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Microbiology ( PCR, Kirby Bauer , ELISA, pGLO, Antibiotics)
Exam Lab 3
Terms in this set (91)
What is the purpose of the kirby-bauer method?
measures sensitivity of bacteria to antibiotics by culturing bacteria on solid growth media surrounding sources of drug
*took the diameter in millimeters
Antibiotic Activity : What is broad spectrum in antimicrobial?
broad spectrum works on Gram+ & Gram- bacteria
Antibiotic Activity: Whats is Narrow spectrum in antimicrobial?
narrow spectrum works ONLY on Gram+ or Gram-
(DOES NOT WORK ON BOTH)
What is Structural Staining?
staining for specific structures like capsules, and spores
What is the Capsule stain Theory?
-2 Part Stain
-Specifically highlights any potential capsules produced by bacteria
-No Heat Fixing
What are the reagents used in capsule stain?
2. Maneval Stain
What are antimicrobials?
What is Antibiotics ?
Naturally Occurring substance that one organism produces to STOP the growth of another organism
What is Chemotheraputics?
Do not occur naturally; made in a laboratory to STOP the growth of an organism
Theory of Kirby-Bauer Test
-Looks for zones of Inhibition
- Looks to see if a bacteris is Resistant, Intermediate, or Susceptible to an antibiotic
- test efficacy if we expose bacteria do we get zones of inhibiton
Explain the purpose behind the simmons citrate media
SImmons Citrate works by identifying Enterics wether or not the produce CITRATE PERMEASE
1. Try to identify unkown organism
2.Helps identify Enterics
- Gram negative lactose fermentor
What is the role of Citrate Permease ?
-Enzyme used as channel protein to allow cell to import Citrate.
-Citrate is only carbon containing compound media
What color does a Media POSITIVE for citrate permease turn?
What color does a NEGATIVE citrate permease media turn?
What is PHENOL RED?
What was the role of phenol red in the experiment ?
To help identify and unknown organism & look for sugar fermentation
What was the durham tube?
Inverted test tube
What was the role of the DURHAM tube in the sugar fermentaiton experiment?
Used to look for carbon dioxide production
Produces: Acids, Alcohol, and Gas
What are the 3 sugars used in the sugar fermentation experiement?
What enzyme breaks down Glucose?
What enzyme breaks down sucrose?
What enzyme breaks down Lactose ?
How do you identify a sugar fermentation test?
Phenol Red Broth
What is the purpose of testing fermentative capabilities of out microorganisms with the 3 different sugar broths?
1. To see is they can breakdown sugar.
2.To se what kind of waste product they produce
What products could be produced through fermentation of these sugars ? Explain how we tested these sugars.
Products that can be produced are: Acid & Gas
We Added our Unknown organism into the broths
If acid is produced the broth turns yellow
If gas is produced the broth will have bubbles
How do you know a sugar fermentation is POSITIVE for acid ?
The broth turns yellow
How do you know a sugar fermantation is POSITIVE for gas ?
The broth will have bubbles
What is the purpose of using an Enterotube?
Used to identify Gram negative bacteria ( specifically Enterics)
How can one determine what organism is in the enterotube?
There are 12 test in the enterotube allowing us to see with which test the bacteria reacts. We also use the score card.
What is the purpose of performing a CATALASE test?
To identify unknown bacteria based on whether the bacteria makes an enzyme known as CATALASE.
What dose Catalase breakdown?
What is the reason behind an organism producing CATALASE?
Organism produces catalase to break down hydrogen peroxide when present. Hydrogen peroxide damages healing tissue.
How can one identify a catalase POSITIVE Organism ?
There will be bubbles present
How can one identify a catalase NEGATIVE organism ?
There will be NO bubbles present
What are the steps to perform a catalase test?
1. Aseptically transfer bacteria onto a slide
2. Add hydrogen peroxide
3. Look for a reaction ( +Bubbles or -Bubbles )
What are the steps when performing differential stain ?
1. Wash & Label Slide
2.Draw a circle
3. Aseptically add H2O onto slide
4.Aseptically add and smear bacteria with the H2O
5.Air dry 2 minutes
7. Apply 1-2 drops of Crystal violet ( let sit for 1-2 minutes)
8.Rinse w/ H2O Shake off excess water
9.Add Mordant ( Iodine) -- Flood with iodine for 1-2 minutes
10. Rinse with H2O
11.Decolorize with Ethanol ( 2 full droppers)
12.Rinse with H2O
13. Add 1-2 drops of Safranin
14. Let sit for 30 seconds
15. Rinse w/ H2O; Blot Dry
What are Gram POSITIVE bacteria characteristics?
