4 - Microbiology (C1)

Question 1

What does aseptic technique prevent?

Answer 1

• Contamination of the environment by the microbes being handled
• Contamination of microbial cultures by unwanted microbes from the environment
• Contamination of the experimentor

Question 2

What is the function of the plasma membrane of prokaryotes?

Answer 2

• Barrier between environment and cytoplasm

- Controls entry and exit of substances in and out of cell

Question 3

What is the function of the peptidoglycan (murein) cell wall of prokaryotes?

Answer 3

Prevents lysis in a hypotonic solution

Question 4

What is the function of the capsule of prokaryotes?

Answer 4

• Outer later of mucopolysaccharide slime which can glue bacteria together and stick them to surfaces
• Protects the bacteria from attack by other cells

Question 5

What is the function of the pili of prokaryotes?

Answer 5

Attaches to surfaces and transfers plasmids by conjugation

Question 6

What is the function of the plasmid of prokaryotes?

Answer 6

• Circular DNA which contains extra bacterial genes for antibiotic resistance
• Can be exchanged between bacteria during conjugation allowing the spread of antibiotic resistance

Question 7

What shape is bacillus?

Answer 7

Rod shaped

Question 8

What shape is coccus?

Answer 8

Sphere

Question 9

What shape is spirillum?

Answer 9

Spiral

Question 10

What are the metabolic requirements for microbes?

Answer 10

• Suitable temp
• Suitable pH
• O2 requirements - controls growth of pathogenic bacteria which grow at a faster rate in anaerobic conditions
• Carbon source - provides energy
• Nitrogen source - required for synthesis of proteins, amino acids, DNA and RNA
• Growth factors e.g. vits and mins - essential for growth

Question 11

What are some important parts of aseptic technique?

Answer 11

• Autoclave all glassware at 121 degrees for 15 mins
• Open petri dish at a small angle
• Keep a roaring blue flame on the bench
• Keep McCartney caps in hand and flame neck of bottle
• Flame loops, wires (until red hot) and glass spreaders (using ethanol) to red heat in a Bunsen flame

Question 12

Why during aseptic technique must all glassware be autoclaved at 121 degrees for 15 mins?

Answer 12

To kill bacteria and spores

Question 13

Why during aseptic technique must petri dish lids be opened at small angles?

Answer 13

To prevent bacteria entering petri dish

Question 14

Why during aseptic technique must a blue flame be kept roaring on the bench?

Answer 14

To create a convection current to uplift air away from cultures

Question 15

Why during aseptic technique must McCartney caps be kept in hand?

Answer 15

To not contaminate the desk

Question 16

Why during aseptic technique must the neck of McCartney bottles be flamed?

Answer 16

To create a convection current to lift air from broth

Question 17

Why during aseptic technique must loops, wires and glass spreaders be flamed?

Answer 17

To kill bacteria

Question 18

How is a viable count of cells done?

Answer 18

Serial dilution is used and colonies are grown on agar plates and counted

Question 19

How is a total count of cells done?

Answer 19

Serial dilution is used, and a haemocytometer is used to count the cells

Question 20

What is the difference between a viable count and total count of cells?

Answer 20

Viable - only living cells are counted

Total - both living and dead cells are counted

Question 21

What are advantages and disadvantages of viable counts of cells?

Answer 21

+ Counts living cells

• Long process
• If culture is mixed some cells may take longer to grow than others

Question 22

What are advantages and disadvantages of total cell counts?

Answer 22

+ Quick
+ Can be used to count several types of cells
- Difficult to count if they clump

Question 23

Why are bacteria cultures diluted before being counted?

Answer 23

As otherwise there will be too many to count

Question 24

Why would it be inaccurate to choose a plate of bacteria to count with less than 50 colonies?

Answer 24

Unrepresentative, unreliable result

Question 25

Why would it be inaccurate to choose a plate of bacteria to count with more than 100 colonies?

Answer 25

Clumping would occur so individual colonies wouldn't be able to be identified

Question 26

Why does the stationary phase occur in bacterial growth?

Answer 26

- Population has reached its maximum size - Division rate = death rate - Lack of nutrients or too many toxins - Unlike wild populations, they DON'T have a carrying capacity and aren't controlled by density dependent factors

Question 27

Why during aseptic technique should cultures be incubated at 28 degrees not 37 degrees?

Answer 27

As this would provide optimum conditions for the growth of a pathogen

Question 28

Why during aseptic technique should the lids of petri dishes be secured with 2 strips of tape but not completely sealed?

