93 terms
Lab Practical II
What is the name of the bacteriophage/virus used in the DNA restriction lab?
Lambda
What is the general purpose of the DNA restriction lab?
DNA from the lambda virus is cut with restriction enzymes which results in the fragmenting of the DNA that is separated using gel electrophoresis
At what temperature are the test tubes with the virus and restriciton enzymes incubated at and for how long?
37 degrees C for 30 minutes
What type of enzymes are used in the DNA restriction lab?
Three different restriction endonucleases
Name all 3 restriction enzymes used in the DNA restriction lab
EcoRI, BamHI, HindIII
What is done with the 4th test tube used in the procedure?
It is the control, it will show the normal DNA pattern of the Lambda virus without any enzymes
Briefly summarize the steps used in the DNA restriction lab
The DNA samples are mixed with a buffer solution and one of the restriction enzymes, each in their own test tube. The 4th test tube is the control. The tubes are incubated at 37 degrees C for 30 minutes. The DNA are then mixed with a loading dye, and are then loaded into the wells of a carefully premade gel agarose via a micropipette. They will be placed under an electrical current causing the DNA to move from the negative to positive side of the gel, leaving behind a "DNA fingerprint." This is visible through a viewing box after you remove your gel and stain it for 20-30 minutes using methylene blue.
What are the two different loading dyes used in the DNA restriction lab?
bromophenol blue (fast moving) and xylene cyanol (slow moving)
What can cause a short run during electrophoresis?
Higher voltage for a shorter period of time
What happens to the electrophoresis when you overload the wells?
The fragmented bands will be smeared, this means there is too much DNA, aka there was more than 4 microliters loaded via the micropipette
What can cause the wells to be poorly formed? What will happen to the electrophoresis if you have poorly formed wells?
The comb may have been removed before the gel was completely set, or there may have been vibrations during the setting of the agarose gel. The fragmented bands will appear to be smeared in all wells that were not formed properly.
What can cause an incomplete digest?
The bands appear faint or there can be extra bands in the lanes, this is a result of not enough enzymes
If there is a bump in the fragmented bands, what does that mean?
That there may have been a bubble in the gel when it set
If the wells are punctured during the loading process, what will happen to the bands?
They can be faint or not appear at all, depending on how much DNA actually stuck in the wells and how much leaked out.
Why is the control lane all one thick band?
Because the control test tube was not cut with an enzyme
Why is the ideal run much more spread out than the short run?
Because the ideal run was set at a lower voltage for a longer amount of time. A decrease in the voltage allows the DNA to separate more.
How can you account for differences in separation and band intensity between your gel and the ideal gel?
Differences in separation and band intensity can have to do with many things like: properly forming your wells, being sure there are no bubbles, that it is completely set before removing the comb; properly micropipetting the contents of the reaction tube into the wells, being sure not to puncture the wells, or under/overload the wells; setting your electrophoresis for the proper time and voltage to create the ideal amount of separation between DNA fragments.
Two small restriction fragments of nearly the same base-pair size appear as a single band, even when the sample is run to the very end of the gel. What could be done to resolve the fragments? Why would it work?
When two fragments appear as a single band, you can correct this process by increasing the length of time and decreasing the voltage of the electrophoresis. DNA samples of similar molecular weight commonly appear as one band causing the bands to be very close together.
Define endonuclease
A restriction enzyme that attaches itself to the center of the DNA/nucleic acid and alters the DNA pattern -- It restricts the replication of the viral DNA by cleaving phosphodiester bonds so that the DNA cannot be read.
What was the first endonuclease discovered and/or islated and from what species of bacteria?
EcoRI from Escherichia coli
What is the most probable reason bacterium evolved endonucleases?
Bacteria genetically evolves to produce endonucleases to become resistant against viral infections -- Survival of the fittest.
What is the purpose of staining?
Stains are required to give bacterial cells pigments so they can be observed using a bright field microscope
What are the 3 major classes of staining?
simple stain
differential stain
specialty stain
differential stain
specialty stain
Define simple stain
A single stain used to study the gross morphological features of bacteria. This staining technique can be helpful because each time you stain a specific bacteria it will have the same exact size every time. Can be crystal violet or methylene blue
Define differential stain
A combination of stains used to study the gross morphological features as well as the chemical composition of the cell wall according to their ability to retain certain dyes
What are the types of differential staining?