60-90% Peptodiglycan layer
Has Teichoic Acid
What are Gream NEGATIVE bacteria characteristics?
10-15% Peptodiglyca layer
NO Teichoic Acid
Lipopolysaccharide layer (LPS)
What is the purpose for the stains and reagents used during differential staining ?
1.Crystal Violet dehydrates peptodiglycan layer trapping in crystal violet.
2.Idoine forms an and insoluble crystal violet iodine
4.Safranin stains bacteria that has been decolorized
(gram negative bacteria can be easily seen Pink)
What is differential stain?
Distiguishes between 2 different organisms
ex: Gram positive & Gram negative
What are the stains used during differential staining?
What is Bactericidal ?
compounds that KILL microorganism directly
What is Bacteriostatic?
compounds that DO NOT kill microorganisms but stops it from dividing and replicating
How did we determine which antimicrobial was MOST effective?
Performed theKirby-Bauer rest to see what antibiotics would work best.
What does the size of a ZONE of inhibition indicate?
Depending on how clear the zone is on the TSA agar the more effective the antibiotic. The Bigger zone the more effective antibiotic is !
What is a Zone of inhibition?
A circular area around the paper antibiotic disc used to determine the susceptibility of bacteria towards an antibiotic.
How did we test the effectiveness of various antimicrobials on microorganism ?
We covered the TSA plate completely with bacteria. Then placed 2 different antibiotic paper disc inside. Then we incubate. Once incubation is complete we measure the zones of inhibition in millimeters.
what are the steps to perform a capsule stain ?
1.Wash & Label 2 slides
2. Add 1 drop of Congo red near label
3.aseptically transfer organism to congo red ( tap it in)
4.Use 2nd slide to smear the congo red drop
5. Air dry for 10 minutes
6. Flood smear with several drops of Maneval stain
7.Gently drip water down the slide
8. Oil view
What dyes were used during capsule staining ?
1. Congo Red
2. Maneval Stain
What are the 4 advantages of an organism producing capsules?
1.Help with attachment to surfaces
3.Resist antibiotic penetration
4. Help protect the cell from suffocation & drying out
Why do we NOT heat fix when performing a capsule stain ?
-Heat will either cause the cell to Shrivel and shrink
-or it will destroy a capsule if the cell actually has one
What are the characteristics of each reagent that help view / observe capsules?
Congo Red- Acidic dye / soluble in water
Maneval stain - Acidic dye
what is the mechanism that is at work allowing us to use ELISA test to look for a particular Antigen ?
The mechanism is ANTIBODY to ANTIGEN specificity, meaning specific antibodies will ONLY bind to the specific antigen if it is present in patient serum.
If Antigen is NOT present, then the antibody has nothing to bind to, resulting in a negative result.
State what the tern ELISA stands for?
ENZYME- LINKED IMMUNOSORBENT ASSAY
What is the role of the PRIMARY antibody in ELISA test?
Primary antibody is specific to the antigen we want to detect.
What is the role of the SECONDARY antibody in ELISA test?
Secondary antibody is specific to the primary antibody
What is the role of the SUBSTARATE in ELISA testing?
Enzyme at the tip of the secondary antibody reacts with the substrate. With the substrate we should see a color change once all antibodies have stuck together.
How does one determine wether a reaction is positive or negative during ELISA testing .
During ELISA testing there will be a color change from clear to blue is the result is positive. If the result is Negative there will be no color change, stays clear
What are the steps to perform an ELISA test?
1.Add patient Serum
2.Let sit for 5 minutes
3.Rinse the side wells with Wash buffer x2
4.Add PRIMARY antibody
5.Let sit for 5 minutes
6.Rinse with wash buffer x2
7. Add secondary antibody
8.Let sit for 5 minutes
9. Rinse with wash buffer x2
10. Add colorizing substrate
11. If enzyme is positive wells turn blue
How and Why are we able to extract DNA from Halobacterium Salinarum cells?
1.Take cells & put them into test tube with diH2O
2.Take swab w/ cells & swirl around Di-Ionized water
3. Cells become hypotonic ( low salt ) solution water rushes into the cells causing them to lyse allowing DNA to float in the water.
