Microbiology

Question 1

What shape are coccus bacteria? Give an example

Answer 1

Spherical
Eg staphylococcus, streptococcus

Question 2

What shape are bacillus bacteria? Give an example

Answer 2

Rod-shaped
Eg E. coli

Question 3

What shape are spirillum bacteria? Give an example

Answer 3

Spiral
Eg vibrio cholerae

Question 4

By which 2 ways can bacteria be classified?

Answer 4

• Shape
• Grouping patterns

Question 5

How can bacteria be distinguished from each other?

Answer 5

By their metabolic features or by antigenic features

Question 6

What is meant by autotrophic bacteria?

Answer 6

Bacteria that synthesise cell constituents using carbon dioxide as the carbon source

Question 7

What is meant by photoautotrophic bacteria?

Answer 7

Either photosynthesise with chlorophyll as an electron donor or alternatives using sulphur or hydrogen gas as an electron donor

Question 8

Describe the Gram stain technique

Answer 8

1. Apply crystal violet
2. Apply Grams iodine solution
3. Alcohol wash (decolourisation)
4. Apply Safranin (counter stain)

Question 9

What colour is a Gram positive bacteria after Gram stain?

Answer 9

Purple

Question 10

What colour is a Gram negative bacteria after Gram stain?

Answer 10

Pink/red

Question 11

Describe a Gram positive bacterial cell wall

Answer 11

• No outer lipopolysaccharide layer
• Thick peptidoglycan cell wall

Question 12

Why do Gram positive bacteria appear purple after a Gram stain?

Answer 12

Thick peptidoglycan cell wall, but no outer lipopolysaccharide layer. They therefore retain the initial crystal violet stain when washed with alcohol.

Question 13

Describe Gram-negative bacterial cell walls

Answer 13

• Have an outer lipopolysaccharide layer
• Thin peptidoglycan cell wall

Question 14

Why do Gram-negative bacteria appear red after a Gram-stain?

Answer 14

Thin peptidoglycan cell wall and outer lipopolysaccharide membrane. When washed with alcohol, they lose this outer layer with the crystal violet stain, appearing colourless. They are then able to take up the counter stain safranin and appear red under a microscope.

Question 15

What is a consequence of Gram negative bacteria having a more complex cell wall?

Answer 15

They are not susceptible to some antibiotics such as penicillin or lysozyme

Question 16

How do bacteria reproduce?

Answer 16

Binary fission
Unicellular yeast may reproduce by budding

Question 17

What temperature to microorganisms require for growth?

Answer 17

Bacterial metabolism is enzyme regulated, with most bacteria thriving between 25 C and 45 C. The optimum temperature for mammalian pathogens is 37 C (human body temp)

Question 18

Microorganisms require nutrients for growth. How and what nutrients are supplied?

Answer 18

Nutrients are supplied in nutrient media such as nutrient agar or liquid broth. The carbon source is usually glucose, while nitrogen for amino acid and nucleic acid synthesis is provided as nitrate ions

Question 19

What is the optimum pH for growth of microorganisms?

Answer 19

Bacteria tend to favour slightly alkaline conditions, while most fungi thrive in neutral to slightly acidic environments

Question 20

What is the oxygen requirement of obligate aerobes?

Answer 20

They can only survive and metabolise in the presence of oxygen. They cannot survive without it.

Question 21

What is the oxygen requirement of obligate anaerobes?

Answer 21

Can only survive and metabolise in the absence of oxygen.

Question 22

What is the oxygen requirement of facultative anaerobes?

Answer 22

They metabolise better in the presence of oxygen but can also survive and metabolise without it

Question 23

Clostridium perfringens are obligate anaerobes that cause gas gangrene. Treatment can involve the use of a hyperbaric oxygen chamber with air pressure 2.5x higher than atmospheric pressure. How would this treatment work to improve the patients health?

Answer 23

Oxygen would inhibit the metabolism and growth of C. Perfringens. Patients immune system can kill any current bacteria. Less toxins produced - patients begin to improve.

Question 24

What happens during the lag phase of population growth?

Answer 24

No/little cell division. Intense metabolic activity such as enzyme synthesis.

Question 25

What happens during the **log phase** of population growth?

Answer 25

Rapid increase in numbers, no limiting factors to growth. Cell division > death rate. Plenty of available glucose.

Question 26

What happens during the **stationary phase** of population growth?

