I have had trouble preparing nutrient agar so would appreciate if anyone has some tips they could give me. I have also seen a suggestion to use potato slices, has anyone else used this technique?
By the way I work at a school in a remote area of WA so I really appreciate having a forum where I can ask questions and just generally comminucate with other Lab tech's
Thanks
Nikki
Nutrient Agar
Hi Nikki
I use this method all the time now and never have a problem. I measure out the quantiy of water required into a large beaker and then add a little of this into a paper cup which already has the measured quantiy of agar in it.I then add a small amount of the water to make a smooth paste. After boiling the larger beaker of water, I then add some of this to the agar paste in the paper cup and give a good stir before pouring it all back into the large beaker of water and continue heating and stirring for about 5 mins. Sorry, I am not in the lab at the moment so I can't give you the exact quantities that I use. I hope this helps.
I use this method all the time now and never have a problem. I measure out the quantiy of water required into a large beaker and then add a little of this into a paper cup which already has the measured quantiy of agar in it.I then add a small amount of the water to make a smooth paste. After boiling the larger beaker of water, I then add some of this to the agar paste in the paper cup and give a good stir before pouring it all back into the large beaker of water and continue heating and stirring for about 5 mins. Sorry, I am not in the lab at the moment so I can't give you the exact quantities that I use. I hope this helps.
-
rae
- Posts: 1045
- Joined: 31 May 2006, 10:00
- School: Oxley College
- Suburb: Burradoo
- State/Location: NSW
Nutrient Agar
Hi
I made some nutrient agar recently and it seemed to work well.
I used a recipe out of a folder called Job Skills for school assistants working in Science.
The recipe is: 15g agar, 5g peptone or 3g of alternative nutrient (I used beef stock) 1000ml of distilled H20. (I didn't need a litre, so i just made up the quantity I needed
I weighed out the dry ingredients and put them in a beaker and then added the water.
I then heated this over a bunsen until it was just about boiling, stirring all the time.
I then transferred this into a conical flask and plugged the top with cotton wool and covered this with foil and sterilised it in a pressure cooker.
I then poured the plates when the agar was still quite hot.
Our year 12 bioloogy classes grew some lovely bacteria and fungi onthis.
Hope this is of some use.
Lorrae
I made some nutrient agar recently and it seemed to work well.
I used a recipe out of a folder called Job Skills for school assistants working in Science.
The recipe is: 15g agar, 5g peptone or 3g of alternative nutrient (I used beef stock) 1000ml of distilled H20. (I didn't need a litre, so i just made up the quantity I needed
I weighed out the dry ingredients and put them in a beaker and then added the water.
I then heated this over a bunsen until it was just about boiling, stirring all the time.
I then transferred this into a conical flask and plugged the top with cotton wool and covered this with foil and sterilised it in a pressure cooker.
I then poured the plates when the agar was still quite hot.
Our year 12 bioloogy classes grew some lovely bacteria and fungi onthis.
Hope this is of some use.
Lorrae
If you buy premixed nutrient agar, make it up as follows.
28g nutrient agar powder/litre boiling distilled water. I usually make up 500ml at a time.
Boil the water first then add the agar powder, stirring vigorously. A magnetic stirrer is invaluable here. Boil for a further few minutes.
After sterilising in a pressure cooker at 15psi (121C, 100kPa) for 30 mins, cool to about 60C then pour the plates.
An alternative recipe is
10g beef extract e.g. Bonox
10g peptone
5g sodium chloride
15g agar powder
1 litre distilled water
Dissolve first three ingredients in the distilled water by heating. Adjust the pH to 8-8.4 with 0.1M sodium hydroxide. Boil for 10 mins, filter and adjust the pH to 7.2 with 0.1M hydrochloric acid. Add the agar powder, stirring vigorously. Sterilise as for nutrient agar above.
The last recipe was taken from a brilliant aid for lab. techs - 'The Laboratory, a Science reference & preparation manual for schools' by Barbara Dungey, ISBN 0 646 38578 X
28g nutrient agar powder/litre boiling distilled water. I usually make up 500ml at a time.
Boil the water first then add the agar powder, stirring vigorously. A magnetic stirrer is invaluable here. Boil for a further few minutes.
After sterilising in a pressure cooker at 15psi (121C, 100kPa) for 30 mins, cool to about 60C then pour the plates.
An alternative recipe is
10g beef extract e.g. Bonox
10g peptone
5g sodium chloride
15g agar powder
1 litre distilled water
Dissolve first three ingredients in the distilled water by heating. Adjust the pH to 8-8.4 with 0.1M sodium hydroxide. Boil for 10 mins, filter and adjust the pH to 7.2 with 0.1M hydrochloric acid. Add the agar powder, stirring vigorously. Sterilise as for nutrient agar above.
