6- microbial techniques

Question 1

what is an aseptic technique

Answer 1

Basic procedures used to prevent unwanted microorganisms from contaminating the culture.

Question 2

why are aseptic techniques needed

Answer 2

This is important as results can be skewed by contamination.

Question 3

types of aseptic techniques

Answer 3

• sterilisation of tools
• media sterilisation
• use of sterile surfaces
• personal hygiene
• protective clothing
• proper waste disposal

Question 4

aseptic techniques- sterilisation of tools

Answer 4

• Sterilise inoculating loops using a
Bunsen burner.
• Autoclave (clean with steam) equipment before and after use.

Question 5

aseptic techniques- media sterilisation

Answer 5

Sterilise media in an autoclave to destroy contaminating organisms.

Question 6

aseptic techniques- use of sterile surfaces

Answer 6

Do work on sterile surfaces or a laminar flow hood to reduce risk of contamination.

Question 7

aseptic techniques- personal hygiene

Answer 7

Wash hands before and after handling cultures.

Question 8

aseptic techniques- protective clothing

Answer 8

Wear lab coats, gloves, and safety glasses to help prevent contamination.

Question 9

aseptic techniques- proper waste disposal

Answer 9

Dispose of biological waste properly to prevent the spread of microbes.

Question 10

microbiological culture media

Answer 10

• Microorganisms are grown in culture media, which provide the necessary nutrients.
• Different types of media are used depending on the organism.

Question 11

inoculation

Answer 11

The process of introducing microorganisms into the media.

Question 12

incubation

Answer 12

After inoculation, cultures are kept in an incubator at optimal conditions for growth.

Question 13

conditions for microbiological growth

Answer 13

• Nutrients (glucose, amino acids).
• Temperature.
• Humidity.
• Light.
• pH.
• Oxygen (anaerobic conditions encourage the growth of pathogens).

Question 14

methods of microbiological culturing

Answer 14

• streak plate method
• serial dilution and plating

Question 15

streak plate method

Answer 15

To isolate a pure strain from a single species of microorganism.

Question 16

serial dilution and plating

Answer 16

To obtain pure cultures by diluting the sample and spreading it on multiple plates.

Question 17

broth cultures

Answer 17

Liquid media used when fresh cultures or large numbers of cells are required.

Question 18

procedure for broth cultures

Answer 18

• Dip an inoculating loop into the stock culture or sample.
• Stir the loop in the broth tube to transfer the organism.
• Observe for turbidity, which indicates microbial growth.

Question 19

agar media

Answer 19

Solid media used for isolating and characterising bacteria.

Question 20

procedure for agar media

Answer 20

• Dip an inoculating loop into the stock culture or sample.
• Streak the loop across the agar plate in a pattern to isolate individual colonies.
• Observe colony growth and colony morphology.

Question 21

selective media

Answer 21

Used to suppress growth of unwanted bacteria and encourage growth of desired ones.

Question 22

procedure for selective media

Answer 22

• Dip an inoculating loop into the stock culture or sample.
• Streak the loop across the selective media plate.
• Observe colony growth. Only microorganisms that can thrive in the conditions will grow (e.g.
MacConkey agar is selective for Gram-negative bacteria).

Question 23

haemocytometer

Answer 23

A thick microscope slide with a grid of standard volume.

Question 24

measuring bacterial growth- cell count (haemocytometry) method

Answer 24

• Dye the broth with trypan blue or methylene blue.
• Count cells under a microscope, except those touching the bottom and left lines.
• Take a mean of counts, repeated at regular intervals throughout growth.

Question 25

measuring bacterial growth- cell count (haemocytometry) advantages

Answer 25

• Provides an accurate estimate of living cells. • Can disregard dead cells from the count. • Direct and accurate if done correctly. • Allows for the observation of cell morphology.

Question 26

measuring bacterial growth- cell count (haemocytometry) limitations

Answer 26

• Time-consuming. • Requires expensive equipment (haemocytometer slides).

Question 27

measuring bacterial growth- dilution plating process

Answer 27

• Dilute the original culture and spread on a plate. • Continue dilution until the number of colonies can be counted.

Question 28

measuring bacterial growth- dilution plating advantages

Answer 28

• Doesn't require complex or expensive equipment. • Provides information on the number of viable cells. • Can be used to isolate individual colonies.

Question 29

measuring bacterial growth- dilution plating limitations

Answer 29

• Slow due to the requirement for an incubation period. • Need for serial dilution. • Sample may not be uniform.

Question 30

measuring bacterial growth- mass method process

Answer 30

A known volume of culture is dried and weighed to find the dry mass of cells.

Question 31

measuring bacterial growth- mass method advantages

Answer 31

• Quick and easy. • Useful for large-scale industrial applications.

Question 32

measuring bacterial growth- mass method limitations

Answer 32

• Difficult to separate organisms from the media. • Does not provide information on individual cells or cell viability. • The presence of extracellular material can affect results. • Small mass being recorded could lead to an increase in error during reading. • Takes time for organisms to grow.

Question 33

measuring bacterial growth- optical method (turbidity) process

Answer 33

• Measure the absorbance of a sample. • Use a calibration graph to obtain the cell count.

Question 34

measuring bacterial growth- optical method (turbidity) advantages

Answer 34

• Measurements can be taken easily. • Can be conducted in the field. • Rapid and non-destructive. • Suitable for continuous monitoring of cell growth.

Question 35

measuring bacterial growth- optical method (turbidity) limitations

Answer 35

• Requires expensive equipment. • Only provides a turbidity reading, an indirect count. • Requires a calibration curve to obtain an actual cell count. • Does not distinguish between live and dead cells. • Assumes that agitation and cell density are equal across the culture. • The presence of particulate matter can interfere with readings.

Question 36

draw a bacterial growth curve

Answer 36

Question 37

bacterial growth curve- lag phase

Answer 37

• Initial phase where there is little or no cell division. • Cells are adjusting to the environment, synthesising enzymes, and preparing for growth.

Question 38

bacterial growth curve- log or exponential phase

Answer 38

• Period of rapid cell division. • Cells are healthy and have ample nutrients, leading to exponential growth. • Growth rate constants can be calculated in this phase using the formula:

Question 39

bacterial growth curve- stationary phase

Answer 39

• Growth rate slows and the total number of viable cells remains relatively constant. • Occurs when nutrient levels decrease and waste products accumulate, leading to cell death at the same rate as cell division.

Question 40

bacterial growth curve- death or decline phase

Answer 40

• Number of viable cells decreases as the rate of cell death exceeds rate of cell division. • Occurs due to the continued depletion of nutrients and accumulation of waste products.