Mico Lab Exam 2
What materials are used in the acid-fast procedure?
Carbol fuchsin acid-alcohol Methylene blue
Which of the following reagents will be used during the acid fast staining technique? Select all that apply.
Carbolfuschin Acid alcohol Methylene blue
Why is acid-alcohol used?
It decolorizes all cells except the acid-fast ones
Heat-fixing
kills the bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins to make them more visible
Cells will Gram stain most reliably when cultures are how old?
less than 24 hours old
how are DNA fragments separated by gel electrophoresis
Because each DNA molecule is negatively charged, it can be pulled through the gel by an electric field. Small DNA molecules move more quickly through the gel than larger DNA molecules
Why is Crystal Violet added to the smear?
Because it penetrates the cell wall and the cytoplasm of both gram positive and gram negative bacteria. BOTH bacteria stain purple when it is added.
Why do Gram + stain purple after decolorizing?
Because of the many layers of peptiglcon that are tightly linked by connecting chains of amino acids.
Why do DNA fragments migrate toward one end of a gel in gel electrophoresis?
Because the DNA fragments are negatively charged, and the electrodes connected to the end of electrophoresis chamber is positve
Why do we heat fix our slides?
it kills the bacterial cells it adheres the cells to the slide so they don't become distorted or wash away
Gram staining procedure
*Heat Fix* 1. Cover smear with Crystal violet 30 seconds *Wash off stain with di-water* 2. Cover smear withMordant- Gram's Iodine (10 seconds to 1 minute) *Wash off the iodine* 3. Decolorization- Acetone-alcohol (hold slide at 45 degree angle and apply decolorizer, do this until it runs clear) *Stop decolorization by washing slide with gentle stream of di-water* 4. Cover the smear withCounterstain- Safranin (30 seconds) *Wash off gently for only a few seconds* 5. Blot dry with bibulous paper & air dry 6.Examine slide under oil immersion
simple stain procedure
1) Place the air dried, heat-fixed slide on the slide holder over the staining pan 2) Cover smear with Crystal Violet 3) Leave the stain on for 30 secs 4) Rinse dye off with lionized water 5) Blot dry
Put the steps of creating a smear in the correct order by matching the number of the step with the action.
1. Clean and label a glass slide 2. Put a drop of water on the slide 3. Using Aspectic technique, transfer a small amount of bacteria to the slide and spread it around 4. Let the slide dry completely 5. Pass the slide through the Bunsen burner several times to heat fix
Why is Gram's Iodine Important?
1. Creates a CV-I complex 2. Keeps Crystal Violet from washing out of Gram (+) 3. Gets washed out of Gram (-)
Match the steps of the Gram stain with the order they should be done.
1. Crystal Violet 2. iodine 3. Alcohol 4. Safrinin
Mycolic Acid
A waxy substance that gives acid-fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution. This waxy wall repels typical aqueous stains - as a result, most acid-fast positive organisms are only weakly G+.
What color will the acid-fast cells be at the end of the acid-fast procedure?
At the end of the staining process, acid-fast cells will be reddish-pink, and non-acid fast cells will be blue.
Which of the following enzymes is needed to replicate DNA in a PCR reaction?
DNA polymerase
Gram positive cells should appear pink once you have finished the Gram stain procedure.
False
What does the Decolorizing Solution, Ethanol, do to Gram-negative bacteria?
Gram-negative: The decolorizing agent dissolves the user membrane passes through and dehydrates the thin peptigolcon layer and begins to dissolve the cytoplasmic membrane. Because the gram negative cell has little peptioglycagon and fewer connecting amino acids to trap the crystal violet complexes, the decolorize easily washes the complexes away. No longer purple
What does the Decolorizing Solution, Ethanol, do to Gram-positive bacteria?
Gram-positive: causes the petigylcogen modules to be more tightly connected making it more difficult for the crystal violet iodine complexes to move through. The decolorizing agent dissolves the cytoplasm membrane causing the dye to wash out of the cytoplasm towards the wall. The dye can not easily pass through the small spaces and multiple layers of petiglycogan and gets trapped by the tetrapeptide chains and amino acid cross bridges. Stains purple
Spore stain procedure
Heat Fix 1. Cover bacteria smear with bibulous paper, should not hang off 2. Apply Malachite Green on the paper towel for 3 minutes with heat being applied. Make sure it stays moist. 3. Rinse stain off the slide with distilled water. Make sure the towel washes off the slide 4. Do not blot after you rinse off stain. 5. Put safranin on the bacterial stain for 30 seconds 6. Rinse the safranin off after 30 seconds. 7. Blot the slide dry Spores will be green and bacteria will be pink
acid fast stain procedure
Heat fix smear 1) Cover entire slide with Carbol Fuschin for 3 minutes 2) Rinse off the carbon fuschin and shake off excess water. 3) Decolorize the smear with Acid-Alcohol mixture, holding slide at 45 degree angle and rinsing until mixture runs clear. 4) Immediately Rinse remaining acid alcohol off with water 5) Counter stain smear with Methylene Blue for 1 minute, do not cover the whole slide 6) Gently rinse smear with water and blot dry with bibulous paper and look at with immersion oil lens. Acid fast will be red nonacid fast will be blue
Why apply a spore stain?
