Mbios 430 Final

Front 1

ISOLATION OF BACTERIOPHAGE FROM SEWAGE.
How were bacteriophage enriched?

Back 1

5 mL DSPB and 5 mL E. coli were added to the flask containing sewage; the flask was placed in a 37C shaking water bath overnight.

Front 2

ISOLATION OF BACTERIOPHAGE FROM SEWAGE.
Why was sample filtered?

Back 2

It was necessary to separate phage from bacteria. Otherwise, overgrowth of bacteria, fungi, and other microorganisms will mask the phage plaque formation. Phage sample is first centrifuged to remove coarse debris and bacterial contaminants. Then the supernatant is passed through a small pore size filter, which retained bacteria and fungi but allowed phage to pass through.

Front 3

ISOLATION OF BACTERIOPHAGE FROM SEWAGE.
How did you determine the host range specificity of your isolated bacteriophage?

Back 3

The host range specificity was determined by culturing phage samples on lawns of several different bacteria.

Front 4

ISOLATION OF BACTERIOPHAGE FROM SEWAGE.
Describe the plaque assay to titrate bacteriophage, including all reagents.

Back 4

1. Overnight nutrient broth cultures of E. coli strains B, C, and K12; Pseudomonas sp., and Salmonella sp.
2. filtered sewage
3. tubes of sterile soft (0.7%) nutrient agar
4. nutrient agar plates
*Serial 10 fold dilutions of the sewage filtrate were made (10-1 to 10-7) in tubes; 0.3mL of host bacterial strain and 0.1 mL of phage dilution were then mixed in soft agar and poured onto plate, rotate to spread.
*Bacterial strains were then added to plates (1 per)
*Controls with PBD and sp. bacterial strain were poured onto plates.

Front 5

ISOLATION OF BACTERIOPHAGE FROM SEWAGE.
How did you calculate the titer of your bacteriophage sample?

Back 5

Count plaques. Only plates yielding 30-300 plaques were considered. The number of plaques was multiplied by the dilution factor of that plate, then that number is divided by the amount of phage plated.

Front 6

ISOLATION OF BACTERIOPHAGE FROM SEWAGE.
What was the purpose of the soft top agar?

Back 6

After cooling, the semi-solid consistency of the soft agar facilitates the growth of a continuous lawn of bacteria and the development of viral plaques, which can be visualized and quantitated.

Front 7

ISOLATION OF BACTERIOPHAGE FROM SEWAGE.
How was contamination prevented in the phage stock?

Back 7

The phage stock was kept sterile by the addition of chloroform.

Front 8

ISOLATION OF BACTERIOPHAGE FROM SEWAGE.
Did your isolated bacteriophage have an envelope?

Back 8

No

Front 9

ISOLATION OF BACTERIOPHAGE FROM SEWAGE.
How did you know that your isolated bacteriophage was unenveloped?

Back 9

After treatment with chloroform, the phage was still able to infect bacteria.

Front 10

DEMONSTRATION OF LYSOGENY
What is a temperate virus?

Back 10

Temperate viruses is integrate their viral nucleic acid into bacterial genome; can persist like this for prolonged periods of time, Information encoded by the viral nucleic acid is partially expressed, as is cellular information. When the bacterium is stressed (heat, environment, impending doom), the virus busts out of the genome, lysing the cell.

Front 11

DEMONSTRATION OF LYSOGENY
What is a lysogen?

Back 11

A lysogen refers to a strain of bacterium that carries a prophage (temperate phage).

Front 12

DEMONSTRATION OF LYSOGENY
What is lysogeny?

Back 12

Lysogeny is when the viral nucleic acid (prophage) is integrated into the bacterial genome and can persist for prolonged periods of time.

Front 13

DEMONSTRATION OF LYSOGENY
How was the lysogen verified?

Back 13

Two sets of plates were divided into 12ths, with each square being streaked w/ a well-isolated colony. This was repeated with 11 other colonies. One set of plates was incubated at 30C and the other at 42C; lysogens won't grow at the higher temp, thus if there was no plaques on the lower temp plate and there were plaques on the 42, you done found yourself a lysogen.

Front 14

DEMONSTRATION OF LYSOGENY
What is unique about the viral genome during lysogeny?

Back 14

Its incorporated into the bacterial genome.

Front 15

IDENTIFY/DESCRIBE WRIGHT'S STAINED BLOOD CELLS
Know what cells are seen in different kinds of infections or disease states (bacterial, viral, parasites)

Back 15

Neutrophils-bacterial
Lymphocytes-viral
Eosinophils-parasite
Basophils-hypersensivity
Mast cell-parasite

Front 16

IDENTIFY/DESCRIBE WRIGHT'S STAINED BLOOD CELLS
In what other tissue were immune cells identified?

Back 16

Spleen, Liver, Skin, Blood

Front 17

IDENTIFY/DESCRIBE WRIGHT'S STAINED BLOOD CELLS
What is the difference between plasma and serum?

