1. microbial techniques

CULTURING MICROORGANISMS

• most microbes can’t be seen by naked eye

• to investigate microbes we need to culture them

• culturing- to grow them in large numbers so they can be measured in some way

• need to be provided w/ nutrients and O2 and have right pH levels and temp for growth

CULTURING MICROORGANISMS- PRECAUTIONS

• equipment sterile before culture started

• once cultures are made they can’t leave lab

• must also be disposed of by sealing them in plastic bags and sterilising at 121˚C for 15 mins under high pressure, before throwing them away- done in an autoclave

• no ethical issues associated with culturing microbes but the danger of infecting people w/ pathogens should always be considered

CULTURING MICROORGANISMS- WHY PRECAUTIONS

• always the risk that a mutant strain could arise which is pathogenic even if microbe is harmless

• risk of contaminating culture w/ pathogenic microbes from env

• when you grow a pure strain of microbe, entry of others from your skin or equipment will contaminate it

ASEPTIC CULTURE TECHNIQUES- MICROBE REQUIREMENTS

• some microbes will grow on pure agar but most need added nutrients

• most need good source of nitrogen, carbon and specific minerals

• most grow when a medium is enriched with a good source of protein like blood or yeast extract

• by producing medium w/ specific ingredients you provide a selective medium- one which only a select group of microbes will grow in

  • important in identifying mutant strains and those bacteria with antibiotic resistance

ASEPTIC CULTURING TECHNIQUES- STEPS

• nutrient medium can be in form of nutrient broth (liquid) or nutrient agar (solid)

• agar is jelly extracted from seaweed- useful as although jelly sets at 50˚C it doesn’t melt until 90˚C

• both media must be kept sterile until ready to use

• once medium is ready, then introduce your microbes e.g. bacteria

• can scrape bacteria from a source or dip loop into a suspension of bacteria

• streak microbe onto agar or into broth- inoculation

GROWING A PURE CULTURE

• to grow a pure culture, isolate organism by:

  • growing microbes under anaerobic conditions- only anaerobic bacteria will survive(same could be done w/ aerobic conditions)- but some can respire in both ways

  • use specific nutritional requirements- produce medium which favours microbes you want to grow and inhibit growth of ones you don't- could also use growth inhibitors, antibiotics or antifungals to do this

  • use indicator media- can cause certain types of bacteria to change colour- ones wanted can then be isolated and cultured

POUR PLATE- WHY USED

• often used to count no. of microorganisms in a mixed sample

• used to perform viable plate counts which let us create growth curves and calculate conc of cells in the tube a sample was plated from

POUR PLATE- TECHNIQUE

• molten agar inoculated before it solidifies

• molten agar should be cooled to 44˚C before plating otherwise it may kill organism

POUR PLATE- RESULT

• colonies uniformly distributed throughout solid medium when appropriate sample dilution is plated

SPREAD PLATE- WHY USED

• also is used to count no. of microorganisms in a mixed sample

• used to help identify microorganisms based on how colonies look

• can add antibiotic etc. to plates to look at selective growth

SPREAD PLATE- TECHNIQUE

• culture is uniformly spread over surface of an agar plate

• if not diluted enough, a lawn of bacteria is produced

• antibiotics or agents can then be added to ‘clear’ zones so their effectiveness can be estimated

SPREAD PLATE- RESULT

• isolated colonies distributed evenly across agar surface if appropriate conc of cells is plated

STREAK PLATE- WHY USED

• used for obtaining a pure culture from a mixed culture or microbes

STREAK PLATE- TECHNIQUE

• inoculating loop is used to add streaks of a culture onto plate

• plate is quarter turned after each streak set

• each new direction streak passes through older streak so sample is diluted as you go

STREAK PLATE- RESULT

• if original culture contained more than one bacterial species, colonies of each individual one can be identified and removed