MIC 304 Final

What does the Kirby-Bauer method test for?

antimicrobial susceptibility

Kirby-Bauer method also known as

standardized filter paper disk agar diffusion method

The sterile filter paper disks are impregnated with

a defined concentration of an antimicrobial agent

Before the sterile filter paper disks are added to the agar plate, it is previously what

inoculated with a standard amount of the organism to be tested

The plates are inverted and incubated after the sides of the disks do what

come into contact with the multiplying organisms

Zones of inhibition are

clear zones around the filter paper disks

What does a zone of inhibition mean?

The bacteria in the plate is susceptible and not resistant to the antimicrobial agent in the disk

What does no inhibition look like?

Growth of the bacteria extends up to the rim of the disk on all sides

What does no inhibition zone mean?

The bacteria in the plate is resistant to the antimicrobial agent in the disk

Size of the zone of inhibition depends on what?

rate of diffusion of the given drug in the medium, degree of susceptibility of the organism to the drug, number of organisms inoculated on the plate, and the organism's rate of growth

Bacteriostatic properties

inhibition of the growth of bacteria without destruction

The filter paper disk method is used when comparing

antiseptics on the basis of their bacteriostatic properties

What plate is used for the filter paper disk method

a seeded nutrient agar plate (a plate that has been inoculated using the spread plate procedure)

The size of the zone of inhibition is an expression for

the agent's effectiveness

Before applying the disks to the plate, the agar surface should be

dry

Sterile forceps are used to press each disk lightly in order to

secure it to the medium

How are the zones of inhibition measured (3)?

diameter, millimeters, bottom of the plate

The zone of inhibition is determined without

magnification

What are some antibiotics used in the experiment?

neomycin, penicillin, streptomycin

Sterile disk is dipped how far into the agent?

Halfway (not completely or it will be too wet)

For the unknown experiment, the tube has two organisms suspended in what

nutrient broth

What type of research project is the unknown experiment?

quasi-research project

Plates should always be stored in what way?

Bottom side up

The purpose of the exercise is to run as _____ tests as possible

few

Traditionally, students begin with the _______ complex tests

least

Most gram positive organisms are glucose ________

positive (acidic)

Most gram negative organisms are glucose ___________

positive (acid-gas)

Which organism is the only variable result for the sucrose test?

E. coli

For the unknowns, the only starch positive organism is

B. cereus

Most organisms are gelatin _________

positive

Most organisms are indole ________

negative

The only H2S positive unknown organism is

P. vulgaris

Most organisms are catalase __________

positive

Antibodies are also called

immunoglobulins

What are antibodies?

human and animal proteins produced by white blood cells in response to antigens (foreign materials)

What isotypes can antibodies take on?

IgG, IgA, IgM, IgE, IgD

What are antigens?

infectious agents and non-self materials

What do antibodies bind to and why?

They bind to antigens to remove them from the body

The binding of antigen to antibody is due to what?

The antibodies ability to to recognize specific chemical charges, sequences, or structural conformational elements of the antigen

Antibodies are composed of how many chains?

2 (heavy and light)

Which region on the chain does the antigen bind? (constant or variable)

variable

Each immunoglobin can bind to two antigens, but the antigens must be what?

identical

Each immunoglobin is specific to how many antigens?

one

What is agglutination or immunoprecipitation?

binding of antigen to antibody

Antigens have relatively ______ surface areas

large

What does ELISA stand for?

Enzyme Linked Immunosorbent Assay

What does ELISA test for?

the presence of IgG molecules directed toward a specific antigen

ELISA utilizes two types of antibodies which are what

primary and secondary antibodies

Primary antibody

specific for the antigen in question

Secondary antibody

raised in rabbits and and goats immunized with "non-self" IgG fractions

Secondary antibodies are purified and cross linked to which enzyme and bind to what?

horseradish peroxidase; bind to the primary antibody

What substrate is added following the addition of the secondary antibody?

hydrogen peroxide and azino-di-ethylbenz-thiazoline sulfonate (ABTS)

ABTS is oxidized by the horseradish peroxidase to produce what color in the wells?

green

The microtiter plate which has the antibodies and antigens should be placed in what direction?

vertically

What are the experimental steps for the ELISA?