Answer 28

To provide the microorganisms with some O2 to grow, otherwise it would provide anaerobic growth conditions for pathogens

Question 29

Why during aseptic technique should the bench be cleaned with Virkon before and after the experiment?

Answer 29

- Before: to prevent bacteria on the bench contaminating the microbes being handled - After: to kill microbes used in the experiment that have contaminated the bench

Question 30

What is the process of serial dilution plating?

Answer 30

- Add 9cm3 sterile water to 6 sterile tubes - Swirl the culture tube to mix the contents and using a sterile syringe add 1cm3 from this tube to the next - Continue until all 6 tubes have been done

Question 31

What is the process of serial dilution?

Answer 31

- Using a sterile syringe transfer 0.5cm3 of each sample onto a sterile agar plate - Use a glass spreader and aseptic techniques to spread over the plate - Repeat this twice to give 3 plates for each dilution - Secure the lids of the plates with 4 strips of tape and label name of bacteria, date, dilution - Incubate plates at 28degrees for 24-48 hours - Once colonies have grown, choose a plate with 50-100 colonies - Count the colonies in the 3 plates for this dilution and calculate mean - Calculate number of microbes in original

Question 32

How can you calculate the number of bacteria in the original sample from a diluted sample?

Answer 32

- Number of colonies x dilution factors e.g. if 1 in 1000, times by 1000 - Correct for different sample sizes, e.g. check if asked for 1cm3 or 0.5cm3

Question 33

What is the process of gram staining?

Answer 33

- Smear bacteria onto slide and 'heat fix' - Apply crystal violet (purple dye) - Apply iodine (mordant) - Wash with alcohol (decolorisor) - Apply safranin (counterstain)

Question 34

Why in gram staining is the bacteria smeared onto a slide and heat fixed?

Answer 34

To prevent it being washed off by staining

Question 35

Why in gram staining is crystal violet applied?

Answer 35

It binds to the peptidoglycan cell wall and so stain all cells purple

Question 36

Why in gram staining is iodine applied?

Answer 36

To ensure the crystal violet fixes firmly to the cell wall

Question 37

Why in gram staining is the bacteria washed with alcohol?

Answer 37

To remove unbound crystal violet and lipopolysaccharide: - Gram neg- lose their stain and become colourless - Gram pos+ remain purple

Question 38

Why in gram staining is safranin applied?

Answer 38

- Stains gram neg- cell wall red/pink | - Gram pos+ remain purple

Question 39

After being gram stained what colour are gram positive+ bacteria?

Answer 39

Purple

Question 40

After being gram stained what colour are gram negative- bacteria?

Answer 40

Red

Question 41

What is the study of small organisms, including bacteria, important for?

Answer 41

- Medicine / healthcare - Food industry e.g. dairy - Water quality

Question 42

What bacterial grouping is diplo?

Answer 42

A pair

Question 43

What bacterial grouping is staphylo?

Answer 43

A clump / 'stack'

Question 44

What bacterial grouping is strepto?

Answer 44

A chain / 'strap'

Question 45

What is the cell wall chemistry of gram positive bacteria?

Answer 45

- Thick peptidoglycan cell wall (penicillin disrupts cross links + causes cell to burst by osmosis = lysis) - Stain with crystal violet and turn purple

Question 46

What is the cell wall chemistry of gram negative bacteria?

Answer 46

- Thin peptidoglycan cell wall - Protective outer layer of lipopolysaccharide - More difficult for antibiotics, enzymes and antibodies to control them - Stain purple but when washed with ethanol, purple outer coat is dissolved, producing clear cells - When a 2nd (counter stain) of safranin is added, the newly exposed peptidoglycan stains red

Question 47

What are the names of bacteria that can survive at extremely high temperatures?

Answer 47

Thermophiles

Question 48

What does penicillin do to gram positive bacteria?

Answer 48

- Prevents the bonds inter-linking peptidoglycan molecules from forming - This is significant when the bacteria make new cell walls when they divide - It makes the cell walls weak, and water uptake by osmosis bursts the cell - Gram neg- bacteria are unaffected as they have a protective outer lipopolysachharide layer in their cell walls

Question 49

Why are human cells not harmed by penicillin?

Answer 49

As animal cells don't have cell walls

Question 50

As gram negative bacteria are resistant to penicillin, how can their number be controlled?

Answer 50

With antibiotics that interfere with the cell's ability to make proteins

Question 51

What does a 'defined' medium contain?

Answer 51

Only known ingredients

Question 52

What does an 'undefined' medium contain?

Answer 52

Components that are not all known

Question 53

What does a selective medium contain?

Answer 53

Only components that allow certain bacteria to grow