2. measuring culture growth

METHODS

• cell counts (haemocytometer)

• optical methods (turbidity)

• dilution plating

• area of fungi

• fungal dry mass

CELL COUNTS

• bacteria and single celled fungi cultured in nutrient broth can be counted directly using a haemocytometer

• haemocytometer- thick specialised microscope slide w/ a rectangular chamber that holds a standard volume of liquid (0.1mm3)

• chamber is engraved with grids

CELL COUNTS- STEPS

• sample of broth is diluted by half with an equal vol of trypan blue- stains dead cells blue so only living cells counted

• each corner of haemocytometer grid has a square divided into 16 smaller squares

• no. of cells on one corner is counted and mean calculated

• haemocytometer is calibrated so that no. of cells in one corner equates to no. of cells x 10^4 per cm3 of broth

• counts can be repeated at regular intervals to show how no. of bacteria changes over time

OPTICAL METHODS (TURBIDITY)

• turbidimetry is a specialised form of colorimetry

• as no. of bacterial cells in a culture increase it becomes increasingly cloudy looking or turbid

• as solution gets more turbid it absorbs more light, so less light passes through it

• a colorimeter measures this

• both dead and living cells will affect turbidity

OPTICAL METHODS (TURBIDITY)- GRAPH

• calibration curve produced by growing a control culture and taking samples at regular time intervals

• turbidity measured and cell count done using a haemocytometer

• shows relationship between no. of cells and turbidity

• using this curve we can then measure turbidity of a sample and look up no. of cells present in it

DILUTION PLATING

• used to find total viable (living) cell count

• based on idea that each colony on an agar plate has grown from a single, viable microbe

• issue is often a solid mass is present after culturing so individual colonies can’t be counted

  • solved by diluting original culture in stages until individual colonies can be seen and counted

  • can then multiply no. of colonies by dilution factor to work out a total viable cell count

• accuracy can be checked using a haemocytometer on a sample of original culture

AREA OF FUNGI

• simple way to assess growth of fungi is to measure diameter of patches of mycelium

• can be used to monitor growth rate in diff conditions

• could be cultured at diff temps and after a period of time, diameters of each final colony measured

• mean colony diameter could be calculated and temp which results in the largest one is he optimum growth temp

• technique could be used on bacteria but bc small, colonies can be difficult to measure

FUNGAL DRY MASS

• another way to find optimum conditions

• best done using liquid growth medium

• samples of broth removed at regular intervals and fungi separated from liquid by centrifugation or filtering

• material then dried to the point that no more loss of mass is recorded e.g. in an oven overnight at around 100C

• gives a measure of dry mass in a certain volume of culture

• increase or decrease in this mass indicates an increase or decrease in mass of mycelia