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MICB 3301 Lab > Quiz 4: Ex. 8B, 9, 10, 11A > Flashcards

Quiz 4: Ex. 8B, 9, 10, 11A Flashcards

1
Q

Biofilms and example

A

multicellular, surface-attached communities

Pseudomonas aeruginosa form biofilms in lungs of patients with cystic fibrosis

Streptococcus and Enterococcus can contribute to endocarditis by growing biofilms on damaged heart valves

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2
Q

3 stages of biofilm production

A

Attachment: planktonic cells use pili or fimbriae to form adherent surface-attached cells

Colonization: form matrix to trap nutrients and protect, communicate w/ quorum sensing, only occurs when critical density is reached

Development: mushroom, nutrient flows thru pores, communicate w/ quorum sensing

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3
Q

What were the plates incubated at in biofilm experiment?

A

plate 1 at 37 deg C

plate 2 at 30 deg C

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4
Q

What are the 4 major components that a good culture medium should provide?

A

carbon source, nitrogen source, energy source, stable pH

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5
Q

Defined/synthetic medium

A

components are known, useful when studying metabolism

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6
Q

Complex/undefined medium

A

contains unknown ingredients, useful if rich nutritional background is required or if microbes needs are unknown

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7
Q

General purpose medium

A

allows growth for many microbes

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8
Q

Enrichment medium

A

selects the microbe of the highest growth rate among all the microbial population to be able to grow

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9
Q

Selective medium

A

favors the growth of a particular organism

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10
Q

Differential medium

A

Distinguishes between different groups of bacteria

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11
Q

Agar

A

comes from red algae, solidifying agent complex carbohydrate, melting point at 100 deg C, solidifies at 44 deg C

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12
Q

Sterilization

A

Complete removal or destruction of all living cells, viable spores, and viruses. Methods include heat, radiation, or filtration

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13
Q

Autoclave

A

moist heat to sterilize– uses saturated steam under high pressure at 121 deg C and pressure of 15 psi for 15 mins

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14
Q

What components were added to the GTYE agar and in what amounts

A 
Glucose 10 g/L
Tryptone 5 g/L
Yeast extract 3 g/L
Beef extract 3 g/L
K2HPO4 6 mM
KH2PO4 1 mM
Agar 15 g/L
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15
Q

What pH should GYTE agar be

A

7.5

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16
Q

For unknowns lab, what tests were performed on the gram negative bacteria?

A

TSI slant, tryptone broth, PAP, PAF, TTC deep, DNase, GTYE

17
Q

For unknowns lab, what tests were performed on gram positive bacteria?

A

TTC deep, DNase, MSA, bile esculin, BAP, GTYE

18
Q

For the unknowns lab, which were used as the 3 initial tests and why?

A

TSA: cultures
MAC: only gram negative
CNA: only gram positive

19
Q

What temperature were the unknown tests incubated at

A

30 for TSA, MAC, CNA, PAF

37 for all else

20
Q

Immunity

A

ability of a host to resist infection/disease

21
Q

Innate immunity

A

Nonspecific/natural

First line of defense

Examples: skin, complement system, cytokines, macrophages

22
Q

Adaptive immunity

A

Specific acquired immunity

in response to antigen

antibodies/immunoglobulins

Memory, B and T cells

23
Q

B lymphocytes

A

produced and mature in bone marrow

differentiate into plasma (secrete antibodies) or memory cells

24
Q

Opsonization

A

Antibodies coating pathogen to make them more easily recognized and engulfed by phagocytosis

25
Q

Describe antibody structure

A

2 identical heavy (longer) chains
2 identical light (shorter) chains
Covalently bound by disulfide bonds
Antibody combining domains is the bent part of all 4 chains

26
Q

What happens when a B cell differentiates into a plasma cell

A

It secretes Ab and no longer expresses surface Ig

27
Q

T lymphocytes

A

from bone marrow, mature in the thymus gland

circulate the lymphatic system

Helper T cells: help B cells respond to antigen, produce cytokines

Cytotoxic T cells: kills cells infected with antigen

28
Q

How do B and T cells differ in antigen recognition?

A

B cells recognize in tact pathogens by antigen recognition

T h cells recognize bound peptide fragments (antigen processing) presented to it with MHC class II

29
Q

What is the difference between MHC class I and II?

A

Class I: presents intracellular antigens to other antigens for cytotoxic T cell killing

Class II: presents extracellular antigens to helper T cells

30
Q

T cell receptors

A

specific for a particular antigen peptide-MHC complex

Similar structure to surface Igs but only have one pair of chains (alpha, beta chains) and therefore only one combining site for antigen

31
Q

Cluster of Differentiation (CD) molecules

A

receptors/coreceptors for intercellular communication

Example: CD4 on helper T cells recognize HIV,
CD8 on cytotoxic T cells also recognize HIV

32
Q

ELISA (enzyme-linked immunosorbent assay)

A

Tests the presence of antibodies in serum

Antigen is stuck to 96 well plate
Washed with horse serum to prevent non-specific binding
Dilutions of ‘antiserum’ containing antibodies are added to wells– they bind to antigens stuck on bottom
Washed
Bound antibodies are detected by adding a secondary antibody with enzymatic activity
H2O2 is added to oxidize and cause color change

Used to screen for HIV to see if Abs envelope gp120 in human serum

33
Q

What enzymes are typically used on the secondary antibody to indicate antigen presence

A 
Alkaline phosphatase (AP)
Horseradish peroxidase (HRP)
34
Q

Cyanogen bromide (CNBr)

A

Cleaves at the carboxyl terminus ends of methionine

35
Q

Trypsin

A

Cleaves at the carboxyl terminus end of lysine and arginine

36
Q 
What is each of the following components in the ELISA experiment? 
OVA
CNBr-cleaved OVA
Trypsin-cleaved OVA
5% horse serum in PBS
Antiserum
Negative control serum
PBS
HRP-conjugated goat anti-mouse IgG
Indicator 
4N H2SO4
A

OVA: antigen

CNBr-cleaved OVA: fragmented antigen

Trypsin-cleaved OVA: even more fragmented antigen

5% horse serum in PBS: uses to block non-specific binding, helps antibody-antigen binding

Antiserum: contains the antibodies

Negative control serum: contains no antibodies: these wells should have no enzymatic activities

PBS: used to wash the plates

HRP-conjugated goat anti-mouse IgG: secondary antibodies with enzymatic activity

Indicator: substrate used to detect presence of antibodies– changed by enzyme

4N H2SO4: substrate used by enzyme to oxidize the indicator

37
Q

What was the indicator substrate used in the ELISA experiment?

A

H2O2 with o-phenylenediamine and citrate-phosphate buffer

38
Q

What absorbance was our ELISA plates read at?

A

490 nm

MICB 3301 Lab (2 decks)

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