Cultures Flashcards
reasons for culturing microorganisms
cant see with naked eye
-investigating to diagnose disease or scientific experiment
what does culturing involve
growing large numbers of microorganisms so they can be measured in some way
conditions for culturing microorganisms
provided with right level of nutrients and oxygen with ideal pH and temperature
why should you take care when culturing microorganisms
-even if microorganisms are planning to culture harmless there is risk of mutant strain arising that may be pathogenic
-risk of contamination of culture by pathogenic microorganism from enviorement
-when you grow strain of microorganism, entry of other microorganisms from air or skin into culture contaminate it
health and safety conditions for culturing organisms
-all equipment must be sterile
-once culture has grown it doesnt leave lab
-all cultures disposed of safely be sealing them in plastic bags and sterilising at 121 for 15 mins under high pressure
-no ethical issues of handling microorganisms but danger of infecting other people with pathogens risk
steps of culturing
-decide on microorganisms you want to culture and obtain them
-need nutrients to grow
-good source of carbon and nitrogen
-nutrient medium can be broth or agar
-solid and liquid media kept sterile until ready for use
what is agar jelly
jelly extracted from seaweed
why is agar jelly useful
although it sets as jelly at 50 degrees id doesnt melt again until heated to 90 degrees
what microorganisms need to grow in culture
-some can grow on pure agar
-most need added nutrients
-medium enriched with protein like blood or meat extract or yeast extract
selective medium
-medium in or on which only select group of microorganisms with those particular requirements will grow
why is selective media important
important in identifying particular mutant strains of microorganism and antibiotic resistance
YM media
low pH which encourages growth of fungi and moulds but discourages bacteria
MacConkey agar
designed to grow gram negative bacteria
how can selective media identify microorganisms that have been genetically modified
-because antibiotic resistance or requirement for particular nutrient engineered with desired gene as marker
2 steps for preparing culture
-once suitable medium prepared introduce microorganisms
inoculation
getting bacteria onto agar and into broth
whats an inoculating loop
scraping off bacteria from one solid media surface either into liquid medium
streaking across another solid medium plate
inoculating broth
-involves making suspension of bacteria to be grown and mixing in known volume with sterile nutrient broth in flask
what to do with flask after inoculation
-stoppered with cotton wool quickly to prevent contamination from air and labelled to make sure broth is aerated allowing oxygen to growing bacteria
inoculating solid media
sterilise inoculating loop by holding it in bunsen burner until glowing hot read and leave it to cool
-dip loop in suspension of bacteria- streak loop across surface of agar avoiding digging into agar
-replace petri dish lid, tape closed and label and turn dish upside down
how to to get pure culture of single type of organism
isolation
pure culture with and without oxygen
-anaerobic conditions ensures only anaerobic bacteria will survive
-growing organisms with oxygen means only aerobic organisms can survive
-some bacteria may grow under both conditions
-not allow to complete separation of microorganisms necessary for pure culture but reduced variety
growing pure cultures and nutrients
produce medium for organism desired and inhibit growth of others
-allows identification of colony you want to re-inoculate for single pure culture
-need to control range of nutrients available or introduce selective growth inhibitors or antifungal chemicals that will reduce growth of microorganisms
indicator media for pure cultures
-indicator media that cause certain bacteria to change colour
-colonies that change colour or dont can be isolated and cultured
what is it important to isolate biologically
-isolate disease causing organisms from those of normal body flora
-so diagnosis can be made and treatment planned
how to count bacteria and single celled fungi cultured in nutrient broth
-directly using microscope and a haemocytometer
whats a haemocytometer
-has specialised thick microscope slide with rectangular chamber that holds standard volume of liquid 0.1mm3
-chamber is engraved with a grid of lines
how to count cells with haemocytometer
grid has square divided into 16 smaller squares
-number of cells in each of four sets of 16 counted and mean taken
haemocytometer being calibrated
number of bacterial or fungal cell in one set of 16 squares equated to number of cells of broth
-enables you to calculate number of microorganisms in standard volume of broth
turbidimetry
specialised form of colorimetry
turbid
cloudy looking as number of bacterial cells in culture increase
what happens as solution becomes more turbid
absorbs more light so less light passes through it
function of colorimeter
-measures how much light passes through sample showing how much light is absorbed and shows how many microorganisms present
how is a calibration curve produced
-by growing a control culture and taking samples at regular time intervals
what can the calibration curve be used for
we can measure the number of microorganisms by using turbidimetry
-also can investigate the effect of different conditions on growth rate of microorganism
