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Isolation technique with aseptic techniques to cultivate bacteria
Prepared BYMs.c: Abed Al Rahman I. Hamad
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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
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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
cultures
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Autoclaving
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
before
after
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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
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Bacteria colonies
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Composition of media
Agar is a polysaccharide obtained from marine algae,
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
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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
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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
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Pouring a plateObjective 1: How to prepare a Petri dishes
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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
Two method to isolate bacteria will discuss:
1) Streak plate procedure
2) Pour plate procedure
How to inoculate a plate?
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Culture media
Plate
Slant
Broth
Deep
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Observation of your plate
You will see individual colonies (hopefully!) Describe using the following criteria:
– Colony shape– Elevation– Color– Texture
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Some vocabulary for colonies morphology
Shape: round, irregular Elevation: convex, flat, raised Color: translucent, shiny Texture: moist, mucoid, dry (or rough)
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Colonial morphology
Margin- edge
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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 samesame stab
Do not use a loop to inoculate the deep*
Objective 3:b How to inoculate a deep
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Bacteria motility
Non motile bacteria will only be found at the site of inoculation
Motile bacteria swim around go everywhere
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Oxygen requirement
Oxygen gradient throughout the tube
Not all bacteria like all oxygen concentration
Some needs a lot of oxygen other are killed by it
No growth
growth
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Deep observation
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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 Only on the surface.
. If you just look to character on slant
Objective 3:c How to inoculate a slant
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Slant observation
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Take a loopful of bacteria with a sterilized loop
Transfer into a new tube Sterilize the loop prior to put back Sterile technique
Objective 3:d How to inoculate a broth
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Broth observation
http://www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/lab_manual/broth_patterns.jpg
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Uses
Broth– High concentration of
bacteria
Slant– Space saving solid
culture
Plate– Individual colonies– Can be used to count
bacteria
Deep– Look at motility & oxygen
requirement
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4.5 Environmental Factors that Influence Microbial Growth