Gram stain
Acid fast stain
Acid fast stain
Define gram staining
By placing your smear in a series of different chemicals, the bacteria will retain certain dyes or be decolorized based on the chemical composition of its cell wall
Gram positive = teichoic and lipoteichoic acid, which will retain the stain and take on a purple color
Gram negative = cell wall contains lipopolysaccarides, which means the counterstain will be absorbed and the cells will take on pink color
Gram positive = teichoic and lipoteichoic acid, which will retain the stain and take on a purple color
Gram negative = cell wall contains lipopolysaccarides, which means the counterstain will be absorbed and the cells will take on pink color
Define acid fast staining
Bacteria with a high lipid content/mycolic acid in their cell walls are difficult to stain, if a bacteria retains the primary stain it IS acid fast. Acid fast=Yes=RED
If the cells are decolorized and retain the counterstain, they are NOT acid fast.
Everything that is NOT acid fast will stain BLUE
Used for Mycobacterium species.
If the cells are decolorized and retain the counterstain, they are NOT acid fast.
Everything that is NOT acid fast will stain BLUE
Used for Mycobacterium species.
What are the different types of specialty stains and what is the purpose of specialty stains?
Flagella stain
Capsule stain
Endospore stain
Fat bodies stain
They are used to study SPECIFIC morphological features of a bacteria
Capsule stain
Endospore stain
Fat bodies stain
They are used to study SPECIFIC morphological features of a bacteria
What is the stain used in a simple staining technique?
Methylene blue (2 minutes)
What is the primary stain used in the gram staining procedure?
Crystal violet (1 minute)
What is the purpose and name of the mordant we use in gram staining?
Gram's iodine (1 minute) -- a substance that intensifies the reaction between bacterial cells and the primary stain
What is the name of the decolorzing agent used in the gram staining procedure?
95% ethanol (repeat dipping for 15-30 seconds until stain is removed)
What is the name of the counterstain used in gram staining?
Safranin (2 minutes)
What is the primary stain used in the acid fast staining technique?
Ziehl's carbol fuschsin (5 minutes)
What chemical is used to decolorize during the acid fast staining technique?
Acid alcohol (10-30 seconds)
What is the counter stain used for the acid fast technique?
Methylene blue (2 minutes)
What is the primary stain used in the endospore staining technique?
Malachite green (5 minutes)
What is the counterstain used in the endospore staining technique?
Safranin (90 seconds)
What chemical is used to stain in a capsule staining technique?
Crystal violet (7 minutes)
What do you rinse the slide with after staining in a capsule staining technique?
20% copper II sulfate solution
Why don't we use water during a capsule staining?
Because heat creates a water halo around the cells
How can you detect the presence of endospores?
If endospores are present, they will turn green. All other cells will be a pinkish color
What happens to the appearance of the smear after you complete a capsule staining procedure?
Capsules will appear as faint blue haloes around dark blue to purple cells; they have a "fried egg" look to them
What is the purpose of the bacterial metabolism lab?
It is the next most reliable thing to differentiate between bacterias that have very similar morphology, cell wall structure and colony characteristics. It will tell you their metabolic processes, ie what their by products are and if they are fermentative or not
What sugars do bacterias have the capability of fermenting?
Dextrose
Lactose
Sugar
DLS
Lactose
Sugar
DLS
Bubbles trapped inside the durham tube inside the test tubes during the metabolism lab means what?
It indicates that CO2 has been created as a by product
What is the medium used for an acid indicator? What happens if an acid is formed?
Phenol red -- it will turn from red to yellow because of the drop in pH
What are the possible by products in the metabolism lab?
Gases (CO2)
Acids
Alcohols
Acids
Alcohols
What does it mean if the tube turns orange?
It means it has turned neutral
Which bacterium produces gas and acid from all 3 sugars?
Citrobacter freundii
Which bacterium did not produce gas or acid from all 3 sugars?
Sarcina lutea
Which bacteriums produced acid but not gas for Dextrose and Sucrose?
Bacillus subtilis
Staphylococcus epidermis
Staphylococcus epidermis
What are the only two bacteriums that produced gases for Lactose?