The last recipe was taken from a brilliant aid for lab. techs - 'The Laboratory, a Science reference & preparation manual for schools' by Barbara Dungey, ISBN 0 646 38578 X
Nutrient Agar
Hi Nikki
Some where in your Lab should be a large grey file, "Science Laboratory Manual" put together by our Regional Technicians group here in WA. In it is a recipe for nutrient agar I have used regularly for 12 years. It is also full of just about everything else you will need to make your life easier. If you can't find this file, would like to know more about our Regional Technicians group, their web site or would just like to bounce some ideas off someone, you can contact me at John Willcock College in Geraldton on 99 658 316.
Bye
Diana
Some where in your Lab should be a large grey file, "Science Laboratory Manual" put together by our Regional Technicians group here in WA. In it is a recipe for nutrient agar I have used regularly for 12 years. It is also full of just about everything else you will need to make your life easier. If you can't find this file, would like to know more about our Regional Technicians group, their web site or would just like to bounce some ideas off someone, you can contact me at John Willcock College in Geraldton on 99 658 316.
Bye
Diana
Agar V's Nutrient Agar
There are some good recepe's and good ideas here, but keep in mind though, that Nutrient Agar can be as much as 5 times more expensive than that of Agar or Agar-Agar.
Dr Robert Crosdale. MRACI. NSS. NSSA. NASA.
Ph.D (Chem), Post Grad Ph.D (Physics), M.Ed, B.Sc (Hons), Dip. Appl. Sc. (Chem)
Lake Munmorah High School.
University of New England.
University of New South Wales.
University of Newcastle.
To understand the Universe from our perspective, we need to look towards our own backyard first for answers.
** AD ASTRA PER ASPERA - SEMPER EXPLORO **
Ph.D (Chem), Post Grad Ph.D (Physics), M.Ed, B.Sc (Hons), Dip. Appl. Sc. (Chem)
Lake Munmorah High School.
University of New England.
University of New South Wales.
University of Newcastle.
To understand the Universe from our perspective, we need to look towards our own backyard first for answers.
** AD ASTRA PER ASPERA - SEMPER EXPLORO **
Hi All,
Over the years I have made up much agar and found when you boil the water first then add the powder it often clumps and is harder to dissolve.
I have 2 methods which I use now. Either sprinkle the agar powder on to cold water and heat while stirring until it boils. A magnetic stirrer/hotplate is good for this. Alternatively at a microbiology session at a conference we were told the easiest way to make an agar solution is to put all the ingresidents in a conical flask, plug with cotton wool (non absorbent) and cover theis with al. foil the put it all into a pressure cooker and heat/sterilize in one.
I usually use plain agar and add some vegemite or bonox to it if I need nutrient agar. Works well and is much cheaper than buying nutrient agar.
Over the years I have made up much agar and found when you boil the water first then add the powder it often clumps and is harder to dissolve.
I have 2 methods which I use now. Either sprinkle the agar powder on to cold water and heat while stirring until it boils. A magnetic stirrer/hotplate is good for this. Alternatively at a microbiology session at a conference we were told the easiest way to make an agar solution is to put all the ingresidents in a conical flask, plug with cotton wool (non absorbent) and cover theis with al. foil the put it all into a pressure cooker and heat/sterilize in one.
I usually use plain agar and add some vegemite or bonox to it if I need nutrient agar. Works well and is much cheaper than buying nutrient agar.
Sue Henderson
Laboratory Technician
Cleeland Campus - Dandenong H.S.
Dandenong 3175
VIC
ph: (03) 8792 7200
fax: (03) 9791 3220
Laboratory Technician
Cleeland Campus - Dandenong H.S.
Dandenong 3175
VIC
ph: (03) 8792 7200
fax: (03) 9791 3220
Nutrient Agar
Hi Nikki
I have been using this recipe for 30 yrs it seems to work well.
20 gm Agar
5 gm Peptone
5 gm Na Cl
2 gm Vegemite
1 gm Beef Stock cube
I000 ml water.
If each plate holds 25ml you should get approx 40 plates.
Heat water, before it reaches boiling slowly add each ingredient and dissolve before adding each one , add agar last slowwly stirring constantly , bring to boil .
autoclave at 15 psi for 20 minutes.
Hope this helps
Michelle Howes
Holy Spirit College
Bellambi
NSW
I have been using this recipe for 30 yrs it seems to work well.
20 gm Agar
5 gm Peptone
5 gm Na Cl
2 gm Vegemite
1 gm Beef Stock cube
I000 ml water.
If each plate holds 25ml you should get approx 40 plates.
Heat water, before it reaches boiling slowly add each ingredient and dissolve before adding each one , add agar last slowwly stirring constantly , bring to boil .
autoclave at 15 psi for 20 minutes.
