Cultivation of bacteria by mikesanye

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									Aseptic techniques and media
  used for bacteria culture

             Lab done over 2 days.
Using sterile techniques

   Bacteria are everywhere
   Media used for bacteria growth  welcoming
    for many bacteria
   We only want specific ones to grow **
    Sterile technique s**
   Sterile remain sterile as long as doesn’t
    touch anything that isn’t sterile
   Also avoid prolonged exposure to air
Sterile techniques: what can you
do in the lab?

   Wash your hands
   Keep your bench clean
   Wear gloves
   Flame loop, neck of tube
   Keep cap facing down
   Work quickly albeit efficiently
   Limit talking when opening

Autoclaving                                                         before
   Apparatus used to
    sterilize liquid and
   Heating up to 121oC at
    15 psi for 15 minutes
   Kill most microbe
   Autoclave tape 
    chemical reaction 
    black stripes if
    autoclaving ok
        Culture media


Slant                                                                           Deep

Bacteria colonies

Composition of media

   NA = Nutrient Agar
    –   peptone, beef extract, salt, agar 1.5%
   TSA = Tryptic soy agar
    –   Peptone from casein, peptone from soymeal,
        sodium chloride, agar 1.5%
   Many other medias available. These 2 will be
    used very often in this lab
   Note: Peptone: enzymatic digest protein
Few notes on agar

   Not degraded by most bacteria
   Is liquified at 100oC and remain liquid until
    about 40oC
   If added to growth medium  medium
    becomes solid
   Semi solid media: 0.5% agar
   Broth: no agar
   Solid media: 1.5-3% agar
How to prepare a Petri plate

   Take liquid agar (in the water bath)
   Pour aseptically into the base of the Petri
    plate (top is larger than the base)
   Wait until solidify (15 minutes)  invert
   ***Plates are kept inverted so condensation
    does not drip onto the agar
Objective 1: How to prepare a Petri dishes
Pouring a plate

Objective 2: Cultivate bacteria sample from the
Pouring a plate                                               Close
Every student: 2 plates
                                                              Open= Environmental

Objective 3:a How to inoculate a plate

   Plate: provide large surface for
    isolation and observation of
   Using a sterile loop or a sterile
    swab streak your sample on
    the petri plate
   Important let your sterilized
    loop cool before you pick up
    your sample
Observation of your plate (Next lab)

   You will see individual colonies (hopefully!)
   Describe using the following criteria:
    –   Colony shape
    –   Elevation
    –   Color
    –   Texture
Some vocabulary for colonies

   Shape: round, irregular, punctiform (tiny dot)
   Elevation: convex, umbonate, flat, raised
   Color: translucent, shiny, dull, white
   Texture: moist, mucoid, dry (or rough)
Colonial morphology

 Margin- edge


   The best descriptions are complete
    –   But short and concise
   Someone else should be able to imagine
    with accuracy what you saw by reading your
How to open a tube

   Hold the loop like a pencil
   Curl the little finger of the same hand around the cap
    of the tube
   Turn the tube with the other hand
   Remove the cap (keep in your hand)
   Flame the opening of the tube
   Remove samples with loop
   Flame the opening of the tube & replace the cap
Objective 3:b How to inoculate a deep

   Semi-solid media (0.5% agar)
    –   Oxygen gradient in the tube
    –   Can be used to look at bacteria motility
   Sterilize the needle (until red hot)  wait a
    few seconds  pick your sample stab the
    needle in the middle of the deep and remove
    it through the same stab
   Do not use a loop to inoculate the deep*
Bacteria motility

   Non motile bacteria will
    only be found at the site
    of inoculation
   Motile bacteria  swim
    around go everywhere

Oxygen requirement
                      No growth
   Oxygen gradient
    throughout the tube
   Not all bacteria like all
    oxygen concentration
   Some needs a lot of growth
    oxygen other are killed
    by it

Deep observation

Objective 3:c How to inoculate a slant

   Provide a solid growth
    surface in a tube format
    (take less space)
   Inoculate as you did for
    the petri plate
   One streak in the
    middle of the surface
    do not dig/ nor stab.
    Only on the surface.
Slant observation
Objective 3:d How to inoculate a broth

   Take a loopful of bacteria with a sterilized
   Transfer into a new tube
   Sterilize the loop prior to put back
   Sterile technique
Broth observation   .jpg

   Broth                          Plate
    –   High concentration of       –   Individual colonies
        bacteria                    –   Can be used to count
   Slant                          Deep
    –   Space saving solid          –   Look at motility & oxygen
        culture                         requirement

   Cultivate a bacterial sample from the environment. Incubate
    27o C
   Inoculate the pure culture provided
    –   Into a broth
    –   Into a slant
    –   Into a deep
    –   Into a Petri plate
    –   *** Using aseptic techniques ***Put all the above in the 37oC
   Describe colonial morphology from a Petri plate and a slant
   Identify growth pattern in broth and deep

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