Micro Lab practical #2

calculate PFU/ml

-Obtain a plate with 25-250 countable colonies. PFU/ml = Number of plaques / dilution x vol. of diluted virus

Describe IgA

-Present in tears, saliva, and mucus, as well as in secretions of the respiratory, reproductive, digestive, and urinary tract -functions to neutralize bacteria and viruses and prevent them from entering the body or reaching the internal organs

Results of lysogenic test

-Prophage DNA incorporated in host DNA -Phage conversion -Specialized transduction

What is the purpose, selective ingredient, differential ingredient and the interpretation of Mannitol Salt Agar (MSA)

-Purpose: If an organism can ferment the sugar, mannitol -Selective ingredient: salt -Differential ingredient: pH indicator (phenol red) -Interpretation: agar turns yellow= positive for mannitol fermentation on MSA. Agar stays red=negative for mannitol fermentation on MSA.

What is the purpose, selective ingredient, differential ingredient and the interpretation of Glucose fermentation tubes?

-Purpose: capable of glucose fermentation. -Selective ingredient: glucose -Differential ingredient: phenol red -Interpretation: Red=more acidic (higher pH), Yellow=more basic (lower pH)

What is the purpose, selective ingredient, differential ingredient and the interpretation of Citrate utilization

-Purpose: citrate as sole carbon source. This depends on the organisms ability to produce the enzyme, citrase. -Selective ingredient: citrate -Differential ingredient: pH indicator, bromothymol blue -Interpretation: increases the pH causing the color to change from green to blue.

What is the purpose, selective ingredient, differential ingredient and the interpretation of Eosin Methylene Blue

-Purpose: ferment lactose. -Selective ingredient: lactose, stains (eosin and methylene blue. -Differential ingredient: stains, eosin and methylene blue Interpretation: Vigorous lactose fermentation results in colonies of a metallic green color.

What is the purpose, selective ingredient, differential ingredient and the interpretation of Esculin Hydrolysis test?

-Purpose: hydrolyze esculin as a carbon source. -Selective ingredient: bile -Differential ingredient: iron -Interpretation: Diffuse blackening of more than half of the slant within 24-48 hours indicates esculin hydrolysis. + blackening is considered positive. All group D streptococci will be bile-esculin positive within 48 hours.

What is the purpose, selective ingredient, differential ingredient and the interpretation of Coagulase

-Purpose: tests an organisms ability to clot blood plasma using the enzyme coagulase. -Selective ingredient: NONE -Differential ingredient: Rabbit plasma -Interpretation: positive test is when the medium solidifies (like jello). A coagulase negative medium results in a liquid broth.

What is the purpose, selective ingredient, differential ingredient and the interpretation of Hemolysis (blood agar)

-Purpose: to determine which genus of Streptococcus are capable of producing extracellular enzymes that can lyse red blood cells. -Selective ingredient: NONE -Differential ingredient: sheep's blood -Interpretation: Beta-hemolysin breaks down the red blood cells and hemoglobin completely. This leaves a clear zone around the bacterial growth. Alpha-hemolysin partially breaks down the red blood cells and leaves a greenish color behind. If the organism does not produce hemolysins and does not break down the blood cells, no clearing will occur. This is called γ-hemolysis (gamma hemolysis).

What is the purpose, selective ingredient, differential ingredient and the interpretation of urease test

-Purpose: used to differentiate organisms ability to hydrolyze urea with the enzyme urease. -Selective ingredient: NONE -Differential ingredient: pH indicator, phenol red Interpretation: When urea is hydrolized, urea hydrolized=pH rises and turns color from yellow/orange to red/pink

What is the purpose, selective ingredient, differential ingredient and the interpretation of Catalase

-Purpose: used to differentiate staphylococci (catalase-positive) from streptococci (catalase-negative) and porduce the enzyme, catalase. -Selective ingredient: NONE -Differential ingredient: NONE -Interpretation: Catalase positive cells will produce bubbles.

