Microbio Lab Final

Front 1

Define Antibiotic

Back 1

made by microbes, that prevent the growth of other microbes, released into the environment and have a low molecule weight (penicillin, streptomycin)

Front 2

Name an antibiotic resistant organism

Back 2

Methicillan resistant S. aureus

Front 3

Define Bacteriocidal

Back 3

Kills bacterial cells

Front 4

Define Bacteriostatic

Back 4

Limits growth of bacterial cells

Front 5

Kirby Bauer Method

Back 5

Tests for antimicrobials that kill or inhibit microorganisms, or antibiotics which are antimicrobials that are produced by microorganisms that inhibit or kill other microorganisms.

If the agent inhibits or kills the test organism, there will be a zone around the disk where no growth occurs (Zone of Inhibition)

Front 6

Three factors used for the Kirby Bauer Method

Back 6

Media used: Mueller Hinton II agar (MH) at a pH between 7.2 and 7.4

Diffusion: turbidity of the broth has to match a standard

High potency disks are used

Front 7

Define Zone of Inhibition

Back 7

Zone around the disk where no growth occurs if the agent inhibits or kills the test organism

Front 8

Oxidase Test- what indicates positive test?

Back 8

Tests for the presence of oxidase- differentiates bacteria that use oxygen as the terminal electron acceptor

Purple indicates positive test

Front 9

Name a bacteria positive for the oxidase test

Back 9

Pseudomonas aerugenosa

Front 10

What does IMViC Stand for?

Back 10

Indole
Methyl Red
Voges-Proskauer
Citrate

Front 11

What does IMViC Differentiate?

Back 11

Erscherichia coli ++--

Enterobacter aerogenes --++

Front 12

What does the I in IMViC Stand for? What does it test for? What is the control? What does the positive test look like?

Back 12

Indole- uses the SIM tube
Control- Kovacs Reagent
Positive test- Red

E coli, P vulgaris tests positive

Front 13

What does the M in IMViC Stand for? What does it test for? What is the control? What does the positive test look like?

Back 13

M for Methyl Red

Tests for- mixed acid fermentation
Control? Methyl Red must be added

Positive- E. coli, red color

Front 14

What does the V in IMViC Stand for? What does it test for? What is the control? What does the positive test look like?

Back 14

V for Voges-Proskauer

Tests for- presence of acetoin
Control? Barritts reagent used as a control

Positive- Enterobacter aerogenes, pink color

Front 15

What does the C in IMViC Stand for? What does it test for? What is the control? What does the positive test look like?

Back 15

C for Citrate

Tests for- bacteria that use Citrate as source of carbon

Control? Contains ammonium salts that will raise the pH if broken down into ammonia

Positive- Enterobacter aerogenes, blue

Front 16

Urea Slant

What does it test for? What is the control? What does the positive test look like?

Back 16

Tests for- presence of urease

Control? Phenol red

Positive test? P. vulgaris, red

Front 17

FTM

What does it test for? What is the control? What does the positive test look like?

Back 17

Tests for - O2 requirements

Control/indicator- fluid thioglycollate medium

Indicator is Resazurin

Facultative- E. coli

Aerobe- Pseudomonas

Front 18

O/F glucose Tube

What does it test for? What is the control? What does the positive test look like?

Back 18

Tests for- determines is bacteria is oxidative or fermentative

Fermentative and Oxidative- Positive for both aerobic and anaerobic- (yellow), E coli

Anaerobic green and aerobic yellow- oxidative metabolism, P. aeruginosa

Anaerobic and aerobic both green- sugar not metabolized

Front 19

Kliger's Iron Agar

(KIA)

What does it test for?
Example of a Positive test? What does it look like?

Back 19

Detects the fermentation of glucose and lactose, as well as the production of hydrogen sulfide (H2S)

Contains ferrous salts that will react and produce BLACK COLOR

P. vulgaris tests positive

Front 20

Nitrate Test

What does it test for?

Reagents added?