Machine green turns the spores green and when combined with heat, drives the stain into the cell
Acid fast stain
Not protected from heat, harder to get antibiotics into. repels heat, clump together (outer layer die but inner layer protected), slow growth rate, hard to get nutrients in and waste products out, structure is gram +, survive harsh environments
Where within the DNA strand to dideoxy-nucleotides incorporate?
Only adjacent to the primer
Why do bacteria form spores?
Serves as a source of protection when environmental conditions are not favorable for survival or growth. -Scarcity of food -Change of water content -Temp changes They do no reproduce, keeps things from going in and out. Do not grow when format, heat resistance, completely dehydrate, not enough food but can still survive.
Materials needed for a spore stain
Spore stain Safranin stain bibulous paper bacterial smear
What is the primary difference between Gram positive and Gram negative cells?
Structure of the cell wall
In addition to the regular components of a PCR reaction, a PCR sequencing reaction also requires dideoxy-nucleotides. What happens when dideoxy-nucleotides are incorporated in to the growing DNA strand.
The DNA strand is terminated
What is the purpose of the Acid-fast stain?
The acid-fast stain is a differential stain which distinguishes organisms with waxy cell walls that can resist decolorization with acid alcohol. ... These bacteria are termed "acid-fast" because they are able to resist decolorization with acid alcohol.
What color is the bacteria in a spore stain
The bacteria is pink from the safranin
Assume that you got the following data from your sequencing reaction. Reading 5' to 3', what are the first 4 bases of your DNA sequence based on the visible results?
The bases are just the letters. This is not a region that codes for a protein so there would be no codons
what dideoxynucleotides are used for in a sequencing reaction.
The dideoxyribonucleotides do not have a 3' hydroxyl group, hence no further chain elongation can occur once this dideoxynucleotide is on the chain. This can lead to the termination of the DNA sequence. Thus, these molecules form the basis of the dideoxy chain-termination method of DNA sequencing
When Safranin is added to Gram +
The light red safranin dye has NO effect on the visible color. Still purple
What causes some organisms to be acid fast (what part of the cell is different and how)?
The presence of mycolic acids in the cell walls of acid-fast organisms is the cytological basis for the acid-fast differential stain.
When Safranin it added to Gram -
The red stain colors the wall and cytoplasm, the thin petioglucon allows them to be decolizered and counter stained red to pink with safranin.
What happens if you put too many cells on your slide (make the smear too thick)? (Check all that apply)
The stained cells may wash away leaving only the lower layers of unstained cells The cells may be too crowded to see the shape and arrangement of the bacteria
Methylene blue is used?
it is applied as to counterstain any cells which have been decolorized.
Why spore stain?
To see if bacteria is capable of producing spores.
You must heat fix your smear before you perform the acid fast staining technique.
True
Why do we use Gram Staining?
Used to stain bacteria as well as distinguish gram positive from gram negative.
PCR reaction
Using PCR, a single copy (or more) of a DNA sequence is exponentially amplified to generate thousands to millions of more copies of the particular DNA segment.
set it aside and let it dry completely
What should you do once you have finished spreading bacteria over the slide to create a smear?
streak plate technique
a loop is used to streak the mixed sample many times over the surface of a solid culture medium in a petri plate
What would happen if you left out the crystal violet when performing the Gram stain?
all the cells would look pink
What would happen if you left out the ethanol when performing the Gram stain?
all the cells would look purple
What are dideoxynucleotides?
are chain-elongating inhibitors of DNA polymerase
Which of the following components needs to be added to a PCR reaction? (Select all that apply)
bacterial DNA template primers DNA Polymerase
The finished reactions are pulled up into a series of long thin tubes. Each reaction mixture is pulled into one of these tubes and an electric current is used to pull the pieces of DNA through the tube to the other side. What do we call this series of tubes?
capillary array
At the end of the spore staining procedure, what color should the spores be?
green
what goes into a regular PCR reaction to amplify DNA
heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus.
Why is Ethanol used in gram staining?
is used as a decolorize or remove the stain from the cells
At the end of the acid fast staining procedure, what color will acid fast cells be?
pink
Which of the following reagents will be used during the spore staining technique? Select all that apply
safranin malachite green
A mordant is a
stabilizer that causes the crystal violet to form large crystals in the peptidoglycan layer of the cell wall.
What would happen if you left out the safranin when performing the Gram stain?
the gram positive cells would be purple but the gram negative cells would not be visible
Why is carbulfuchin stain used?
the primary stain in this procedure, and it contains phenol to help solubilize the cell wall
When the DNA fragments are separated by size using an electric current. Which pieces run the fastest/come out of the tube first.
the smallest pieces
Purpose of bibulous paper in the spore stain
to hold the spore stain over the bacterial smear
Purpose of streak plate
to isolate discrete colonies from which a pure culture can be grown
Spores are typically difficult to stain. How will we get our stain into the bacterial spores?
we will use heat to drive the stain into the spores