Describe IgE

-antibodies are present in tiny amounts in serum - responsible for allergic reactions -bind to the surface of certain cells called mast cells, which contain strong chemicals, including histamine -When an allergen such as pollen binds with its specific antibody, it stimulates the release of histamine from the mast cell.

What are the common characteristics of Enterobacteriales?

-gram negative -short rods -They are non-sporulating -facultative anaerobes

Described IgG

-most common and the most important. -circulate in the blood and other body fluids, defending against invading bacteria and viruses -activates other immune cells that engulf and destroy the antigens -smallest of the antibodies -moves easily across cell membranes -pass through the placenta to her fetus, providing a temporary defense to her unborn child.

Describe IgD

-present in small amounts in the blood. -found mostly on the surface of B cells—cells that produce and release antibodies. -assists B cells in recognizing specific antigens.

Describe IgM

-present in the blood -largest of the antibodies -combining five Y-shaped units -defending against antigens but cannot cross membranes because of its size -main antibody produced in an initial attack by a specific bacterial or viral antigen

What are the shared characteristics of the pathogenic cocci?

-spherical or oval shape -ability to live on its own or in colonies. -can be positive or negative, depending on the strain.

Describe the ELISA method:

1. Antibody is absorbed into the well 2. Patient sample is added; complimentary antigen binds to antibody 3. Enzymed-linked antibody specific for test antigen is added and binds to antigen, forming sandwich 4. Enzyme substrate is added and a reaction produces a product that causes a visable color change.

What are the steps of the lytic cycle?

1. Attachment-Phage attaches to host cell 2. Penetration-Phage penetrates host cell and injects its DNA 3. Biosynthesis-Phage DNA directs synthesis of viral components by the host cell 4. Maturation-Viral components are assembled into virions 5. Release-Host cell lyses, and new virions are released.

Which 2 tests can be performed by conducting a Methyl Red/Voges-Proskauer broth test?

1. Methyl Red test 2. Voges-Proskauer test

What are the steps of the latent cycle (lysogenic)?

1. Phage attaches to host cell and injects DNA 2. Phage DNA circularizes and enters lytic cycle or lysogenic cycle. 3b. Phage DNA integrates within the bacterial chromosome by recombination, becoming a prophage. 4b. Lysogenic bacterium reproduces normally 5. Sometimes the prophage may excise from the bacterial chromosome by another recombination event, initiating a lytic cycle.

What are the steps in Albert's stain?

1. Prepare a smear and fix it by gently heat over a flame. 2.Cover the smear with Albert stain (Albert I) staining solution. Allow it for 5 minutes. 3. Pour off the stain after 5 minutes. Do not wash. 4. Cover the smear with Albert's iodine (Albert II) solution for 2 minutes. 5. Pour off the stain after 2 minutes. Do not wash 6. Blot to dry the smear. 7. Examine the smear on oil immersion objective lens (x 100) of a microscope

What are 2 Voges-Proskauer reagents

1. alpha-naphthol 2. potassium hydroxide

How many classes of antibodies are there?

5

What is the purpose of resazurin?

A color indicator which turns pink when oxidized providing a reliable indicator of oxygen penetration of the media. A positive result = pink band

What is the mechanism of action for bleach (halogen)

Acts to remove electrons from cellular material which disrupts cellular membranes and denatures proteins.

What are dichotomous keys, and how/why do we use them.

An efficient method of organizing biochemical tests. It is a series of questions, each involving a biochemical test, which can be answered with a yes or a no.

Were any disinfectants more effective against certain types of organisms than others?

B. subtilis = more susceptible to bleach and ethanol at a 1:4 strong concentration E. Coli= more susceptible to bleach and ethanol at a 1:4 strong concentration S. aureus= more susceptible to bleach at at 1:4 strong concentration and amphyl at a 1:1 low concentration.

Which disinfectant was most effective?

