Isolation technique with aseptic techniques to cultivate bacteria

Prepared BYMs.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

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

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

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 plateObjective 1: How to prepare a Petri dishes

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?

Culture media

Plate

Slant

Broth

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

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

Bacteria motility

Non motile bacteria will only be found at the site of inoculation

Motile bacteria swim around go everywhere

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

Deep observation

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

Slant observation

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

Broth observation

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

4.5 Environmental Factors that Influence Microbial Growth