Answer 26

Limiting factors prevent further growth of the population eg competition for glucose. Carrying capacity has been reached.

Question 27

What happens during the **death phase** of population growth?

Answer 27

Limiting factors cause the population size to decrease. Death rate > cell division. This could be due to the build up of toxic waste. Or no glucose left in the medium.

Question 28

State 4 aseptic techniques which prevent contamination of pure cultures by *environmental microbes*

Answer 28

- Sterilise all media and equipment (eg inoculating loops) before use - Handle cultures carefully, flaming the neck of culture bottles before opening and closing - Use a lit Bunsen burner to create a convection current - Disinfect workbenches beforehand

Question 29

State 4 aseptic techniques which prevent *contamination of the environment* by cultures being grown

Answer 29

- Sterilise all work surfaces before and after experiments using a disinfectant - Lift agar dish no more than 45 degrees - Seal agar dishes with adhesive tape but not all the way around - Flame the neck of the culture bottle without placing the cap on the work surface

Question 30

Describe how to inoculate an agar dish (including aseptic techniques)

Answer 30

1. Pass the metal inoculating loop through a flame until red hot, then allow it to cool 2. Hold the bacterial culture in one hand then remove the cap with the little finger of the other hand without placing it down 3. Flame the neck of the culture bottle for 2-3 seconds and dip the inoculating loop into the bacterial culture 4. Lift the petri dish lid to 45 degrees, allowing entry of the inoculating loop, and streak the agar with the bacterial culture 5. Secure the Petri dish with adhesive tape, but do not seal completely to avoid anaerobic conditions that could promote pathogenic growth 6. Incubate at a suitable temperature (25 or 37 degrees Celsius) for 24-48 hours 7. Sterilise all equipment after use

Question 31

How does an autoclave sterilise equipment? (Glassware and metal equipment)

Answer 31

- Seal in an autoclave bag - Heat to 121 degrees Celsius in steam - Apply high pressure for 15 minutes

Question 32

How is plastic equipment sterilised?

Answer 32

Gamma irradiation

Question 33

What is the difference between the total cell count and the total viable count?

Answer 33

Total cell count is living and dead cells in a bacterial sample whereas total viable count is living cells in a known volume of liquid medium

Question 34

How do you count the number of cells in a yeast culture using a haemocytometer?

Answer 34

Count a cell if it is in a square or touches the left or upper line

Question 35

What does serial dilution assume?

Answer 35

A single bacterial cell will reproduce asexually to form a visible colony. Number of colonies therefore represents the number of bacteria in the original sample.

Question 36

Describe how to carry out a serial dilution (total viable count technique)

Answer 36

- Place 9cm3 of sterile distilled water into 5 sterile test tubes using a sterile pipette - Place 1cm3 of the original bacterial culture into the first tube and gently mix. The bacterial culture has now been diluted 10 times - Transfer 1cm3 of this 10-1 dilution from the 1st tube into the 2nd tube and gently mix with 9cm3 of sterile distilled water. Bacterial culture has now been diluted 100 times (10-2) repeat this procedure for the remaining tubes. - Transfer 1cm of each diluted sample onto a sterile nutrient agar plate. Use a sterile spreader to distribute the sample evenly on each agar plate - Repeat twice more to give a total of 3 plates per dilution - Seal each agar plate with tape (not all the way round) and incubate at 25 degrees C for 24-48 hours - After incubation, identify the dilution with distinct, non-merging colonies. Count the number of distinct colonies on each plate - Multiply the number of colonies by the dilution factor to give the number of bacteria in the original 1cm3 bacterial culture sample

Question 37

What inaccuracies may occur when carrying out a serial dilution if the original bacterial culture is under diluted?

Answer 37

Colonies might merge and counting may be inaccurate, resulting in an underestimate of cell numbers

Question 38

What inaccuracies may occur when carrying out a serial dilution if the original bacterial culture is over diluted?

Answer 38

If dilution is too great then there will be too few colonies on each plate to count to be statistically sound, leading to inaccuracies

Question 39

Describe how you could indirectly count cells

Answer 39

Use a colorimeter to measure the turbidity of a culture as cell numbers increase. Bacterial population measurements are obtained by finding the suspension’s absorbance value and referencing a standard graph of light absorbance plotted against the number of bacterial cells. This results in a total cell count as the colorimeter cannot differentiate between living and dead cells