4.Add ICE cold Ethanol slowly down side of tube
5.Ethanol Phase causes precipitation
6.DNA does not dissolve in Ethanol
7. Precipitate of DNA will begin to rise into the ethanol
8. Bromothymol Blue gets added ( ph Indicator)
What are the characteristics of Halobacterium Salinarum ?
Survives in HIGH salt environment
can import salt into cells
What is the purpose of using ICE COLD ethanol and bromothymol blue during DNA extraction ?
- Makes DNA Soluble precipitate by adding Ice cold Ethanol
-DNA does not dissolve in Ethanol
-Bromothymol Blue is the ph indicator
*yellow = Acid
* Blue = Alkaline
What is insoluble?
Can't be dissolved
what is soluble?
able to dissolve
What is the purpose of performing the polymerase chain reaction (PCR) test?
PCR allows us to make multiple copies of a single DNA sample.
What is the role of the FreeNucleotide in PCR testing ?
(dNTPs) Deoxy Nucleo Typhosphate
Act as the building blocks
What is the role of TAQ DNA in PCR testing ?
-TAQ DNA polymerase enzyme
- runs the action of copying
What is the role of the BUFFER in PCR testing?
Has Magnesium Chloride , helps keep the enzymes functioning properly
What is the role of PRIMERS in PCR testing?
Target specific areas that need to be copied
What occurs during PCR DENATURATION?
- This is step 1
-Opens/ unzips the DNA strand
-Happens by increasing temperature to the test tube to 95 degrees celcius
-Breaks hydrogen bonds to unzip DNA
What occurs during PCR ANNEALING?
- This is step 2
-cool the test tube to 50-60 degrees celcius
-allow primers to target specific areas in DNA
* primers are made in laboratory
What occurs during PCR EXTENSION ?
-This is step 3
-Increase temp to 72 degrees celcius
-TAQ DNA polymerase finds starter sequence, reads it, and builds new strand .
What is the purpose of performing electrophoresis ?
Allows us to separate & visualize DNA sample
How does electrophoresis work?
The gel box has an electric field with a positive and negative end. DNA is negatively charged so it migrates to the positive end.
How can one determine if a patient teste positive for a virus by looking at a electrophoresis image?
compare bands between virus and patient. Choose the patient with matching band to the virus.
What is transformation?
the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s).
What is the mechanism by which our bacteria were transformed ?
Electroporation: bacteria is mixed with DNA & apply electric shock to culture causing temporary holes.
CaCl2 & Heat Shock: Chemical Transformation
What is a competent cell?
a bacterial cell that can take up DNA
What are the 3 genes found on pGLO?
State the function and role of the bla gene
The bla gene includes a promoter at the 5' end of the gene. This is a weak constitutive promoter (always "on" at a low-level). It will instruct RNA polymerase to continually make a low-level of mRNA for this gene. The mRNA will be translated to produce low-levels of the b-lactamase protein
What is the function and role of araC gene?
Arabinose -C (Operon) Series of genes that work together.
Responsible for metabolism of arabinose
Promotor sequence for transcription
State the function & role of GFP gene?
Green Fluorescent Protein- used as a marker protein to allow scientist to view particular proteins
Isolated from Aquarea victoria jelly fish
Glows in the dark
What is the role of Luria Broth?
Nourish the cells
What is the role of the Luria Broth Plates?
Luria plates are the control for the experiment
What is the role of the Luria broth/ampicillin plate?
This plate has antibiotic, when adding Negative pGlO they wont grow because they do not have antibiotic resistance gene.
No Growth - No Glow
What is the role of the Luria broth/ Ampicillin/ Arabinose plate?
This plate has growth but NO glow, grew in presence of ampicillin because this plate received the antibiotic resistance gene. it does not have arabinose sugar to turn on Arainose-C operon. Without the operon there is no glow
Explain why you would see more growth on the LB/-pGLO plate VS. +LB / Ampicillin +pGlo plate.
LB/-pGLo = theres nothing preventing the bacteria from growing. Lawn growth , Not every cell gets transfromed.
+LB/Ampicillin +pGlo= has the +pGLo but does not have the inducer to turn on Operon
Colonies are visible
What is the role of COLD CaCl2 ( Calcium Chloride) ?
Promotes the binding of plasmid DNA to Lipopolysaccharides ( LPS)
What is the role of pGLO?
Procedure used to transform bacteria with a gene that codes fro green fluorescent protein (GFP)
What is the role of the water bath ?
Heat shock puts temporary holes into the cells, allowing plasmids to enter..
how does the term competent refer to our experiment ?
The ability for the bacterial cells to take up GFP or pGLOW.
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