Back 17

Blood plasma is the yellow liquid component of blood, mostly water (90% by volume) and contains dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide Blood serum is blood plasma without fibrinogen or the other clotting factors

Front 18

IMMUNOCYTOCHEMICAL STAINING OF LYMPHOCYTES
What surface marker is involved?

Back 18

Mouse BCR-IgM

Front 19

IMMUNOCYTOCHEMICAL STAINING OF LYMPHOCYTES
What is the primary Ab?

Back 19

anti-mouse IgM-HRP conjugate

Front 20

IMMUNOCYTOCHEMICAL STAINING OF LYMPHOCYTES
What is the conjugate?

Back 20

HRP

Front 21

IMMUNOCYTOCHEMICAL STAINING OF LYMPHOCYTES
To what does the HRP conjugate bind?

Back 21

any mouse IgM present

Front 22

IMMUNOCYTOCHEMICAL STAINING OF LYMPHOCYTES
What is the substrate?

Back 22

DAB

Front 23

IMMUNOCYTOCHEMICAL STAINING OF LYMPHOCYTES
What do positive and negative cells look like?

Back 23

B cells have brown halo around the membrane with blue-violet nuclei.
T cells have blue-violet nuclei.
Neutrophils have blue-violet nucleus
RBCs are clear

Front 24

IMMUNOCYTOCHEMICAL STAINING OF LYMPHOCYTES
What are the controls and why are they important?

Back 24

Endogenous peroxidase control
Morphology control-Determine morphology of cells present w/ hematoxylin stain

Front 25

IMMUNOCYTOCHEMICAL STAINING OF LYMPHOCYTES
Why were cells quenched?

Back 25

Cells were quenched to satiate endogenous peroxidase in RBCs and neutrophils so that they didn't end up w/ a brown halo like the B lymphocytes

Front 26

IMMUNOCYTOCHEMICAL STAINING OF LYMPHOCYTES
What organs were identified in the mouse?

Back 26

spleen, lungs, heart, stomach, intestines, thymus

Front 27

AGGLUTINATION AND PRECIPITATION
What is the difference between the two?

Back 27

*Agglutination involves the clumping together of insoluble molecules.
*Precipitation involves the aggregation of soluble molecules.

Front 28

AGGLUTINATION AND PRECIPITATION
Double immunodiffusion; be able to design an AGID.

Back 28

FUCK!

Front 29

TISSUE CULTURE
Why is MEM with 2% serum used some time and 10% serum at other times?

Back 29

10% for growth
2% for infection
for mammalian cells (BT)

Front 30

TISSUE CULTURE
What cells did we use?

Back 30

Bovine Turbinate Cells

Front 31

TISSUE CULTURE
What viruses did we use?

Back 31

IBRV
Infectious Bovine Rhinotracheitis Virus

Front 32

TISSUE CULTURE
What is the difference between animal viruses and bacteriophage?

Back 32

CPE

Front 33

TISSUE CULTURE
What is CPE?

Back 33

Cytopathic Effect
Visual outcome of virus infecting the cell

Front 34

TISSUE CULTURE
How do you determine a TCID50?

Back 34

Median tissue culture infective dose; that amount of a pathogenic agent that will produce pathological change in 50% of cell cultures inoculated.

Front 35

TISSUE CULTURE
Know how to set up an assay to determine if a virus has an envelope.

Back 35

Solvent sensitivity assay (sensitivity to lipid solvent)

Front 36

TISSUE CULTURE
What are quantal and quantitative assays and how are they different?

Back 36

Quantal-Immunocytochemistry (DAB)
Quantitative-ELISA (ABTS)

Front 37

TISSUE CULTURE
Know the differences between insect and mammalian tissue culture (at least 4 differences).

Back 37

1. Air exchange
2. Medium (SFM v. MEM)
3. Sf9 finicky, BT dying-just change medium and reincubate

Front 38

BACULOVIROLOGY
What is the cell line used to grow baculovirus?

Back 38

Sf9-Spodoptera frugiperda from fall armyworm ovary

Front 39

BACULOVIROLOGY
Describe the resulting CPE. Be very specific.

Back 39

Refractile, pearlescent polyhedra; multiple polyhedra per cell
bigger

Front 40

BACULOVIROLOGY
What is the natural host of baculoviruses?

Back 40

caterpillar, armyworm-non-vertebral cells

Front 41

BACULOVIROLOGY
Why is baculovirus important in molecular biology?

Back 41

recombinant expression vector, no polyhedra formed; post-translational modification possible, glycosylation, etc.

Front 42

BACULOVIROLOGY
What is the specific name of the baculovirus used in lab?

Back 42

AcMNPV-Autographa californica MultiNucleopolyhedrosis virus

Front 43

ELISA
Describe/diagram an ELISA

Back 43

Controls: +, -, conjugate, treatment
1. Load Ag onto wall.
2. Wash
3. Add NFDM to block
4. Wash
5. Add serum
6. Wash
7. Add goat anti-serum Ig-HRP conjugate
8. Wash
9. Add substrate (ABTS) and observe colorimetric change.

