Practical 1. Differentiation of Bacteria and the Induction of Bacterial Gene Expression During Infection

Question 1

How can a rough phenotypic differentiation of bacteria be obtained ?

Answer 1

1. The source of bacteria
2. Cell shape and colony colour and morphology
3. Gram status
4. Metabolic features
5. Phenotypic tests for presence of characteristic genes
   6.

Question 2

What test will identify if a bacteria is gram positive or gram negative ?

Answer 2

The potassium hydroxide test

Question 3

What can potassium hydroxide do to bacteria ?

Answer 3

Quickly lyse gram negative cells as they have a weaker cell envelope and liberate DNA

Question 4

What do gram positive cells have ?

Answer 4

1. Wall technoic acids
2. Lipotechnoic acids
3. Capsular protein

Question 5

What do gram negative cells have ?

Answer 5

1. Lipopolysaharide
2. Outer membrane
3. Periplasm
4. Outer membrane protein

Question 6

What is the composition of LPS ?

Answer 6

1. O-antigen
2. Core saccharide
3. Lipid A

Question 7

What does a gram negative bacteria result of a potassium hydroxide test ?

Answer 7

Slimy stringy substance

Question 8

What may gram negative bacteria be further characterised by ?

Answer 8

Whether they possess the ability to grow in the presence or absence of oxygen

Question 9

What is the test used to show the presence of oxygen in gram negative bacteria ?

Answer 9

Oxidative fermentative test

Question 10

What is used to seal the test tube in the oxidative fermentative test ?

Answer 10

Paraffin

Question 11

What does paraffin prevent ?

Answer 11

Gas exchange with the environment

Question 12

What does the growth medium in the oxidative fermentative test contain ?

Answer 12

A pH indicator

Question 13

What will the pH indicator in the growth medium in the oxidative fermentative test do when bacteria have acidified/presence of oxygen ?

Answer 13

Will turn the colour of the growth medium yellow

Question 14

What is the oxidase test used for ?

Answer 14

If a bacterium possesses certain cytochrome c oxidases

Question 15

What does the possession of certain cytochrome c oxidases mean for bacteria ?

Answer 15

They can utilise oxygen for energy production via an electron transport chain

Question 16

What does the oxidase test use as a redox indicator ?

Answer 16

N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD)

Question 17

What happens when the reagent is oxidised is the oxidase test ?

Answer 17

Becomes dark blue

Question 18

What colour is the reagent in the oxidase test in its initial reduced state ?

Answer 18

Colourless

Question 19

What are many gram negative pathogens ?

Answer 19

Oxidase positive

Question 20

What are some examples of gram negative, oxidase positive bacteria ?

Answer 20

1. Pseudomonas aeruginosa
2. Helicobacter pylori
3. Vibrio cholera
4. Campylobacter jejuni

Question 21

What does the oxidase test show about the Enterobacteriaceae ?

Answer 21

They are oxidase negative

Question 22

What do the results of the oxidase test show about staphylococci and streptococci ?

Answer 22

Oxidase negative

Question 23

What will the catalase test do ?

Answer 23

Distinguish gram positive bacteria

Question 24

What do most obligate aerobic and faculative anaerobic bacteria produce ?

Answer 24

Hydrogen peroxide as an oxidative end product of the aerobic breakdown of sugars

Question 25

What do bacteria do to protect themselves from the toxic hydrogen peroxide ?

Answer 25

Produces catalase which converts hydrogen peroxide to harmless water and oxygen

Question 26

What do you see if a bacteria possesses catalase ?

Answer 26

Bubbles of oxygen

Question 27

What do bacterial infections occurring via the oral route involve ?

Answer 27

Bacteria passing through the stomach prior to entering the intestinal tract

Question 28

What about the stomach kills most bacteria ?

Answer 28

The low pH (1.5-3.5)

Question 29

What have bacteria like salmonella enterica serovar typhimurium evolved ?

Answer 29

The ability to survive acidic conditions for long enough to reach the intestine

Question 30

What is the asr gene ?

Answer 30

highly acid-inducible and is up regulated during salmonella infection

Question 31

What does the asr gene encode ?

Answer 31

A small basic periplasmic protein of 102 amino acids

Question 32

What is the small basic periplasmic protein that the asr gene encodes required for ?

Answer 32

Growth of bacteria under acidic conditions

Question 33

Where is the asr gene highly expressed ?

Answer 33

During the growth at acidic pH 4.5 and 4.0

Question 34

What does the asr mRNA indicate ?

Answer 34

asr gene is not highly expressed at pH of 7.2 or 5.0

Question 35

What have genetic tools been developed for ?

Answer 35

To enable scientists to visualise when a gene is expressed

Question 36

What is a commonly used genetic tool ?

Answer 36

Green fluorescent protein

Question 37

What were E.coli cells transformed with ?

Answer 37

A plasmid carrying the genetic reporter

Question 38

What does the this plasmid carry ?

Answer 38

A genetic fusion between the aid responsive promoter of the asr gene of salmonella enterica

Question 39

What does the genetic fusion between the acid-responsive promoter of the asr gene of salmonella control ?

Answer 39

The expression of the asr gene and the gene encoding green fluorescent protein

Question 40

What happens when transcription is initiated from the asr promoter in response to acidic conditions ?

Answer 40

mRNA is transcribed from the downstream green fluorescent protein gene

Question 41

What does the translation of green fluorescent protein mRNA lead to ?

Answer 41

Production of the fluorescent green fluorescent protien

Question 42

What does the production of the fluorescent green fluorescent protein make ?

Answer 42

The bacteria fluoresce when exposed to light of the appropriate wavelength

Question 43

What does the excitation of the green fluorescent protein protein with blue light lead to ?

Answer 43

The generation of green fluorescence

Question 44

What does the origin of replication on plasmid required for ?

Answer 44

Maintenance and replication of the plasmid

Question 45

What does the beta lactamase encoding gene provide the bacteria with ?

Answer 45

Ampicillin resistance

Question 46

What is ampicillin ?

Answer 46

A beta lactam antibiotic

Question 47

What is the beta lactamase encoding gene required for ?

Answer 47

To select for cells that carry the plasmid and for plasmid maintenance

Question 48

What is the potassium hydroxide test an alternative to ?

Answer 48

Gram staining

Question 49

What are some shapes of bacteria ?

Answer 49

1. Coccus 2. Coccoid 3. Rod 4. Comma shaped 5. Spirillum 6. Club shaped 7. Rod with spore

Question 50

How many genes does salmonella have ?

Answer 50

4500

Question 51

How many salmonella genes can change in a few minutes to react to a new environment ?

Answer 51

Hundreds