Escherichia coli
Citrobacter freundii
Citrobacter freundii
Define differential media
a nutrient agar that will contain a specific chemical that results in a kind of growth or change that will allow you to differentiate between different bacterias
Define MacConkey agar
An agar that is used to isolate and differentiate between lactose fermenting and non lactose fermenting gram negative enteric bacteria
What happens to the bacteria on the MacConkey agar if it is lactose fermenting?
The colonies will turn a pinkish color
What happens to the bacteria on the MacConkey agar if it is non-lactose fermenting?
The colonies will be colorless
What two bacterias are lactose fermenting?
Escherichia coli
Citrobacter freundii
Citrobacter freundii
Which bacteria is non lactose fermenting?
Proteus vulgaris
Define enzyme
A catalytic agent that is protien in nature and capable of speeding up biochemical reactions. They decrease the activation energy barrier, are only needed in small amounts, and are reaction/substrate specific.
Exoenzymes are AKA....
hydrolases
What is the purpose of a hydrolase?
To split complex organic compounds into smaller units for easier absorption
What is the purpose of the enzymatic activity lab?
Biochemical tests are used to determine the presence of certain enzyme systems responsible for the decomposition of lipids, starches and protiens.
For starch hydrolysis, what is the differential media used?
Potato dextrose agar
The cells ability to hydrolyze starch is dependent upon what enzyme?
Amylase
What is the chemical used as an indicator of starch being present?
Iodine
When iodine comes in contact with a medium that contains starch, what color will it turn?
Blue-black or deep purple
If the starch molecules have been hydrolyzed, what color will the medium be?
Clear
For protien hydrolysis, what differential media is used?
Skim milk agar
What is Casein?
The principal protien in milk, it also gives milk its opaque/whiteness. Many bacteria produce enzymes to hydrolyze this protien into a more transparent derivative.
During protien synthesis, if casein hydrolysis occurs, what will happen to the skim milk agar?
The skim milk agar will have a fading of the white color/opaqueness
During lipid hydrolysis, what differential media is used?
Spirit blue agar
During lipid hydrolysis, what will happen to the cells and what happens to the agar if hydrolysis occurs?
The cleavage of the phospho ester bonds will form a water soluble lipid, creating a opalescence around the growing bacteria. The agar will have a whiteish/fading color if hydrolysis occurs
Only one bacteria hydrolyzed starch, which one was it?
Bacillus subtilis
Only one bacteria hydrolyzed lipids, which one was it?
Bacillus cereus
Which bacterium did not hydrolyze on any of the differential mediums?
Bacillus megaterium
Bacillus subtilis hydrolyzed which mediums?
Starch
Protien
Protien
Bacillus cereus hydrolyzed which mediums?
Protien
Lipid
Lipid
Why did we use compartmentalized agar plates for the spirit blue and potato dextrose but not for the skim milk?
Because you would not be able to tell the difference between the fading of the skim milk between compartments/bacteriums
What is the purpose of the microbial sensitivity lab?
To determine which chemical substances are the most effective in controlling the growth of bacteriums
What are the 3 categories of chemicals that are used to control microbial growth?
Antiseptic
Disinfectant
Antibiotic
Disinfectant
Antibiotic
The sensitivity lab used assay mediums. Define assay media.
Promotes or inhibits the growth of bacterias by the use of different chemical substances
What is a clear zone and what does it indicate?
A clear ring around the chemical discs that indicates how effective the chemical is at inhibiting a specific bacterias growth based on the measurement of the clear zone
At what temperature and for how long do you incubate the petri dishes with the chemical discs on them?
22-25 degrees C for a period of 24 hours
What ANTIBIOTIC was the MOST effective against both species?
Chloramphenicol
Which ANTIBOITIC was the LEAST effective against both species?
Penicillin
Which ANTISEPTIC was the MOST effective against both species?
Tincture of iodine
Which ANTISEPTIC was the LEAST effective against both species?
Listerine
Which DISINFECTANT was the MOST effective against both species?
Bleach
Which DISINFECTANT was the LEAST effective against both species?
Ammonia
Did any chemical affect the two species differently? Which ones?
Yes
Scope > E. coli - No; B. cereus - Yes
Listerine > E. coli - Yes; B. cereus- No
Scope > E. coli - No; B. cereus - Yes
Listerine > E. coli - Yes; B. cereus- No
What was the purpose of the controls in the sensitivity lab?
To show normal bacterial growth without any chemicals