow in a selective media what conclusions can be drawn pretaining to the differential media?
No conclusions can be drawn. When using media that are selective and differential, it is important to remember that the selective assay takes precedence over the differential assay
What type of test is the Mannitol Salt Agar (MSA)? What does it test for?
- MSA is a selective and differential media
- MSA selects for halotolerant organisms
- Bacterial growth = halotolerant
- No growth = not halotolerant
- MSA is differential for the ability of bacteria to ferment mannitol
- Growth & Yellow = halotolerant organism that ferments mannitol
- Growth & Colorless = halotolerant organism that can’t ferment mannitol
- No growth = no conclusion can be draw about fermentation
What type of test is PEA? What does it test for?
- PEA is selective for Gram-positive bacteria
- Not a differential test
- The presence of alcohol suppresses the growth of Gram-negative bacteria
- The presence of alcohol doesn’t suppress the growth for Gram-postive bacteria
- Growth = Gram positve
- No growth = Gram negative
What type of test is the MacConkey Agar (MAC)? WHat does the MAC test for?
MAC is a selective and differential medium
- MAC is selective for Gram-negative bacteria
- Growth = Gram negative
- No growth = Gram positive
- MAC is differential for lactose fermentation
- Growth & pink = Gram negative bacteria that can ferment lactose
- Growth & colorless = Gram negative bacteria that cannot ferment lactose
- No growth = Gram positve but no conclusions can be drawn about fermentation
What is the purpose of the KOH test?
The KOH test is a differential test used to confirm the results of the Gram stain.
KOH causes lysis of Gram-negative bacteria (gooey)
KOH will not cause lysis of Gram-positive bacteria (watery)
Why does KOH become gooey in the presence of Gram-negative cells?
KOH causes lysis of Gram negative bacteria cells. After lysis, proteins and other macromolecules from the lysed cells denature, and form a thick, gooey, sticky liquid.
What is denaturation?
Denaturation is the break down of protein molecules.
As a beginning microiologist, which do you think is more reliable, a Gram stain or the KOH test?
THe KOH, because there is less steps to cause error and the results are instanteous.
What happens to Gram positve bacteria cell after the KOH test?
The Gram-positve cultures do not lyses, and the KOH remains watery.
What happens to Gram negative bacteria after the KOH test?
KOH causes lysis of the Gram-negative bacterial cells, causing the proteins and other macromolecules to denature and form a thick, gooey, sticky liquid.
What oxygen indicator is used in thioglycollate broth?
Resazurin acts as an oxygen indicator
(Thio-gly-coll-ate)
(Re-saz-ur-in)
Why is resazurin used as an oxygen indicator for thioglycollate?
Oxygen diffuses into the top portion of the thioglycollate media, which turns the resazurin pink, indicating that the top area of the thioglycollate broth is aerobic.
What component of the thioglycollate broths maintains the anaerobic conditions in the lower portion of the broth?
Sodium thioglycollate acts as a oxguen scrubber, maintaining an anaerobic environment in the thioglycollate medium below the pink band of resazurin.
Where in a thioglycollate broth tube would you expect to find growth for the following types of bacteria?
Facultative anaerobes
Throughout the tube (top and bottom)
Facultative anaerobes can use either aerobic respiration or fermentation, so can use oxygen but does not require it.
Where in a thioglycollate broth tube would you expect to find growth for the following types of bacteria?
Obligate aerobes
Top of the test tube only (where the resazurin is)
Obligate aerobes require oxygen for their metabolism, and are incapable of growing in an anaerobic environment, as they cannot use fermentation or anaerobic respiration.
Where in a thioglycollate broth tube would you expect to find growth for the following types of bacteria?
Obligate anaerobes
Obligate anaerobes are poisoned by oxygen, so they gather at the bottom of the tube where the oxygen concentration is lowest.
Where in a thioglycollate broth tube would you expect to find growth for the following types of bacteria?
Aerotolerant anaerobes
Aerotolerant anaerobes typically use fermentation to make ATP, so they do not require nor utilize oxygen.
Aerotolerant anaerobes will grow within the lower, anaerobic portion of the broth
What type of metabolism is used by obligate aerobes?
Obligate aerobes use (oxygen) aerobic respiration
What type of metabolsim is used by facultative anaerobes?
Facultative anaerobes can use both (oxygen) respiration and (anaerobic/without oxygen respiration) fermentation
What type of metabolism is used by obligate anaerobes?
Obligate anaerobes can only use fermentation
What type of metabolism is used by aerotolerant anaerobes?
Aerotolerant anaerobes use fermentation
Would you expect an obligate aerobe to be capable of fermentation?
No, because obligate aerobes can only use oxygenic/ aerobic respiration, not fermentation to survive.
What is the substrate of gelatinase?
Gelatin (collagen)
Do all types of bacteria have the same oxygen requirements?
No, different types of bacteria have different oxygen requirements.
_______________ require oxygen for their metabolism and are incapable of growing in an anaerobic environment, as they cannot use fermentation or anaerobic respiration.
Obligate aerobes
Both ____________ and __________ will grow in an anaerobic environment.
Facultative anaerobes
Aerotolerant anaerobes
____________ can use either aerobic respiration or fermentation, so they can use oxygen but do not require it
Facultative anaerobes
_______________typically use fermentation to make ATP, so they do not require nor utilize oxygen.
Aerotolerant anaerobes
____________ require low levels of oxygen.
Microaerophiles
What type of medium is used to determine the requirements of bacteria?
Thioglycollate broth
What does thioglycollate both contain?
Sodium thioglycollate, resuzurin, and 0.05% agar
What in thioglycollate both acts as an oxygen indicator?
Resazurin
__________ is a differential nutrient broth used to determine the oxygen requirements of bacteria
Thioglycollate
Organisms that cannot grow in anaerobic conditions, ___________, will grow only in the ________ portion of the tube
- Obligate aerobes
- Upper/ top
Organisms that can grow anaerobically, ___________and ________, will grow within the _______, anaerobic portion of the thioglycollate broth.
- Aerotolerant anaerobes and facultative anaerobes
- Lower/ bottom
What type of enzyme is secreted by bacteria (and other organisms) into their environment?
Exoenzymes
(Exo-enzymes)
What is another name for gelatinase?
Gelatinase is sometimes called collagenase
Gelatinase/collagenase is a type of what?
Protease
What enzyme catalyzes the hydrolysis of gelatin/collagen?
Gelatinase/ collagenase which is a type of protease
Why type of enzyme is used by a wide variety of bacteria to break down proteins for nutrient acquisition?
Exoenzymes
Gelatinase/ collagenase is what type of enzyme?
Exoenzyme
What type of differential medium is used to determine if an organism produces gelatinase?
Nutrient gelatin
What are the ingredients for nutrient gelatin?
Gelatin (collagen)
Peptone
Beef extract
If an organism produces ______, the exoenzyme will break down gelatin to its component amino acids and the solid gelatin will be liquid at room temperature
Gelatinase/ collagenase
How is gelatinase used as a virulence mechanism?
Pathogens may use enzymes like gelatinase to break down connective tissues as part of their invasion process, an important virulence mechanism
Why do you think agar is used to solidify media?
Agar has a higher melting point than gelatin. Gelatin is liquid at 28oC and the incubator’s temperature is at 35oC, so gelatin media would not be good to track growth, like agar would.
How do you interpret the nutrient gelatin assay?
If an organism produces gelatinase, the exoenzyme will break down gelatin to its component amino acids and the solid gelatin will be liquid at room temperature
Why must observations for nutrient gelatin be done at temperatures lower that 28oC?
Gelatin is a liquid at 28oC or above