6- microbial techniques Flashcards
what is an aseptic technique
Basic procedures used to prevent unwanted microorganisms from contaminating the culture.
why are aseptic techniques needed
This is important as results can be skewed by contamination.
types of aseptic techniques
- sterilisation of tools
- media sterilisation
- use of sterile surfaces
- personal hygiene
- protective clothing
- proper waste disposal
aseptic techniques- sterilisation of tools
• Sterilise inoculating loops using a
Bunsen burner.
• Autoclave (clean with steam) equipment before and after use.
aseptic techniques- media sterilisation
Sterilise media in an autoclave to destroy contaminating organisms.
aseptic techniques- use of sterile surfaces
Do work on sterile surfaces or a laminar flow hood to reduce risk of contamination.
aseptic techniques- personal hygiene
Wash hands before and after handling cultures.
aseptic techniques- protective clothing
Wear lab coats, gloves, and safety glasses to help prevent contamination.
aseptic techniques- proper waste disposal
Dispose of biological waste properly to prevent the spread of microbes.
microbiological culture media
• Microorganisms are grown in culture media, which provide the necessary nutrients.
• Different types of media are used depending on the organism.
inoculation
The process of introducing microorganisms into the media.
incubation
After inoculation, cultures are kept in an incubator at optimal conditions for growth.
conditions for microbiological growth
• Nutrients (glucose, amino acids).
• Temperature.
• Humidity.
• Light.
• pH.
• Oxygen (anaerobic conditions encourage the growth of pathogens).
methods of microbiological culturing
- streak plate method
- serial dilution and plating
streak plate method
To isolate a pure strain from a single species of microorganism.
serial dilution and plating
To obtain pure cultures by diluting the sample and spreading it on multiple plates.
broth cultures
Liquid media used when fresh cultures or large numbers of cells are required.
procedure for broth cultures
• 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.
agar media
Solid media used for isolating and characterising bacteria.
procedure for agar media
• 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.
selective media
Used to suppress growth of unwanted bacteria and encourage growth of desired ones.
procedure for selective media
• 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).
haemocytometer
A thick microscope slide with a grid of standard volume.
measuring bacterial growth- cell count (haemocytometry) method
• 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.
measuring bacterial growth- cell count (haemocytometry) advantages
• 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.
measuring bacterial growth- cell count (haemocytometry) limitations
• Time-consuming.
• Requires expensive equipment (haemocytometer slides).
measuring bacterial growth- dilution plating process
• Dilute the original culture and spread on a plate.
• Continue dilution until the number of colonies can be counted.
measuring bacterial growth- dilution plating advantages
• Doesn’t require complex or expensive equipment.
• Provides information on the number of viable cells.
• Can be used to isolate individual colonies.
measuring bacterial growth- dilution plating limitations
• Slow due to the requirement for an incubation period.
• Need for serial dilution.
• Sample may not be uniform.
measuring bacterial growth- mass method process
A known volume of culture is dried and weighed to find the dry mass of cells.
measuring bacterial growth- mass method advantages
• Quick and easy.
• Useful for large-scale industrial applications.
measuring bacterial growth- mass method limitations
• 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.
measuring bacterial growth- optical method (turbidity) process
• Measure the absorbance of a sample.
• Use a calibration graph to obtain the cell count.
measuring bacterial growth- optical method (turbidity) advantages
• Measurements can be taken easily.
• Can be conducted in the field.
• Rapid and non-destructive.
• Suitable for continuous monitoring of cell growth.
measuring bacterial growth- optical method (turbidity) limitations
• 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.
draw a bacterial growth curve
bacterial growth curve- lag phase
• Initial phase where there is little or no cell division.
• Cells are adjusting to the
environment, synthesising enzymes, and preparing for growth.
bacterial growth curve- log or exponential phase
• 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:
bacterial growth curve- stationary phase
• 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.
bacterial growth curve- death or decline phase
• 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.
