🧫Biology Unit 8 Microbes

• Microbiology experiments require special techniques and precautions, for two good reasons: Health and Safety - to prevent the escape of any experimental microbes to the surrounding environment, where they may cause disease; Validity - to prevent contamination of experiments by microbes from the environment
• Safety Precautions for Practical Microbiology


  • Never eat or drink in a microbiology laboratory
  • Avoid putting your hands in or near your mouth during a microbiology practical
  • Ask for a plaster to cover any cuts or scratches on your hands or arms
  • Never open a Petri dish or culture bottle unless you have been specifically told to by your teacher
  • Never touch cultures with your fingers, clothing or any part of your body
  • Practice aseptic technique before attempting any transfer for real
  • If you drop or spill a culture do not touch it or attempt to clean it, report the accident immediately
  • Dispose of glassware and other apparatus in the labelled receptacles provided
  • Never pour any medium or culture down the sink
  • Wash your hands with a disinfectant soap before you leave the laboratory
  • You are responsible for your own safety and the safety of everyone else in the lab
• Sterilise


  To kill or remove all microbes from somewhere
• Medium


  The mixture that the microbes are grown (or cultured) on

  
• Selective Medium


  A synthetic medium with specific composition to allow the growth of only one group of microbes and stop the growth of all others
• Nutrient Medium


  A cheap general-purpose complex medium used for growing bacteria in most school experiments
• Broth


  A liquid medium (i.e. without agar) in a test tube, universal bottle or flask

  
• Agar


  Agar is mixed with a liquid medium to make a solid medium
• Inoculate


  To add cells to a medium, so that they may grow
• Incubate


  To leave a culture to grow under defined conditions
• Culture


  A growth of microbes in a medium
• Colony


  A visible circular growth of bacteria on an agar plate containing many millions of cells
• Streak Plate


  A method of inoculating an agar plate with bacteria so that the bacteria are gradually diluted

  
• Bacterial Lawn


  A smooth even layer of bacteria covering the surface of an agar plate

  
• Serial Dilution


  Microbial cultures can have a very large concentration of cells in them, so you often start by making a serial dilution of your culture to give you a large range of concentrations

  
• Cell Counting with a Haemocytometer

  • Counts the total cells in a broth culture by observing the individual cells under the microscope
  • Useful for large cells like yeast, but more difficult for bacterial cells since they are so small
• Turbidometry


  Measures the density of cells indirectly by measuring the absorbance of light through a sample of the liquid culture
• Dilution Plating


  Counts viable cells by taking a small sample from a broth culture, diluting it, and spreading it on an agar plate to grow into colonies that can be counted

  
• Dry Mass


  A method for measuring fungal growth by drying and weighing the fungal biomass
• Visible light is not actually absorbed by the cells (as it is by pigment molecules), but scattered
• Cells in suspension will settle over time, so it is important to shake the culture flask before collecting a sample so that the mixture is homogenous
• The turbidity reading should be taken quickly before the cells settle to the bottom of the cuvette
• If the absorbance reading is too high, the original culture will have to be diluted using serial dilution to obtain a reading below 0.5
• If a range of culture samples are counted in a haemocytometer and also in a colorimeter, then a calibration curve can be plotted

  
• Generally, if the absorbance is below 0.5, the calibration curve is a straight line
• From the calibration curve the concentration of cells in a new sample can be read off for any absorbance, without having to use a haemocytometer
• Dilution Plating


  1. Take a small sample from a broth culture using aseptic technique and a sterile pipette

  2. Spread the sample evenly onto an agar plate using a sterile glass or plastic spreader

  3. Incubate the agar plate at 30ºC for 24h

  4. Count the number of colonies on the agar plate

  
• Each colony arose from a single living cell, so the number of colonies is the number of viable cells in the sample
• For most cultures there will be too many colonies to count, so a serial dilution is used to get a range of cell concentrations, and a sample from each dilution is spread on an agar plate

  
• For most of the agar plates there will be too many colonies to count, and in some agar plates there will be too few, so any count would be unreliable
• But in one of the dilutions there will be a good number (20-200) of individual colonies, and from this we can calculate the concentration of viable cells in the original culture
• This method is very accurate, but tedious. It is the only good way to count viable cells, because only those cells that grow into living colonies are counted
• Fungi


  • Unicellular fungi (yeasts) can be treated just like bacteria and any of the cell counting methods can be used
  • Multicellular fungi (moulds) form a large mass called a mycelium, made of thread-like hyphae

  
• Measuring Mould Growth in Solid Cultures

  1. Moulds growing on a solid agar plate tend to form fairly flat circular mycelia, so the area of the mycelium is an easy measure of fungal mass

  2. If the mycelium is not circular it can be treated as an ellipse, and the area calculated using: area = π x r1 x r2, where r1 and r2 are the longest and shortest radii

  
• Measuring Mould Growth in Liquid Cultures

  1. Take a sample of the culture and filter or centrifuge to separate the fungi from the solution

  2. Dry the mould fully by heating in an oven at 100ºC to evaporate the water and then weigh

  3. Repeat the cycle of heating and weighing until the mass is constant, which means that all the water has been driven off

  4. This gives the dry mass (i.e. fungal cell mass) in the original sample volume

  
• Comparison of Counting Methods

  • Haemocytometer
  • Turbidity
  • Spread Plate
  • Dry Mass

  
• Viable or total count

  Haemocytometer - total (or viable with vital stain)

  Turbidity - total

  Spread Plate - viable

  Dry Mass - total
• Speed


  Haemocytometer - Slow (hour)

  Turbidity - Fast (minutes)

  Spread Plate - Very slow (24 h)

  Dry Mass - Slow (hours)
• Used for


  Haemocytometer - bacteria

  Turbidity - bacteria

  Spread Plate - bacteria

  Dry Mass - Moulds

  
• Bacterial Growth Curve


  • Lag phase - cell division and cell death rates are both low, so the population does not change much

  Exponential phase - cells divide without any external limiting factors, population doubles in a set time known as the generation time

  Stationary phase - cell division slows down and cell death increases until the two rates are approximately equal, so the population of viable cells is roughly constant

  Death phase - cell death rate is greater than cell division rate, so number of viable cells decreases