Bacterial identification
plating
streaking
how to inoculate
how to observe
Using sterile techniques
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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:
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Wash your hands
Keep your bench clean
Wear gloves
Flame loop, neck of tube
Keep cap facing down
Work quickly and efficiently
Limit talking when opening
cultures
http://www.parentsguidecordblood.com/vita34cleanroom50.jpg
Autoclaving
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before
after
Apparatus used to
sterilize liquid and
instrument
Heating up to 121oC at
15 psi for 15 minutes
Kill most microbe
Autoclave tape 
chemical reaction 
black stripes if
autoclaving ok
http://tea.armadaproject.org/Images/stoyles/stoyles_before_autoclaveJPG.JPG.jpg
http://www.sterilizers.com/Images/Tecno-gaz-autoclave.jpg
http://www.fibermark.com/images/prod_imgs/ds_m_01_Autoclave2.jpg
Inoculation of bacteria in to
Culture media
Plate
Broth
Slant
Deep
http://student.ccbcmd.edu/courses/bio141/lecguide/unit2/control/images/broth.jpg
http://www.mushmush.nl/images/methods/working_with_agar/slant.jpg
http://82.43.123.182/globalplantclinic/images/Bacteria_plate.jpg
http://student.ccbcmd.edu/courses/bio141/labmanua/lab7/images/negmotility.JPG
Uses
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Broth
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High concentration of
bacteria
Slant
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Space saving solid
culture
Plate
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Individual colonies
Can be used to count
bacteria
Deep
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Look at motility & oxygen
requirement
Pure vs mixed culture
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Pure: originate from 1
bacteria strain
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All colonies look the
same
Mixed: originate from
many bacteria strains
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Colonies have different
size/shape
http://smccd.net/accounts/case/biol240/streakplate.html
Bacteria colonies
http://textbookofbacteriology.net/growth.html
Composition of media
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NA = Nutrient Agar
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peptone, beef extract, salt, agar 2%
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Many other medias available. These 2 will be
used very often in this lab
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Note: Peptone: enzymatic digest protein
Few notes on agar
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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: 2% agar
How to prepare a Petri plate
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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
Pour plate method
Pouring a plate
http://www.biotopics.co.uk/microbes/pourp2.gif
Objective 3:a How to inoculate a plate
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Plate: provide large surface for
isolation and observation of
colonies
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
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You will see individual colonies (hopefully!)
Describe using the following criteria:
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Colony shape
Elevation
Color
Texture
http://faculty.mc3.edu/jearl/ML/ml-9.htm
http://www.bact.wisc.edu/themicrobialworld/Prop.acnes_colonies.jpg
Colonies morphology
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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
http://www.rlc.dcccd.edu/mathsci/reynolds/micro/lab_manual/Coloniy_morph.jpg
How to open a tube
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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
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Semi-solid media (0.5% agar)
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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
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Non motile bacteria will
only be found at the site
of inoculation
Motile bacteria  swim
around go everywhere
http://www.bact.wisc.edu/bact100/Motility.jpg
Objective 3:c How to inoculate a slant
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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. on
the surface.
http://www.liddil.com/beer/culture/slant.gif
Slant observation
http://www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/lab_manual/slant_patterns.jpg
Broth observation
http://www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/lab_manual/broth_patterns
.jpg