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

2. 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

3. 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

4. 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

5. 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

6. Bacteria colonies

7. 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

8. 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

9. 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

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

11. 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?

14. Culture media
Plate
Broth
Slant
Deep

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

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

17. Colonial morphology
Margin- edge

18. 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*

20. Bacteria motility
Non motile bacteria will only be found at the site of inoculation
Motile bacteria  swim around go everywhere

21. 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

22. Deep observation

23. 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

25. Slant observation

26. 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

28. Broth observation

29. 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

30. 4.5 Environmental Factors that Influence Microbial Growth