Lab Exam 3 Flashcards
What is transformation
Type of horizontal gene transfer - recipient cell take up dna from donor
Why is it useful for bacteria to preform transformation
Allows bacteria to acquire new traits
How is a bacterial cell made competent in the transformation lab?
It is chemically induced with an ice shock and calcium chloride and then a heat shock to let the dna pass through the membrane
What is a vector
A plasmid that Carries foreign dna into bacterial cells
Only a small percentage of bacterial cells will take up plasmid dna
TRUE or FALSE
True
Recombinant plasmid
Plasmid that successfully gets inserted into MCS
What color colonies will a recombinant plasmid be
White
MCS (multiple coding site)
Where the dna of interest is inserted by researcher
Which plasmid was used in the transformation lab
Puc18
What are the 3 important sites we looked at in the transformation lab
Lac z gene
Bla gene
Multiple cloning site
The bla gene
Allows us to determine which cell has a plasmid
What does the bla gene produce
Enzyme penicillinase - breaks down beta lactam ring
What happens once ampicillin has broken the beta lactam ring
It can not inhibit the growth of bacteria therfore it will continue to grow
Lac z (b- galacoside) gene
Allows us to determine which cell has a recombinant plasmid
What is the purpose of beta galactosidase
Metabolizes xgal on the plate
What occurs when the lac z gene (beta galactosidase) metabolizes xgal
A blue color is produced
Where is the multiple cloning site (mcs) on the puc18 plasmid
In the lac z gene
If new foreign dna is able to be inserted into the multiple cloning site of a plasmid, the plasmid is
Recombinant
If the new foreign dna is not able to insert into the multiple cloning site of the plasmid, then the plasmid is
Non recombinant
Non recombinant plasmid
No dna in the multiple cloning site
What color would a non recombinant plasmid be
Blue!! Due to the xgal not being interrupted
Why did we use water as the control in the transformation lab
To see if e. Coli will grow
What was in the lb ++ plates used in the transformation lab
Xgal
Ampicillin
What is a mutagen?
A chemical substance or physical agent that can induce changes in the base sequence of dna
What does salmonella need in order to grow?
Histidine
Histidine
Used by bacteria to grow and survive
What type of media plate does not contain histidine
Minimal media
Purpose of the Ames test
To show rather or not a certain chemical substance is a mutagen
Back mutagen
Mutation that reverts the bacteria genome to a while type (Normal)
Wild type
What is found in nature ( normal type)
What type of mutation is present in salmonella F in the Ames test?
Frameshift mutation
What type of mutation was present in the salmonella M strain?
Base pair mutation
What was the negative control plate used in the Ames test?
Minimal media plate with disc soaked in water
What is the outcome for the negative control plate possibly having colonies? _ the water
Spontaneous mutation occurring during dna replication allowing salmonella to grow w/o histidine
What was the positive control plate in the Ames test?
Minimal media plate w/ disc soaked in NaAz
What is sodium azide (NaAz) and what does it cause?
It is a mutagen
- causes: base pair mutation
4-Nitro is? And causes what?
Mutagen that causes a frameshift insertion mutation
4- nitro is capable of fixing what salmonella strain
F
NaAz is capable of fixing what salmonella strain
M
What would a positive control plate look like in the Ames test?
Growth of salmonella due to presence of known mutagen
What does no growth indicate on a minimal media plate during the Ames test
The chemical is not a mutagen
What type of plate was used in the toxicity test?
Complete media
What does complete media contain that minimal media doses not?
Histidine
If a zone of inhibition is present during a toxicity test that indicates
The chemical is toxic to the bacteria therfore they die which causes the clearing
What are the 6 metabolic tests used to identify bacterial species
Catalase
Coagulase
Mannitol salt agar
Triple sugar iron test
Coli form test
Entertube test
What type of bacteria does the catalase test identify?
Bacteria that can preform aerobic respiration
What type of bacteria does the coagulase and mannitol salt agar test identify?
Staphylococcus species
What type of bacteria does the coliform test identify?
Bacteria, usually e. Coli present in water as fecal contamination
What type of bacteria does the triple sugar iron test identify ?
Enteric bacteria
What is catalase
An enzyme that breaks down hydrogen peroxide to water and oxygen
Why would a bacteria need to produce catalase?
To breakdown hydrogen peroxide since it is toxic as byproduct in aerobic respiration
What type of bacteria needs catalase
Ones that can preform aerobic respiration
What type of bacteria preform anaerobic respiration
Obligate aerobes
Facultative anaerobes
If a bacteria did not produce bubbles in the catalase lab what does that mean?
Catalase reaction did not occur
What locations on the body might you find bacteria with the catalase enzyme?
Mouth or skin
Where on the body might you find catalase enzyme not present?
Intestines
Difference between fermentation and anaerobic respiration
Electron transport chain not present in fermentation
What is coagulase?
Enzyme that catalyzes conversion of fibrinogen into fibrin
Why does a bacteria need to produce coagulase
Helps to evade hosts immune system
How does clotting the blood help. A bacteria envied the host’s immune system?
Fibrin creates a protective barrier by surrounding saureus cells protecting it from immune cells which will kill them
Why is coagulase considered a virulence factor?
Causes bacteria to survive and continue to replicate in the host cell
What type of blood plasma did we us in the coagulase test in lab?
Rabbit plasma
What would a positive coagulase test look like? negative?