Test positive? Test Negative

Back 20

Tests for - nitrate respiration

Reagent A- Sulfanilic acid
Reagent B- Dimethyl-alpha-naphthylamine

Positive test- E. coli
Negative test- RED

Front 21

Catalase Test

Tests for?

Reagents/indicators?

Positive example?

Back 21

Tests for- presence of enzyme catalase

Control- H2O2, hydrogen peroxide- causes bubbling

Positive test- Staph aureus

Front 22

Durham Tubes

Tests for?

Control?

Positive test?

Negative test?

Back 22

Tests for- fermentation of sugar (glucose, lactose, mannitol)

Control? Phenol red in media

Positive test- yellow color, E. coli

Negative test- red color

Front 23

SIM Media

Tests for?

Controls used?

Positive tests?

Back 23

H2S (hydrogen sulfide)- turns BLACK- P. vulgaris

Indole- turns RED, Kovac's Reagent used

Motility- E. coli

Front 24

Catabolism

Back 24

Enzymes that function in breaking down food material

Front 25

Anabolism

Back 25

Synthesis of complex molecules from smaller molecules, and the storage of energy

Front 26

Exoenzymes

Back 26

Excreted outside of the cell that function in the degradation of large macromolecules.

Front 27

Examples of exoenzymes

Back 27

Protease
DNase
Amylase

Front 28

Coenzymes

Example

Back 28

Assist enzymes in the functioning of catalytic reactions

Example- NAD+ and FAD

During Oxidative Phosphorylation, reduced coenzme NADH generated in metabolic reactions is converted back to NAD+ because oxygen acts as a terminal electron acceptor and is converted to water.

Front 29

What is the role of tryptophanase?

Back 29

Tryptophanase is a hydrolytic enzyme that split tryptophan into pyruvate and indole. Pyruvte is metabolized and Indole is not, so tests for indole indicate whether or not bacteria do or do not produce tryptophanase.

Positive test is red- indole

Front 30

What is the full name for the classification manual

How does it differentiate organisms?

Back 30

Bergey's Manual for Determinative Bacteriology

-What tests can be performed
-What they infect

Front 31

How are chapters in Bergey's Manual organized?

Back 31

-O2 requirements
-Morphology (physical characteristics)
-Gram+ or Gram-

Front 32

Define Facultative Aerobe

Back 32

Bacteria can grow aerobically or anaerobically depending on the culture conditions.
Aerobic growth in the presence of oxygen is by respiration, but if oxygen is absent, they can grow by other modes of metabolism such as fermentation.

E. coli is a facultative anaerobe

Front 33

Define Aerotolerant Anaerobes

Back 33

Can grow in the presence of O2 and are not usually harmed by its presence in the environment. Metabolism does not require oxygen but rather involves fermentation.

Example, S pyogenes

Front 34

Three enteric pathogens of primary medical importance

Back 34

Salmonella
Shigella
E. coli

Front 35

What is an enteric organism?

Back 35

Breaks down things we can not allowing us to use it

Front 36

Seratype definition

Back 36

Different types of antigens that present of surface cells

Front 37

• What was the characteristic that separated Salmonella/Shigella from other gram-negative pathogens?

Back 37

Salmonella/Shigella do not ferment lactose like E. coli

Front 38

• What coliform is the most difficult to distinguish from Salmonella/Shigella? How are they differentiated?

Back 38

Proteus is most difficult to distinguish

color change distinguishes the two

Front 39

HE (Hektoen Enteric)
Explain how it is Selective & Differential

Back 39

Used to inhibit gram-postive bacteria. Differentiate lactose & non-lactose fermenters

Front 40

MacConkey Agar
Explain how it is Selective & Differential

Back 40

• Enterics: fermentation of lactose, red/pink
• Selective/Differential: Bile salts/ crystal violet, inhibits grm +
• E. coli: red/pink
• P. vulgaris/Salmonella/Shigella: colorless

Front 41

XLD or Xylose Lysine Desoxycholate

Back 41

•Bile salts/ sodium desoxycholate and inhibits gram +
•Salmonelloa :red, black centers (H2S producer)
•P. vulgaris: black reduce H2S
•E. coli yellow

Front 42

Name the media used in unknown 2

Back 42

HE
MacConkey
XLD

Front 43

• Explain the purpose of the Miniaturized multitest system (Enterotube). Give examples of tests it can perform.

