BY 206 Lab Tope Week 9 (EX: 4-2, 5-6, 5-8, 7-3)

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COLUMBIA CNA w/ 5% SHEEP BLOOD AGAR

staphylococci, streptococci, enterococci

COLUMBIA CNA

Columbia CNA agar is used to isolate ___, ___, and ___ (primarily from clinical specimens).

hemolysis

COLUMBIA CNA

The inclusion of 5% sheep blood allows differentiation based on a _____ reaction.

undefined, differential, and selective

COLUMBIA CNA

What type of medium is Columbia CNA?

Gram-positive

COLUMBIA CNA

Does this medium allow the growth of Gram-positive or Gram-negative organisms?

yeast extract

COLUMBIA CNA

What ingredient in this medium provides B-vitamins?

colistin and nalidixic acid (CNA)

COLUMBIA CNA

What are the antibiotics that act as selective agents against G- organisms by affecting membrane integrity and interfering with DNA replication?

Klebsiella, Proteus, and Pseudomonas

COLUMBIA CNA

Colistin and Nalidixic acid (CNA) are particularly effective against what species of bacteria (3)?

α-hemolysis

COLUMBIA CNA

Which hemolysis reaction partially hemolyzes RBCs?

α (alpha), β (beta), or γ (gamma)?

β-hemolysis

COLUMBIA CNA

Which hemolysis reaction completely hemolyzes RBCs?

α (alpha), β (beta), or γ (gamma)?

γ-hemolysis

COLUMBIA CNA

Which hemolysis reaction does NOT hemolyze RBCs?

α (alpha), β (beta), or γ (gamma)?

-organism is inhibited by CNA

-probable G- organism

COLUMBIA CNA

Poor/NO growth

-NOT inhibited by CNA and partially hemolyzes RBCs

-probable α-hemolytic Staphylococcus, Streptococcus, or Enterococcus

COLUMBIA CNA

Good growth with greening of medium (α-hemolysis)

-NOT inhibited by CNA and completely hemolyzes RBCs

-probable β-hemolytic Staphylococcus, Streptococcus, or rarely Enterococcus

COLUMBIA CNA

Good growth with clearing of medium (β-hemolysis)

-NOT inhibited by CNA and does NOT hemolyze RBCs

-probable γ-hemolytic Staphylococcus, Streptococcus, or rarely Enterococcus

COLUMBIA CNA

Good growth with no change of color (γ-hemolysis)

positive

*because the bacteria is expected to grow

COLUMBIA CNA

Was the control in this test positive or negative?

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CATALASE TEST

Micrococcaceae, Streptococcaceae

CATALASE TEST

This test is used to identify organisms that produce the enzyme catalase.

It is used to differentiate members of the catalase-positive ____ from the catalase-negative _____.

flavoprotein

*FADH₂ is also capable of this reaction

CATALASE TEST

an ETC carrier molecule that can bypass the next carrier in the chain and transfer electrons directly to oxygen, producing hydrogen peroxide (H₂O₂)

Organisms that produce hydrogen peroxide and the superoxide radical are toxic because they oxidize biochemicals and make them non-functional. But, organisms that produce these can also produce enzymes to break them down.

CATALASE TEST

note:)

superoxide dismutase

CATALASE TEST

What enzyme catalyzes the conversion of superoxide radical to hydrogen peroxide?

catalase

CATALASE TEST

What enzyme converts hydrogen peroxide into water and gaseous oxygen?

positive

CATALASE TEST

If bubbling occurs after adding hydrogen peroxide, is the organism positive or negative for catalase?

negative

CATALASE TEST

If bubbling does NOT occur after adding hydrogen peroxide, is the organism positive or negative for catalase?

Obligate aerobe

superoxide dismutase: present

catalase: present

CATALASE TEST

Obligate aerobe

superoxide dismutase: _____

catalase: _____

Facultative anaerobe

superoxide dismutase: present

catalase: present

CATALASE TEST

Facultative anaerobe

superoxide dismutase: _____

catalase: _____

Microaerophile

superoxide dismutase: present

catalase: present in small amounts

CATALASE TEST

Microaerophile

superoxide dismutase: _____

catalase: _____

Aerotolerant anaerobe

superoxide dismutase: present

catalase: absent (alternative mechanism)

CATALASE TEST

Aerotolerant anaerobe

superoxide dismutase: _____

catalase: _____

Obligate anaerobe

superoxide dismutase: absent

catalase: absent

CATALASE TEST

Obligate anaerobe

superoxide dismutase: _____

catalase: _____

ur doing great <3

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NITRATE REDUCTION TEST

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NITRATE REDUCTION TEST

What type of medium is nitrate broth?

anaerobic respiration

*the reduction of an inorganic molecule other than oxygen

NITRATE REDUCTION TEST

Nitrate reduction is an example of . . .

nitrate reductase

NITRATE REDUCTION TEST

Many G- bacteria contain the enzyme ____ ____ and perform a single step reduction of nitrate to nitrite.

