Microbiology Lab Final

Front 1

What is a plaque?
What occurred to result in the formation of the plaque?

Back 1

A plaque is an area of clearing on a lawn of host cells that is formed as a result of infection of one cell by a single viral particle.

Front 2

How do you calculate the titer of viruses?

Back 2

PFU/mL = (# of plaques on plate x dilution factor)/amount of phage plated in mL

Front 3

What is the purpose of the starch hydrolysis (amylase) test?
How are the results read?

Back 3

This test looks for the production of amylase: amylose gets broken down into maltose, which is broken down into glucose subunits by maltase.

A positive result appears as clearing following the addition of iodine to plate.

Front 4

What is the purpose of the urease test and how are the results read?

Back 4

The urease test is looking for production of urease. Urea is broken down into 2 ammonia molecules + CO2 which increases pH and shows a positive result.

A positive result shows a color change from light peach to bright pink.

Front 5

I mannitol salt agar selective or differential medium? Why?

How do you determine the results?

Back 5

Mannitol salt agar is selective and differential.

It is selective because it contains 7.5% NaCl that inhibits the growth of most organisms except some species of Staphylococcus and Micrococcus. Lack of growth means that the organism cannot tolerate NaCl.

It is differential because it contains mannitol and phenol red (pH indicator). The fermenting of manniitol results in an acid environment which will generate a yellow halo around the bacteria. Non-fermentors don't cause a change in pH and so there is no yellow appearance.

Front 6

What is the Kirby-Bauer assay and what is its purpose?
How do you read the results?

Back 6

The Kirby-Bauer method determines the sensitivity of a microbe to a specific antimicrobial agent.

To read these results, the diameter of each zone is measured in mm. Next you will look at the chart provided and determine if the bacterium is susceptible, intermediate susceptibility, or resistant. The disc symbol can be read on each disk.

Front 7

How do you know if a bacterium is sensitive or resistant to an antimicrobial substance?

Back 7

If the bacterium is sensitive to an antimicrobial substance, the substance will kill some, all , or most of the bacterium. If the bacterium is resistant it will grown regardless of the presence of the antimicrobial.

Front 8

What is the difference between a narrow-spectrum antibiotic and a broad-spectrum antibiotic?

Back 8

Narrow-spectrum antibiotic - an antibiotic that is effective only against either gram positive or gram negative organisms.

Broad-Spectrum antibiotic - antibiotics that are effective against both gram-positive and gram negative bacteria.

Front 9

What are the steps of the biochemical pathway of the catabolization of Cysteine Desulfhydrase?

Back 9

The first step in the biochemical pathway used to catabolize cysteine is the removal of part of the R-group. The enzyme that catalyzes this reaction is called cysteine desulfhydrase and it removes the -SH portion of the R-group from cysteine converting it into another amino acid, alanine, releasing hydrogen sulfide (H2S) gas. H2S is the foul smelling gas that gives the characteristic smell to rotten eggs and to sulfur hot springs.

Front 10

How does the cysteine test work? What color changes does it go through and why? How are the results read?

Back 10

Cysteine digestion is indirectly determined by detecting the production of H2S. H2S reacts with iron ions (Fe2+) in the medium to produce iron sulfide (FeS), which turns the media black.

The medium used in the cysteine digestion test starts out being the regular color of microbial medium, yellow to cream. After incubation with bacteria that are capable of cysteine digestion, the medium turns black.

No color change is recorded as negative result for cysteine digestion.

Front 11

What reaction ends the hydrolysis of cysteine?

Back 11

The complete hydrolysis of cysteine required another reaction, The next step in the catabolism of cysteine is the deamination (removal of the amino group) of alanine, a reaction performed by the enzyme alanine deaminase in which the amino group is removed from alanine, producing pyruvate and ammonia (NH3). Ammonia is a by-product of the reaction and pyruvate is further catabolized in the TCA cycle to produce ATP.

Front 12

Why is blood agar nutrient and differential media?

Back 12

Nutrient - contains nutrients that will allow most bacteria to grow.

Differential - contains blood and differentiates bacteria by their ability to lyse red blood cells.
Beta hemolytic - complete lysis of red blood cell resulting in a complete curing around the bacterial colony
Alpha hemolytic - partial lysis of red blood cell resulting in a green clearing around the bacterial colony
Gamma hemolytic - no lysis of red blood cell: no clearing around the colony.

Front 13

What produces DNase and why?

Back 13

Pathogenic Staphylococcal species and other pathogenic bacterial species produce DNase to aid in dissemination throughout the body.
Pathogenic species (S. aureus) produce a clearing on DNase plate, nonpathogenic (S. epidermidis) do not.