Positive : clot formed - thick and cloudy
Negative : no clot - liquid
What type of media is mannitol salt agar?
Selective - halo tolerant bacteria
Differential - staphylococcus aureus
What ingredient in MSA plates make it selective?
7.5% sodium chloride
Ph indicator
How is MSA plates differential?
They distinguish between bacteria that can ferment and those that can not
What ingredients in a MSA plate make it differential?
Mannitol
Ph red indicator
Mannitol is
A carbohydrate broken down via fermentation
What is the important by product of fermentation of mannitol?
Lactic acid
What does the phenol red indicator measure in the MSA test?
Presence of lactic acid indicating bacteria can do fermentation
If phenol red indicator turns yellow, what does that mean
Ph level = low
- lots of lactic acid therefore can ferment mannitol
If the phenol red indicator turns pink on the MSA plate, what does that mean?
Ph is high
- no lactic acid therefore an not ferment mannitol
MSA -/- plate
Can not grow in high salt environment and can not breakdown mannitol to lactic acid
MSA +/+ plate
Can grow in high salt environment and can breakdown mannitol
- will be yellow
MSA +/- plate
Can grow in high salt environments and can breakdown mannitol but can not break down to lactic acid (pink)
Enteric bacteria are gram+ or gram-?
Gram -
Enteric bacteria are coccus or bacillus?
Coccus
Are enteric bacteria facultative aerobes or facultative anaerobes?
Facultative anaerobes
What are the two abilities of enteric bacteria?
Able to ferment sugars and breakdown amino acids
What is the triple sugar iron test used for?
To distinguish between enteric bacteria
What makes up a TSI agar plate
Peptone base
Glucose
Sucrose
Lactose
Why are glucose, sucrose and lactose used in the triple sugar iron test?
To see if the bacteria can ferment them
What sugar does bacteria prefer to use the most?
Glucose!!!
What ingredient allows us to see if a bacteria can ferment any of the 3 types of sugar?
The red phenol indicator
What does the red phenol indicator measure in the TSI test?
Acid by product being used for fermentation
How can you tell gas was produced via fermentation?
Bubbles occurred
What occurs on the slant of the tube?
Aerobic respiration
What occurs in the butt of the tube in a TSI test?
Fermentation
No fermentation of sugars has what color slant/butt
Slant = pink
Butt = red
If a bacteria can only ferment glucose what color will the slant/butt be
Slant = pink
Butt = yellow
If a bacteria can ferment glucose, lactose and sucrose, what color will the slant/butt be?
Slant = yellow
Butt = yellow
If a bacteria is able to metabolize cysteine, what color will the slant/butt be?
Slant = yellow
Butt = black precipitate
What ingredient is added to the TSI test to see if the bacteria can break down the amino acid cysteine?
Feso4
Obligate aerobe tube in TSI test will have
Red slant w/ red butt
- NO FERMENTATION
What does cysteine (amino acid) get broken down to?
Hydrogen sulfide
The ph indicator in the TSI test detected the breakdown of what amino acid
Cysteine
What does the coliform Analysis test for?
Fecal contamination in water
Coliform means
Having E. Coli like characteristics
E. Coli characteristics
Gram-
Bacillus
Can ferment lactose
What are the three steps (tests) in Coliform analysis?
- Presumptive test
- Confirmed test
- Completed test
What type of tube is used in the presumptive test?
Lactose broth with small Durham tube inside
Why is lactose broth used in the coliform analysis lab?
To make sure it selects e.coli - it is needed to ferment
The purpose of the first test - presumptive test?
To select bacteria that can ferment lactose
What type of tube was used in the 2nd test - confirmed test?
Brilliant green lactose bile broth w/ Durham
What makes the brilliant green lactose broth selective?
Brilliant green dye and bile salts
How does the brilliant green dye and bile salts make it selective?
Inhibits growth of gram+ making it promote the growth of gram-
What was the purpose of the 2nd test - confirmed test?
To select for bacteria that can ferment lactose and are gram negative
What type of plate was used in the completed test in coliform analysis?
EMB - eosin methylene blue agar plates
what type of media was the methylene blue agar (EMB) ?
Selective & differential
What made EMB plates selective?
The methylene blue inhibited the growth of gram+
What made the EMB plates differential?
Eosin dye - making the copies look metallic green due to high acid production
Whatwas the purpose of the last - completed test in the coliform analysis?
To select gram negative bacteria that can ferment lactose by producing lots of acid
Does a presumptive test indicate evidence of fecal contamination in a water sample?
No- only the presumptive test tells if a bacteria can ferment lactose not if it is gram -
Does a confirmed test indicate definitive evidence of fecal contamination in a water sample?
No- further testing is needed
- it confirms the test is positive for fermenting lactose and being gram- but not how well it can ferment
Carcinogen
Chemical substance or physical agent that is capable of causing cancer
What type of bacteria (group) was being tested in the rapid one lab?
Enteric bacteria
What types of breakdown can enteric bacteria do?
Ferment sugar, breakdown amino acids
Name a sugar that was used in rapid one
Sorbitol , adonitol
Name an example of an amino acid in rapid one lab
Lysine, arginine
- ends in ine
Name an example of another molecule used in rapid one
Urea, fatty acids
How could you tell a bacteria was able to ferment sugar, breakdown an amino acid or a molecule in rapid one?
Color change occurred - indicating ph change
When sugars are broken down in rapid one, does the product become more acidic or basic?
Acidic - ph lowers