Back 43

o Multiple tests can be performed in one tube. Faster, less media

Front 44

What is alkaline reversion? Why is it important?

Back 44

A condition in which the medium turns yellow during the first part of the incubation period then changes to red later due to increased alkalinity.

Important because the color change occurs after periods of time longer then 18-24 hrs and can alter the results.

Front 45

Define coliform, give two examples

Back 45

rod, facultative anaerobe, ferments lactose, gram-

E. coli and E. aerogenes

Front 46

List three characteristics of a good sewage indicator.

What is an example of a good sewage indicator?

Back 46

1) The best example is human waste. Total coliforms are good, fecal coliforms narrow down and are a better indicator
2) The organism can be easily identified in microbial tests
3) It is not as fastidious as the intestinal pathogens, and hence it survives a little longer is water samples

E. coli is the best sewage indicator because it occurs primarily in the intestines of humans and some warmblooded animals and it is not found routinely in soil or water

Front 47

Name a good sewage indicator that is NOT a coliform

Back 47

Enterococcus faccalis

Gram+ cocci, found in human intestines but NOT a coliform

Front 48

Membrane Filter Method vs Most Probably Number Method

Back 48

MPN is complicated, takes a long time

Membrane filter method- allows greater amount of water to be filtered

Front 49

How is MPN determined?

Back 49

coliform count * 100/Amt. water Filtered Materials

Front 50

What is EMB?

Back 50

inhibits gram-positive bacteria. Gram-neg lactose fermenters have dark center. Coliforms appear metallic

Front 51

What is ENDO?

Back 51

Coliforms appear red. Non-fermenters of lactose appear colorless

Front 52

Define Transformation

Back 52

Uptake of foreign DNA, often a circular plasmid

Front 53

Name the genes found in the pGLO plasmid

What is the purpose of each?

Back 53

GFP

b-lactamase= ampicillan resistance

araC regulator protein= regulates GFP transcription

Front 54

What two methods can be used to transform cells?

Why are these methods used?

Back 54

-Electroporation
-CaCl2/Heat Shock

Used to make the membrane more permeable

Front 55

What are the requirements for heat shock?

Back 55

50 seconds

42 degrees celsius

Front 56

What happens to the cell if it is heated too long? too short?

Back 56

DNA and other particles leave the cell

Circular DNA can not enter the cell

Front 57

Explain Transformation Protocol

Back 57

A competent E. coli cell can take up DNA by treating it with CaCl2 which makes membranes more permeable. Heat shock at 42 degrees Celsiuis for 50 seconds initiates travel of DNA into the cell. Then immediate ice is necessary to close the pours.

Front 58

If a plate has

LB
A
and
+pGLO

What would you expect?

Back 58

Growth because had plasmid for amp R

Front 59

If a plate has

LB
A
and Arabinose
and
+pGLO

What would you expect?

Back 59

GFP is transcribed and ara makes it grow

Front 60

If a plate has

LB

and
-pGLO

What would you expect?

Back 60

Lawn, no amp so nothing to resist

Front 61

If a plate has

LB
A
and
-pGLO

What would you expect?

Back 61

No growth

Front 62

How is the GFP gene regulated?

Back 62

Arabinose!

Front 63

What does ELISA stand for?

Back 63

Enzyme linged immunosorbent assay

Front 64

What is an antigen?

Back 64

High molecular weight
Complex 3D shape
Stimulates production of Antibodies
Specific reaction to the antibody
Foreign object to your body

Front 65

What is an antibody? How are they specific?

Back 65

Parts of the antibody are specific- called variable regions- have different amino acid sequences that cause specific binding to antigen

Made by B cells in response to antigen

Mount immune response against foreign agent

Front 66

Explain the ELISA test

Back 66

Step 1
Add antigen

Step 2 wash with substrate to get rid of excess

Step 3
Block and allow time to bind

Step 4
Wash excess block

Step 5
Bind primary antibody- PBS

Step 6
Wash

Step 7
Add second antibody with enzyme located at the end

Step 8
Wash

Step 9
Add substrate

Front 67

What part of ELISA do you know?

