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Bacterial identification plating streaking how to inoculate how to observe Using sterile techniques 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: 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 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 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 Pure vs mixed culture Pure: originate from 1 bacteria strain – All colonies look the same Mixed: originate from many bacteria strains – Colonies have different size/shape http://smccd.net/accounts/case/biol240/streakplate.html Bacteria colonies http://textbookofbacteriology.net/growth.html Composition of media NA = Nutrient Agar – peptone, beef extract, salt, agar 2% 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: 2% 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 Pour plate method Pouring a plate http://www.biotopics.co.uk/microbes/pourp2.gif Objective 3:a How to inoculate a plate 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 You will see individual colonies (hopefully!) Describe using the following criteria: – – – – 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 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 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 http://www.bact.wisc.edu/bact100/Motility.jpg 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. 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