BIOCHEMICAL TESTS LAB QUIZ

Question 1

Motility - Test Medium with TTC (MTM-TTC)

Answer 1

The medium contains agar 2,3,5 Triphenyl-Tetrazolium Chloride
Viable bacteria reduce TTC to form a red precipitate called formazan. The distribution of formazan indicates the location of bacteria in the tube.

Motile organisms will grow away from the stab. The medium will be red and turbid (cloudy).

Non-motile organisms will be grow only along the stab. The tube will be red along the stab. No cloudiness.

Question 2

Oxygen Requirements - Fluid Thioglycollate medium (FTM)

Answer 2

FTM contains glucose (energy) and cystine and sodium thioglycollate.

This creates an oxygen gradient. High at the top of the tube (for aerobes). Low at the bottom (for anaerobes).

A dye called Rezaurin turns the medium pink in hte presence of oxygen.

Obligate Aerobe - Growth at the top of the medium
Facultative Anaerobe - Growth throughout the medium
Obligate Anaerobe - Growth at the bottom of the medium

Question 3

Carbohydrate fermentation

Purpose: To detect the ability of an organism to ferment a specific carbohydrate with or without the production of gas.

Glucose
Lactose
Mannitol
Maltose
Sucrose

Answer 3

Phenol red is an indicator in each medium

If the microbes do not ferment the specific carbohydrate (negative) in the broth, the medium stays red

If fermentation occurs (positive), acid is produced which changes the indicator from red to yellow.

The inverted tube (called a Durham tube) absorbs some of the gas (if any). A bubble in the tube indicates the presence of gas).

Question 4

Gelatin Hydrolysis

The medium contains gelatin (semisolid). Gelatinase hydrolyzes gelatin, a large protein, into smaller molecules which can be metabolized by the organism.

Answer 4

Positive - Gelatin liquefies

Negative - Gelatin remains semisolid

Question 5

Catalase (TSA Slant)

Hydrogen peroxide is decomposed by the enzyme catalase to water and oxygen. Hydrogen Peroxide is toxic to organisms. This allows the organism to survive.

Hydrogen peroxide is a product of aerobic respiration of sugars.

Answer 5

Positive (Bubbling)

Nagative (No Bubbling)

Question 6

UV Range Lethal to bacteria

Answer 6

260 nM

Question 7

Lethal effects of UV light

Answer 7

Creating thymine dimers (adjacent thymines bond covalently) also frameshift mutation.

(Frameshift mutations are deletions or additions of 1, 2, or 4 nucleotides that change the ribosome reading frame and cause premature termination of translation at a new nonsense or chain termination codon)

Question 8

Why are lids removed before exposure in the UV light experiment?

Answer 8

UV light is not very penetrating, and will not penetrate petri dish lids.

Question 9

Why are exposure times different between the two lab microbes used in the UV experiment?

Answer 9

S. Aureus does not form endospores, so no more than 10 minutes of exposure was required to kill all of the bacteria in the dish.

Bacillus megaterium is an endospore foming bacteria. It required about an hour to kill all of the bacteria and the endospores.

Question 10

How can bacteria repair damage from UV light?

Answer 10

Light repair. An enzyme called photolyase uses light energy to break the bonds of pyrimidine to pyrimidine dimers.

Question 11

What is a sign that damage from UV light has been repaired in bacteria?

Answer 11

Regrowth on cleared areas on the petri dish.

Question 12

Which agar is used for a Kirby-Bauer test?

Answer 12

MHA Mueller-Hinton Agar

It is a non-selective, non-differential medium. This means that almost all organisms plated on here will grow. Additionally, it contains starch.

Question 13

What are NCCLS standards?

Answer 13

National Committee for Clinical Laboratory Standards

Question 14

Which tool should be used to do lawning on an agar plate?

Answer 14

A cotton swab. DO NOT use a loop.

Think of a big cotton swab over a grass lawn.

Question 15

How are zones of inhibition measured?

Answer 15

Using a ruler, in millimeters. This is compared to a table.

Question 16

What are the differences between antibiotics and antimicrobials.

Answer 16

Antibiotics are produced from living sources (eg: mold).

Antimicrobials are artificially made.

Question 17

Difference between bacteriostatic and bacteriocidal

Answer 17

Bacteriostatic drugs inhibit the growth of bacteria, but do not kill the bacteria. Bacteriocidal drugs kill the bacteria.

Question 18

What is PCR?

Answer 18

Polymerase chain reaction

polymerase chain reaction (PCR) is a fast and inexpensive technique used to “amplify” - copy - small segments of DNA.

Question 19

How does PCR work?

Answer 19

three major steps are involved in a PCR. These three steps are repeated for 30 or 40 cycles. The cycles are done on an automated cycler, a device which rapidly heats and cools the test tubes containing the reaction mixture. Each step – denatauration (alteration of structure), annealing (joining), and extension – takes place at a different temperature:

Denaturation: At 94 C (201.2 F), the double-stranded DNA melts and opens into two pieces of single-stranded DNA.

Annealing: At medium temperatures, around 54 C (129.2 F), the primers pair up (anneal) with the single-stranded "template" (The template is the sequence of DNA to be copied.) On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.

Extension: At 72 C (161.6 F), the polymerase works best, and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.

With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.

Question 20

Advantages of PCR over conventional methods

Answer 20

Fast and simple. However it can be an expensive test.

Question 21

What it Taq Polymerase?

Answer 21

A DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates. It is used because it is heat tolerant.

Question 22

What is the purpose of the primer in PCR?

Answer 22

Taq polymerase requires a starting point. A short sequence of nucleotides that provides a starting point for DNA synthesis. The primer determines which DNA will be synthesized.Two primers are used in each PCR reaction, and they are designed so that they flank the target region (region that should be copied). That is, they are given sequences that will make them bind to opposite strands of the template DNA, just at the edges of the region to be copied.

Question 23

What is the mecA gene, its product, and its occurence in MRSA?

Answer 23

mecA gene, which encodes an alternative penicillin-binding protein, PBP 2. It occurs in S. Aureus, which makes Methicillin-resistant Staphylococcus aureus.

Question 24

What are clinically important staphylococci?

Answer 24

S. Aureus - Found in the nose. Various infections (skin, bone, food poisoning).
S. Epidermis - Found on skin. Catheter infections.
S. Saphrophyticus - Urinary Tract Infections

Question 25

Principle in catalase test, Reagents used, applications

Answer 25

All of the clinically important staphylococci will test catalase positive, unlike other cocci

reagent 3% Hydrogen Peroxide

bubbling or effervescence indicates a positive result

Question 26

MSA on staphylococcus

Answer 26

Test for mannitol fermentation

S. Aureus - Yellow (fermentation)
S. Epidermis - Pink
S. Saphrophyticus - Yellow

Question 27

Hemolytic reactions on blood agar for staphylococci

Answer 27

S. Aureus - B Hemolytic (full clearing)
S. Epidermis- Gamma (No clearing)
S. Saphrophyticus - Gamma (No clearing)

Question 28

Hemolytic Reactions - Streptococci

Answer 28

S. Pneumoniae - Alpha (partial, greenish)
S. Fecalis - Gamma (no clearing)
S. Pyogenes Beta (full clearing)