Enterobacteriaceae

NITRATE REDUCTION TEST

Virtually all members of _____ perform one-step reduction of nitrate to nitrite.

denitrification

*formation of gas/bubbles

NITRATE REDUCTION TEST

Other bacteria perform ____, a multi-step process by which bacteria convert nitrate into molecular nitrogen.

Durham tube

*if gas is produced, then denitrification has occurred and the test is complete/positive

NITRATE REDUCTION TEST

What is placed inside the broth to trap a portion of any gas produced?

sulfanilic acid, α-naphthylamine

NITRATE REDUCTION TEST

If there is no visual evidence of denitrification, ____ ____ (nitrate reagent A) and ______ (nitrate reagent B) are added to test for nitrate reduction to nitrite.

nitrous acid (HNO₂)

*it reacts with the reagents to produce a red color (positive test)

NITRATE REDUCTION TEST

If nitrite is present after adding the reagents, ____ ____ will form in the medium.

zinc dust

NITRATE REDUCTION TEST

If no color change takes place after the addition of the reagents, another test must be performed. What is added to the broth to catalyze the reduction of any nitrate to nitrite?

negative

NITRATE REDUCTION TEST

If the medium turns red after adding zinc, then nitrate is still present. Would this be a negative or positive result?

positive

NITRATE REDUCTION TEST

If the medium doesn't change color after adding zinc, then the organism has reduced nitrate to nitrite. Would this be a negative or positive result?

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ANTIMICROBIAL SUSCEPTIBILITY TEST: DISK DIFFUSION (KIRBY-BAUER) METHOD

The disk diffusion test is a standardized method used to measure the effectiveness of antibiotics and other chemotherapeutic agents on pathogenic microorganisms.

DISK DIFFUSION (KIRBY-BAUER) METHOD

application:)

antibiotics

DISK DIFFUSION (KIRBY-BAUER) METHOD

natural antimicrobial agents produced by microorganisms

antimicrobials, antimicrobics

DISK DIFFUSION (KIRBY-BAUER) METHOD

Because many agents that are used to treat bacterial infections are synthetic, the terms ____ or ____ are used to describe all substances used for this purpose

zone of inhibition

*does not mean that the bacteria have been killed

DISK DIFFUSION (KIRBY-BAUER) METHOD

Antimicrobic-impregnated paper disks are placed on the agar plate inoculated to form a bacterial lawn. If the organism is susceptible to the drug, a clear zone, _______, will appear around the disk where the concentration is high enough to stop growth.

minimum inhibitory concentration (MIC)

DISK DIFFUSION (KIRBY-BAUER) METHOD

At the junction of the zone of inhibition, the concentration of the antimicrobic has become too low to effectively stop growth. What represents this junction?

bactericidal

DISK DIFFUSION (KIRBY-BAUER) METHOD

Drugs that kill the organism are said to be . . .

bacteriostatic

DISK DIFFUSION (KIRBY-BAUER) METHOD

Drugs that stop bacteria from dividing, but do not kill them are said to be . . .

McFarland turbidity standard

DISK DIFFUSION (KIRBY-BAUER) METHOD

corresponds to a cell density between 1 and 2 x 10⁸ CFU/mL

resistance, susceptibility

DISK DIFFUSION (KIRBY-BAUER) METHOD

The zone diameter below which all resistant strains fall is the _____ breakpoint. The zone diameter above which all susceptible strains fall is the ____ breakpoint.

-altered target such that the antibiotic can no longer interact with the cellular process

-an alteration in how the drug is taken into the cell

-enzymatic destruction of the drug

-development or increased activity of an efflux mechanism

DISK DIFFUSION (KIRBY-BAUER) METHOD

Not all antibiotics affect cells in the same way. Resistance mechanisms can be broken down into four main categories:

Chloramphenicol

-prevents peptide bond formation during translation

-poor uptake and inactivation of drug

DISK DIFFUSION (KIRBY-BAUER) METHOD

Chloramphenicol

-cellular target?

-resistance mechanism?

Ciprofloxacin

-interferes with DNA replication

-altered target, poor uptake of drug

DISK DIFFUSION (KIRBY-BAUER) METHOD

Ciprofloxacin

-cellular target?

-resistance mechanism?

Penicillin

-inhibits cross-linking of the cell wall's peptidoglycan

-altered target, poor uptake of drug, or production of β-lactamases

DISK DIFFUSION (KIRBY-BAUER) METHOD

Penicillin

-cellular target?

-resistance mechanism?

Streptomycin

-blocks initiation complex formation in protein synthesis

-altered target

DISK DIFFUSION (KIRBY-BAUER) METHOD

Streptomycin

-cellular target?

-resistance mechanism?

Tetracycline

-blocks attachment of aminoacyl tRNA to A site on ribosome

-efflux mechanism

DISK DIFFUSION (KIRBY-BAUER) METHOD

Tetracycline

-cellular target?

-resistance mechanism?

Trimethoprim

-inhibits purine and pyrimidine synthesis

-altered target

DISK DIFFUSION (KIRBY-BAUER) METHOD

Trimethoprim

-cellular target?

-resistance mechanism?