Front 14

Is the DNase test selective or differential? Why?

Back 14

It is differential because it differentiates the ability to break down DNA and differentiates between pathogenic and non-pathogenic staphylococcal species.

Front 15

How is the DNase test read?

Back 15

The clearing is only observed after 1N HCl has been added to the plate to precipitate the DNA.
Clearing of DNA around the bacteria is positive.
No clearing around the bacteria is negative.

Front 16

What does the Bacitracin sensitivity test test for? How are the results read?

Back 16

It utilizes the susceptibility to the antibiotic bacitracin, contained in the A disc, to test for the differentiation of B-hemolytic group A streptococci (such as Streptococcus pyogenes), which are bacitracin sensitive, from the non-group A, B-hemolytic streptococci, which are resistant

(B- stand for beta)

Sensitive bacteria have a clearing in bacterial growth around the A disc.
Resistant bacteria have growth all around the A disc.

Front 17

How does the decarboxylase (lysine) test work?

Back 17

In the case of lysine, decarboxylation leads to the production onf CO2 gas and the amine, cadaverine. This reaction only occurs in anaerobic conditions. to test for the decarboxylation of lysine, the organisms are grown in lysine decarboxylase broth, which contains the amino acid lysine, a fermentable sugar, and thepH indicator bromcersol purple.
In addition, in order for the enzyme to be active, anaerobic conditions need to be created: therefore, facultative anaerobes must be driven to use anaerobic fermentation. To do this after inoculating the broth, a few drops of mineral oil and overlayed on top of it in order to keep air out and to create an anaerobic environment.
The bottom line is this test looks for the ability to decarboxylate amino acids. The result in a pH increase causes a change in color of the medium to purple.

Front 18

How does the gelatinase test work?

Back 18

Pathogens that attack connective tissue often produce the extracellular enzyme, gelatinase, which hydrolyses gelatin into short chains of amino acids called peptides. The peptides are then non=specifically hdrolyzed by extracellular peptidases and the reesulting amino acids are transported across the cell membrane by specific amino acid permeases. The gelatinase liquefies the medium to give a positive result and if the medium is solid it is a negative result.

Front 19

What is an antibody?

Back 19

An antibody is a protein produced by the B-lymphocytes of the immune system that have many functions in the protection against pathogens. It has a high specificity for antigens (something foreign that activates an immune response)

Front 20

How can bacterial infections be rapidly diagnosed using antibody-antigen testing?

Back 20

Since antibodies are specific to one kind of antigen and are produced only after exposure to the antigen, antibody titer can also be used in disease diagnosis. (a titer is the amount of antibody in the serum sample).

Front 21

What are the steps of an ELISA and what is done during each step?

Back 21

Enzyme-Linked Immunoabsorbant Assay

1. Transfer purified antigen in to the wells and allow to bind to the well
-wells have been treated with the antigen HIV capsid protein p24
2. Wash unbound antigen out of the wells
3. Add positive control, negative control, and patient samples to their own wells and allow samples to bind to the antigen
-anti-p24 antibodies bind if present in the serum
4. Wash the wells using buffer
-Unbound antibodies that are not anti-p24 are washed out
5. Add secondary antibody and allow to bind to primary antibody
-These are enzyme-linked anti-human antibodies
6. Wash two times to avoid a false positive
-excess, unbound secondary antibodies are washed out
7. Add enzyme substrate to the wells. No color change is negative for HIV and a blue color is positive.
-Chromogenic substrate for the enzyme is added (chromogenic means that when the substrate is reacted on by the enzyme, the products are colored)
-Enzyme cleaves substrate and color develops indicating positive result.

Front 22

What is agglutination?

Back 22

The clumping of cells such as bacteria or red blood cells in the presence of an antibody. The antibody or other molecule binds multiple particles and joins them, creating a large complex. An example occurs when people are given blood transfusions of the wrong blood group.

Front 23

What is a primary immune response and a secondary immune response?

Back 23

Primary response- occurs upon the first exposure to an antigen. Body has to learn how to deal with the antigen and make antibodies for it.
-Includes a latent period during which little antibody is detected in the blood and the antigen is being processed and concentrated in lymphoid organs where the correct B-cell clone is activated and converted into an antibody secreting plasma cell.
-Includes an active period where the infectious agent is being neutralized and cleared most effectively

Secondary immune response- when the body has seen the same antigen more than once. The immunological memory has been established and the immune system can start making antibodies immediately. The antigen is often quickly killed and the person might be unaware that they were attacked.