Front 44

ELISA
Describe/diagram an Ag capture ELISA

Back 44

Abs on wall, rest the same

Front 45

ELISA
What are the two different kinds of ELISAs and what do they detect?

Back 45

1. Direct ELISA-monoclonal Ab w/ conjugated enzyme/marker
2. Indirect ELISA-2 Abs, first directed against type of Ag detecting, 2nd directed against 1st, while having marker/enzyme attached

Front 46

ELISA
Why are indirect immunoassays versatile?

Back 46

Don't need expensive monoclonal Ab

Front 47

MAMMALIAN TISSUE CULTURE/VIROLOGY
What cell type was used?

Back 47

Bovine Turbinate (BT)

Front 48

MAMMALIAN TISSUE CULTURE/VIROLOGY
What virus was used?

Back 48

IBRV
Infectious Bovine Rhinotracheitic Virus

Front 49

MAMMALIAN TISSUE CULTURE/VIROLOGY
Describe CPE for this virus.

Back 49

big ball of motherfuckin cells that form a conglomerate

Front 50

MAMMALIAN TISSUE CULTURE/VIROLOGY
Know the steps to trypsinize cells.

Back 50

1. Discard spent medium
2. Rinse monolayer
3. Add min. volume of trypsin; inc 37C
4. Cells round up + detach
5. Monitor w/ inverted microscope.
6. Triturate cells and subdivide in 10% MEM
**Serum inactivates trypsin
**Cells can be digested by trypsin

Front 51

VIRUS CHARACTERIZATION
What does the treatment of virus with chloroform tell you?

Back 51

Whether or not the virus is enveloped

Front 52

VIRUS CHARACTERIZATION
What controls are needed for this assay?

Back 52

known enveloped virus
known naked virus
chloroform control
experimental

Front 53

Does IBRV have an envelope?

Back 53

yes

Front 54

VIRUS CHARACTERIZATION
Were other enveloped viruses studied this semester?

Back 54

yes; AcMNPV (autographa californica multiple nucleopolyhedrosis virus)

Front 55

MAMMALIAN TISSUE CULTURE
Protocol for infecting cells?

Back 55

1. Cells should be 70-80% confluent
2. Remove medium
3. Add minimal volume 2% MEM (enough to cover monolayer)
4. Add inoculum and adsorb for 1 hr @ 37C
5. Discard inoculum, add minimal volume 2% MEM
6. Monitor for CPE

Front 56

MAMMALIAN TISSUE CULTURE
Protocol for passing cells?

Back 56

1. Cells have to be dissociated from plastic w/ trypsin
2. Discard spent medium
3. Rinse monolayer w/ small volume trypsin
4. Add minimal volume trypsin, incubate @ 37C
5. Cells will round up and detach
6. Monitor progress w/ inv. mic
7. Triturate cells and subdivide in 10% MEM
*Trypsin is inactive at lower pH
8. If cells don't round up, trypsin may need to be changed
*Serum inactivates trypsin
*Never leave cells in trypsin longer than necessary, cell will be digested

Front 57

MAMMALIAN TISSUE CULTURE
Type of cells?

Back 57

Bovine turbinate cells (BT cells)

Front 58

MAMMALIAN TISSUE CULTURE
Type of virus?

Back 58

Infectious bovine rhinotracheitis virus IBRV
Herpes virus

Front 59

MAMMALIAN TISSUE CULTURE
IBR aka red nose...

Back 59

URI can lead to fatal pneumonia
animals infected for life, virus can recrudesce when stressed (shipping fever)
animals are vaccinated, reduce stress

Front 60

MAMMALIAN TISSUE CULTURE
If cells don't grow, what could be the reason?

Back 60

1. pH too acidic
2. pH too basic
3. bacterial infection/contamination

Front 61

VIRUS CHARACTERIZATION
What does treatment of virus with chloroform tell you?

Back 61

Enveloped or no

Front 62

VIRUS CHARACTERIZATION
Does IBRV have an envelope?

Back 62

yes

Front 63

VIRUS CHARACTERIZATION
Were other enveloped viruses studied this semester? How was that determined?

Back 63

Sensitivity to lipid solvent test

Front 64

COMMERCIAL TEST
Pregnancy test-what type of assay was it?

Back 64

ELISA

Front 65

COMMERCIAL TEST
Pregnancy test-what does it detect?

Back 65

HCG
Human Chorionic Gonadotropin

Front 66

Be able to write a hypothesis fucktard

Back 66

done and done

Front 67

When picking plaques or bacterial colonies, why is it important to select one that is well isolated?

Back 67

so that you don't get more than one... you want to isolate ONE type of bacteria or virus

Front 68

Counting cells on hemocytometer...

Back 68

Average the count of 4 squares
Multiply average by dilution factor
(Dil. fact. = reciprocal of dilution)
Multiply by 10^4
Answer is cells/mL

Front 69

Determining 50% end point (Reed Meunch Calc)...

Back 69

[(% positive above 50%)-50%]/[(% positive above 50%)-(% positive below 50%)]