Isolation technique with aseptic techniques to cultivate bacteria Prepared BY Ms.c: Abed Al Rahman I. Hamad

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 cultures

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

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

Bacteria colonies

Composition of media NA = Nutrient Agar TSA = Tryptic soy agar 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

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

Pouring a plate Objective 1: How to prepare a Petri dishes

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 Two method to isolate bacteria will discuss: Streak plate procedure Pour plate procedure How to inoculate a plate?

Culture media Plate Broth Slant Deep

Observation of your plate You will see individual colonies (hopefully!) Describe using the following criteria: Colony shape Elevation Color Texture

Some vocabulary for colonies morphology Shape: round, irregular Elevation: convex, flat, raised Color: translucent, shiny Texture: moist, mucoid, dry (or rough)

Colonial morphology Margin- edge

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 oxygen other are killed by it growth

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. . If you just look to character on slant

Slant observation

Objective 3:d How to inoculate a broth Take a loopful of bacteria with a sterilized loop Transfer into a new tube Sterilize the loop prior to put back Sterile technique

Broth observation http://www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/lab_manual/broth_patterns.jpg

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

4.5 Environmental Factors that Influence Microbial Growth