Bleach

What does a positive Albert's stain test look like?

Body of Corynebacterium diphtheriae appears green and metachromatic granules appear blue black.

Describe the results of the SIM medium motility test

Can be determined by observing growth relative to the stab line. Nonmotile organisms will grow around the stab line only. Motile organisms will grow throughout the medium, producing turbidity.

What is the purpose of Fluid Thioglycollate Broth?

Contains a small amount of agar to slow diffusion of oxygen in the media is used for both anaerobic and microaerophilic organisms. It can also help distinguish organisms relationship to oxygen. Capable of supporting a wide varied of organisms. Strict anaerobic grow in the bottom of the media and strict aerobes will only grow on the surface.

What organism is positive microbe for VP test and negative for MR?

Enterobacter aerogenes

What does ELISA stand for

Enzyme Linked Immuno- Sorbent Assay

Name 1 bacteria that is indole positive

Escherichia

What organism is a positive microbe for MR test and negative for VP?

Escherichia coli

What is the mechanism of action for hydrogen peroxide (oxidizing agent)

Have a strong affinity for electrons. Oxidizing agents will act to pull electrons off cellular material such as proteins and membranes which will result in lysing the cell.

Which disinfectant was least effective?

Hydrogen peroxide

What do Gamma (y) hemolytic bacteria cause?

If an organism does not induce hemolysis, the agar under and around the colony is unchanged, and the organism is called non-hemolytic

Name the 5 different classes of antibodies? (GAMED)

IgG IgA IgM IgE Ig D (Ig = antibody)

How are antigens tested for and describe the results. (Lab 19-ELISA)

In an ELISA it can be determined whether an antigen binds to an antibody through the use of a reporting system. This system is an enzyme that is covantently linked to the antibody and the enzyme has the ability to produce a colored product. When an antibody binds to an antigen you can determine the interactions by looking for a color change.

In Lab 13, Virus Plaque Assay, what was the purpose of E.coli?

It was the control

Name 2 pathways of virus replication

Lytic lysogenic (latent)

What is a test that detects an organism's ability to perform a mixed acid fermentation which overcomes the phosphate buffer in the medium, lower the pH, and produce stable acid?

Methyl Red test

Results of Lytic testing

Phage causes lysis and death of host cell

What is PFU

Plaque Forming Units

What are the limitations of UV light as a disinfectant?

Poor penetration power

What is the purpose of Methyl Red Vosges-Proskauer

Purpose: distinguishes homolactic from heterolactic acid-producing fermentation and identifies those glucose fermenters which convert their fermentation products to acetoin and 2, 3-butanediol. An organism will not be both MR and VP positive.

What is the purpose, selective ingredient, differential ingredient and the interpretation of indole (SIM medium)

Purpose: split amino acid tryptophan to form the compound indole. Selective ingredient: tryptophan Differential ingredient: Kovac's reagent Interpretation: If indole has been produced, the addition of Kovac's reagent will produce a bright red cherry layer floating on top of the culture.

what color is a positive result of the methyl red test?

Red

Name 2 bacteria that are positive for H2S production

Salmonella Proteus

What three things are tested with the SIM medium?

Sulfar reduction Indole production Motility

What variable was manipulated in Lab 13, using UV light to control bacterial growth?

TIme of exposure

What are some applications for ELISA?

The detection of infectous diseases and antibodies for such diseases as Rocky Mt. spotted fever, syphillis, rotovirus, varcilla-zoster, HIV and Lyme disease.

Why use soft agar while performing virus plaque assays?

The semi-solid medium restricts virus spread by diffusion and ensures that newly released virus can only spread from cell to cell causing a plaque to be formed.

Describe the results of the Eosin Methylene Blue Agar (EMB) test

The stains, eosin and methylene blue, act to inhibit gram positive bacteria anf they are also the differential ingredient.. Lactose fermenters metabolize the lactose in the media and produce acid byproducts, causing a color change in the colony. Thus, EMB is also a differential medium.-

What was diluted in Lab 13, Virus Plaque Assay?