Antigen, Primary Antibody, Secondary Antibody, Substrate

The ELISA incubation period is ___ minutes

5

Salicylate is oxidized to produce what color

brown

Which two biologists discovered specific agglutination in 1896?

Herbert Edward Durham and Max von Gruber

French physician Fernand Widal used the agglutination reaction as the basis for a test for

typhoid fever

Blood serum from a typhoid-free person did NOT agglutinate which bacteria?

Salmonella Typhi

Austrian physician Karl Landsteiner was able to

categorize human blood into four types

When particles are red blood cells, clumping is called

hemagglutination

The slide agglutination procedure is also called the

flocculation test

What is the flocculation test?

a procedure where a loop full of a heavy bacterial suspension is mixed with antiserum

Instead of serum for the control, what is used?

saline

How many blood group systems are known to exist in the human species?

33

Which first three major blood factors were discovered by Karl Landsteiner in 1900?

A,B,O

Blood group A has antigen __ and the antibody against __

A; B

Blood group B has antigen __ and the antibody against _

B; A

Blood group AB has antigens __, __ and the antibody against _

A, B; none

Blood group O has antigen __ and the antibody against __, _

O; A, B

Chemically, ABO antigenic factors are composed of what

polysaccharide amino acid complexes

A and B antigens are found in

red blood cells and other tissues like sperm and the liver

Blood group substances AB are also found in

body fluids like saliva, gastric juice, perspiration, and seminal fluid

Individuals producing blood-group substances like AB are referred to as _____________, which is a ___________ controlled property

secretors; genetically

AB individuals are universal _____________ for red blood cells and universal __________ for serum

recipients; donors

One determines the blood type of an individual by

observing agglutination reactions that incorporate sera from A and B individuals

The sera are mixed separately with unknown blood-cell specimens either on a _____ ______ or in a ____ ___

glass slide; test tube

The Rh factor was designated for

rhesus

What reaction was recognized as the principal cause of erythroblastosis fetalis?

the antibody response of sensitized Rh-negative individuals to the administration of Rh-positive antigens

Do Rh antibodies occur naturally in humans?

No

How can Rh antibodies develop?

transfusing Rh-negative individuals with Rh-positive blood or Rh-negative women bearing a Rh-positive fetus

What is bacterial transformation?

the process in which bacteria take up free DNA from the environment

The number of different bacterial strains from bacterial transformation produce the ability to grow specifically on

selective media

The plasmid pGal makes the colonies transformed appear what color?

blue

pGal imparts transformed colonies with

amp resistance

Competency refers to

the state in which bacteria can be transformed

Competency can be induced by treating bacteria with what?

chloride salts and temperature change

Treatments affect the permeability of what?

the cell wall and membrane so that DNA molecules can pass through

Supercoiled plasmid DNA generates ________ transformation efficiencies than relaxed plasmid DNA

higher

If E. coli is successfully transformed, it will be able to

grow on media containing the antibiotic ampicillin

To suspend cells in the solution for a bacterial transformation, what should you do?

vortex them until no clumps are visible

PCR stands for

polymerase chain reaction

PCR was conceived by

Kary B. Mullis

PCR is used to make

copies of scarce samples of DNA

The PCR process makes use of a heat stable enzyme isolated from what organism?

a thermophile: Thermus aquaticus

What enzyme is a major component in the PCR?

Taq polymerase

Taq and other enzymes like it allows researchers to run multiple cycles using

the same enzyme

The three phases of PCR

denature, annealing, and polymerization (elongation)

Each PCR phase is conducted how?

for a set amount of time at a specific temperature