#1 Science Forum For Lab Technicians

Hi All,

I'm not sure if I read about agar plates and sterilising in the microwave on this site last year.
I was wondering if anyone can remember talking about this. I have gone into the forum, but can't seem to find anything
I hate it when you can't remember where you read or heard good ideas. :-k
But most of the great ideas I have implemented have come from this site.
Keep up the great work.

Di

I too would be most interested in read how people do their agar plates.

Regards

Hi all

My question about Agar plates is; whats the secret to making up good nutrient agar plates. My petri dishes were brand new I used the following recipe: 3gm beef extract, 5gm peptone, 15gm agar, 1L distilled water. I tried to be so careful when pouring the agar into the plates but it looks like there are spores on them. Is there a trick to getting good agar plates?

Cheers Nicky

Nicky

Did you sterilise the agar before you poured the plates? I use an old pressure cooker and 500ml glass bottles (Schott Duran Reagent bottles) but you should only half fill them and loosen the lids before hand. Thats the recipie that i use as well

Andrew

A great way to keep plates clean is to pour in a still air chamber. A cheap and easy version of this is a fish tank - clean well, wipe out with ethanol, roll it over on its side so you can pour the plates while looking through the glass top. If you sterilize your agar, pour in a still air chamber, use good aseptic techniques, contamination is vertually nil. Works a charm.

cheers Kim

Hey Nicky are you sure they are not lumps of undisolved agar? I always sprinkle in agar till no more will disolve then skim surface to remove any lumps

Thanks guys I will take all you suggestions on board. Gotta love this website.

Nicky

hi all

Treat all cultured organisms as potentially pathogenic and therefore a potential source of infection.

Avoid contact between the hands and the mouth and other skin surfaces, e.g. touching the plate with fingers or pencils or pens, or touching an un sterilized loop.

Hands must be washed before leaving the laboratory.

EXPOSURE OF AGAR PLATES

• Agar plates should not be exposed in situations where pathogenic organisms may exist, e.g. toilets, near persons coughing or sneezing etc.
• Once agar plates have been exposed or inoculated they must be taped securely closed. Do not remove the tape from plastic Petri dishes and only remove the tape from glass dishes after they have been sterilized.

STERILISATION

• Inoculating loops should be heated to red-hot before and after use.
• Equipment and specimens may be sterilised by autoclaving or heating in a pressure cooker for 15 minutes.
• All equipment and work surfaces must be disinfected after use.

DISPOSAL
• Plastic Petri dishes must be autoclaved or sterilized in a pressure cooker before disposal.
• Glass Petri dishes and culture tubes must be sterilised before cleaning.
• Unsterilised cultures must not be disposed of down the sink.
• Sterilised culture wastes may be disposed of in the industrial waste bin.

Hi again,
i copy this from internet, i use this website a lot
http://www.sciencestuff.com/nav/instructions/agar1.htm

Agar is a gelling agent extracted from red seaweed. Nutrient agar is a commonly used food medium for microbial cultures. Nutrient agar contains:
beef extract (provides carbohydrates, nitrogen, vitamins, salts)
peptone (helps control pH)
agar (a carbohydrate used as a solidifying agent)
distilled water (an agent for distributing food materials to growing colonies of micro-organisms)
Materials: Agar Powder
distilled water
flask or beaker
glass stir rod lab thermometer
sterile petri dishes (plastic)
flame or boiling mixture
heat resistant hand protection

Note: Keep sterile petri dishes closed until ready to pour agar into them.
Air-borne contaminants can easily invade an open petri dish.
Procedure:
Measure agar and distilled water into clean flask or beaker.
Recipe: Agar + Distilled Water = Yield Agar
23 g
11.5 g
9.2 g
4.6 g + Distilled Water
1000 ml
500 ml
400 ml
200 ml = Yield
50 plates
25 plates
20 plates
10 plates

Flame sterilize a clean glass stir rod to stir the medium as it melts.
While wearing heat resistant hand protection, hold the flask or beaker over the flame. Swish or stir the mixture constantly while heating.
Boil the mixture for 1 minute. Remove from heat.
Place a sterile lab thermometer. in the mixture and monitor the temperature until it falls to approximately 45 - 50° C or if a lab thermometer is not available, cover and let stand a few minutes.
Pour enough melted agar into each sterile plastic petri dish to cover the bottom - about 1/8" to 1/4" deep. Replace the lid immediately.
Place agar plates on a counter top to cool and set. Agar medium will set like stiff gelatin at room temperature.
The agar medium is now ready for storage or use.
Storage: Stack agar plates upside down in the refrigerator. Do Not Freeze! The purpose of placing the plates upside down is to prevent condensation from dripping down onto the agar surface which could then facilitate movement of organisms between colonies.

Preparing the Plates
If plates have been refrigerated, set them out and allow them to warm to room temperature.
Sterilize the loop
To sterilize the loop, hold the handle with a pot holder and place the tiny looped wire in a flame until it turns bright red
Allow the loop to cool for 3 - 5 seconds before touching the collection area.
Resterilize the loop after each inoculation.
Do not allow the loop to touch any surface other than the collection area and the agar.
Uncover each agar plate just long enough to inoculate the medium. hold the petri dish lid directly over the petri dish (or tilt the lid just enough to allow the loop inside) while inoculating the medium to help prevent contamination from air-borne particles. Do not allow the loop to touch the petri dish.
Do not Dip the loop in the agar, let it glide over the surface.
Make a pattern of inoculation lines (parallel lines, tic-tac-toe, zig-zag, initials, etc.) to help determine that what is growing is what you put there and not an air-borne contaminant.
Place the cover back on the plate immediately.
Incubation:
Turn the plates upside down and put them in a warm place. The ideal temperature for incubation is 32° C or 90° F. Bacterial growth should start to become visible in about 2 -3 days

Thanks everyone,

What a great response - I'm looking into them all.

Jazz wrote:• Once agar plates have been exposed or inoculated they must be taped securely closed. Do not remove the tape from plastic Petri dishes and only remove the tape from glass dishes after they have been sterilized.

Just to clarrify: "taping securely closed" does not mean airtight. That will kill your areobic culture durring the experiment, and encourage growth of anaerobic pathogens instead; these are not wanted in schools nor waste.

The culture lid just needs to be secured firmly so that it does not come off by accident or studepidity. Two or three strips running across the edge (from top to bottom, not along the rim), so that the lid is held tightly to the dish, are sufficient for the task. Air will diffuse through the gap, but spore migration is blocked.

A strip of Parafilm is excellent for sealing plates as it stretches as you slowly pull it around the edge of the plate.

JudyM wrote:A strip of Parafilm is excellent for sealing plates as it stretches as you slowly pull it around the edge of the plate.

Much more to the point is that, quite unlike Cellotape ("sticky" tape), Parafilm is specifically designed to allow oxygen to difuse across its membrane while sealing in water vapour and volatile organic gasses. So, okay, a Parafilm seal can be used to keep aerobic cultures from drying out, if that proves a problem in drier seasons.

However, do note that it is not safe to autoclave Parafilm and it must be removed prior to sterilization.