The virus

What are the 2 parameters that were controlled in Lab 15, Control of Microbes by Chemical Agents:Disinfectants

Time Concentration

an addition of these substances into the medium oxidizes acetoin to diacetyl, which then reacts with guanidine nuclei from peptone to create a red color, the positive result

VP reagents

How do antigens and antibodies interact?

When an antigen enters the body, it stimulates the immune system to produce antibodies. ... The antibodies attach, or bind, themselves to the antigen and inactivate it.Each one is highly specialized to recognize just one kind of foreign substance. Antibody molecules are typically Y-shaped, with a binding site on each arm of the Y. The binding sites of each antibody, in turn, have a specific shape. Only antigens that match this shape will fit into them.

Define Antibody

Y shaped proteins that are produced by our immune system and used to mark antigens for destruction

What does a plaque represent?

a cell that has been infected with a single replicating virus

What is Albert's stain used for?

a technique is a type of special staining technique since it is used to demonstrate a special structure in bacteria. It is chiefly used to demonstrate metachromatic granules found in Corynebacterium diphtheriae.

What is the mechanism of action for amphyl (phenols)

acts to denature proteins and disolve lipids

Indirect ELISA test detects

antibodies (in patients serum)

Direct ELISA test detects

antigens (in patients sample)

What does PFU signify?

areas of cell lysis (CPE) in monolayer cell culture, under overlay conditions, initiated by infection with a single virus particle.

How did we evalute disinfectant effectivenss in Lab 15,Control of Microbes by Chemical Agents:Disinfectants

assesment of growth on a scale of 1-3

Describe the results of the lytic cycle

clear zones on media because the virus had ruptured the host cells, killing them.

What do Alpha (A) hemolytic bacteria cause?

colonies that oxidize the iron in the hemoglobin (in red blood cells) producing a greenish color

What do Beta (B) hemolytic bacteria cause?

complete lysis of red blood cells, hence they form clear zone around their colonies.

What is the color of a negative result of the Voges proskauer test?

copper/no color change

Define antigen

foriegn particles recognized by our immune system antibodies

Name a genus of bacteria that is motile. Name a genus of bacteria that is non-motile

gram positive cocci, Enterococci. E. gallinarum and E. casseliflavus/E. flavescens generally are motile. Enterococcus faecium and E. faecalis are non-motile

What is the mechanism of action for ethanol (alcohol)

kills bacteria mainly through 2 mechanisms: protein denaturation and dissolving the lipid membrane.

which pH indicator verifies mixed acid fermentation; it is red at 4.4, yellow at 6.2, and orange in between?

methyl red pH

what is a combination broth that tests an organism's capability to follow specific fermentation pathways?

methyl red-voges proskauer broth

What defense mechanisms were observed in the organisms tested in Lab 13, using UV light to control bacteria growth

pigment formation

Describe the results of the SIM medium Indole production test

produced when the amino acid tryptophan is deaminated by the enzyme tryptophanase. If produced, the addition of the Kovac's reagent will produce a red cherry top floating on top of the medium.

Describe the results of a SIM medium Sulfar reduction test.

reduction results from the anaerobic donation of electrons to sodium thiosulfate present in the media. The result is the production of hydrogen sulfide gas (H2S). This gas reacts with an iron containing compound in the media to produce ferric sulfide. This is visible as a black precipitate in the media.

Why don't the antigen-antibody complexes wash out of the wells?

the antibody in the well sticks to the bottom of the well. When the antigen is added to the well, the antigen sticks to the antibody holding it in the well.

What is the mechanism of UV light

thyamine dimmers damage DNA.

What is a test that detects if an organism can ferment glucose and then quickly convert their acid products to acetoin and 2,3-butanediol?

voges proskauer test

What color is a negative result of the methyl red test?

yellow/orange