Back 67

ONLY THE ANTIGEN

Front 68

What does the ELISA positive test look like?

Back 68

Green!

Front 69

What human diseases can the ELISA test be used to detect?

Back 69

-HIV
-Influenza testing

Front 70

If you test water and get a positive water test, it is safe to assume..

Back 70

that it is safe to drink until other tests are performed

Front 71

Explain how to determine a positive result for a Durham tube (Lactose/Mannitol/Dextrose) and how to tell if gas is produced during the reaction

Back 71

-phenol red indicator- color change to yellow

Durham tube traps gas during fermenttion

Front 72

When reagent is used for the phenylalanine test?

Name positive organism

Back 72

Ferric Chloride

P. vulgaris

Front 73

What was the negative control in the pGLO experiment?

Back 73

- pGLO
cells plated on AMP or
AMP/ARA plates

Front 74

Explain the results during the ELISA experiment

Back 74

Antibody 1 bound better than Antibody 2 which could be seen by comparing the columns. The less dilute solutions (1:400) produced a brighter green color-positive control

Front 75

1000ug = ____ mg

Back 75

1

Front 76

If 1 mL of water is 1g, how many kg is 10 L?

Back 76

1 L= 1000 mL = 1000 g/ kg = 1000*10 L= 10000 g = 10 kg

Front 77

Explain how positive ions and heat assist in bacterial transformation

Back 77

o Positive ions bind to negatively charged plasmid DNA and bring it to negatively charge cell membrane. Heat dilates pores in membrane to allow plasmid DNA into cytoplasm

Front 78

What is the purpose of adding mineral to O/F glucose?

Back 78

Keeps oxygen out creates an anaerobic environment

Front 79

What reagent is used to perform a catalase test on an unknown organism?

Back 79

Hydrogen peroxide

Front 80

A red color after the addition of nitrate reagent A&B indicates what?

If no color change, what reagent is then added to check for denitification?

Back 80

Nitrate reduction to nitrite

Zinc powder is added

Front 81

What is indicated by a Durham tube?

Back 81

Gas production by fermentation

Front 82

How are slants that show glucose fermentation indicated?

How are slants that show both glucose and lactose fermentation indicated?

Back 82

- yellow butts with red slants

- All yellow no red

Front 83

During water testing, what might cause a false positive test?

Back 83

If gas is present only after 48 hours, the gas is probably not due to coliforms and further testing is necessary

Front 84

Presence of coliforms is indicated by a water test, why might you perform the IMViC tests on these isolates?

Back 84

Can't distinguish between E. coli and E. aeruginosa.

Front 85

Why is it important to have a working slant?

Back 85

Spreads culture to indicate absolute growth

Front 86

Describe a real life application of a membrane filter

Back 86

Tests for purity

Front 87

Motility +

Back 87

E coli

Front 88

Positive test for catalase test

Back 88

Staph aureus

Front 89

Control for the catalase test? What does it do?

Back 89

Hydrogen peroxide causes bubbling

Front 90

Control for the durham tube?

Back 90

Phenol red in media

Front 91

What color is the positive test for the Durham Tube? Organism positive for Durham Tube?

Back 91

Yellow, E. coli

Front 92

What color is the negative test for the durham tube?

Back 92

Red!

Front 93

What does the Durham tube test for?

Back 93

Fermentation of sugars

Front 94

What is an example of an aerotolerant anaerobe?

Back 94

S. pyogenes

Front 95

What reagent is used for the phenylalanine test?

Back 95

Ferric chloride

Front 96

What organism shows positive tests for the phenylalanine tests?

Back 96

P. vulgaris

Front 97

What colors are coliforms from P. vulgaris on the XLD agar? Why?

Back 97

Black indicating H2S producer

Front 98

What color are coliforms form salmonella on the XLD agar? Why

Back 98

Red with black centers, H2S production