Hope this helps
Michelle Howes
Holy Spirit College
Bellambi
NSW
-
Mother
- Posts: 275
- Joined: 17 May 2006, 10:00
- Job Title: Science lab. technician
- School: Dubbo College/Senior Campus
- Suburb: Dubbo
- State/Location: NSW
petrie dishes
HI There
I am just wondering if you need to autoclave the nutrient agar if you use disposable sterilised petrie dishes???
I am just wondering if you need to autoclave the nutrient agar if you use disposable sterilised petrie dishes???
Thanks to everybody for your suggestions , I have a few to try now. The main problem I have is with sterilising as I dont have an autoclave. I have a pressure cooker, is this sufficient and what is the best way to do it in a pressure cooker. Is it safe to put something glass straight on the bottom of the pressure cooker which is then put on a gas flame.?
In regard to purchasing nutrient agar, cost is definatley a factor for us as we are a small district high and the budget I have to work with borders on the ridiculous especially with the price of chemicals and freight to get things up here. So that being said I am usually looking for the most cost effective ways of doing things.
, I have a few to try now. The main problem I have is with sterilising as I dont have an autoclave. I have a pressure cooker, is this sufficient and what is the best way to do it in a pressure cooker. Is it safe to put something glass straight on the bottom of the pressure cooker which is then put on a gas flame.?In regard to purchasing nutrient agar, cost is definatley a factor for us as we are a small district high and the budget I have to work with borders on the ridiculous especially with the price of chemicals and freight to get things up here. So that being said I am usually looking for the most cost effective ways of doing things.
Nikki,
using a pressure cooker for sterilization is fine. Heat it until the pressure builds up. If the pressure cooker has a guage it will usually show you what the pressure is. If it does not have a guage there is some other way of indicating pressure. Mine has a weight and a pop up indicator. Keep the pressure up for 15 to 20 minutes and anything will be sterilized. If you want more assurance of this you can purchase autoclave tape. Put some of this on the item and it will change colour (mine gets stripes on it) when sterilization has taken place.
I have a metal slotted shelf - for want of a better term, which came out of an old pressure cooker that is about 5mm high and sits in the bottom of the pressure cooker and then the items sit on that. I think sitting a glass item on the base may not be the best idea as sometimes "bumping" may occur during heating.
A tip which I'm sure is well known - when sterilizing contaminated disposable petri dishes put them into an oven bag before putting in the pressure cooker. They will all melt into a mass which can then be wrapped and disposed of without any further handling.
using a pressure cooker for sterilization is fine. Heat it until the pressure builds up. If the pressure cooker has a guage it will usually show you what the pressure is. If it does not have a guage there is some other way of indicating pressure. Mine has a weight and a pop up indicator. Keep the pressure up for 15 to 20 minutes and anything will be sterilized. If you want more assurance of this you can purchase autoclave tape. Put some of this on the item and it will change colour (mine gets stripes on it) when sterilization has taken place.
I have a metal slotted shelf - for want of a better term, which came out of an old pressure cooker that is about 5mm high and sits in the bottom of the pressure cooker and then the items sit on that. I think sitting a glass item on the base may not be the best idea as sometimes "bumping" may occur during heating.
A tip which I'm sure is well known - when sterilizing contaminated disposable petri dishes put them into an oven bag before putting in the pressure cooker. They will all melt into a mass which can then be wrapped and disposed of without any further handling.
Sue Henderson
Laboratory Technician
Cleeland Campus - Dandenong H.S.
Dandenong 3175
VIC
ph: (03) 8792 7200
fax: (03) 9791 3220
Laboratory Technician
Cleeland Campus - Dandenong H.S.
Dandenong 3175
VIC
ph: (03) 8792 7200
fax: (03) 9791 3220
Hi Mother,
I beleive that you do need to sterilize the nutrient agar if you are using sterile disposable agar plates, because otherwise the nutrient agar will contain microbes from the distilled water and surrounding environment.
Also at a recent lab assistants meeting that I attended it was recommended that we do not sterilize the used agar plates using a pressure cooker due to the risk of aerosol microbes. Instead I now put them in a solution of cheap bleach (approximately 50:50) and water for a few days before I dispose of the plates.
I beleive that you do need to sterilize the nutrient agar if you are using sterile disposable agar plates, because otherwise the nutrient agar will contain microbes from the distilled water and surrounding environment.
Also at a recent lab assistants meeting that I attended it was recommended that we do not sterilize the used agar plates using a pressure cooker due to the risk of aerosol microbes. Instead I now put them in a solution of cheap bleach (approximately 50:50) and water for a few days before I dispose of the plates.
Quick and easy Agar plates
For the level of microbilogy we undertake in high schools I find the best method is for every 100ml of water add 1g agar pawder, 1.5 g nutrient broth and microwave it in a concile flask with some boiling chips on the bottom, 500ml flask for 300ml volume is best. This makes about 15 plates of small volume.
Start with cold deionised water and keep an eye on the flask, it will start bublining after 90 sec, and you want to stop start with the bubble until they become large.
Have had no proble with contanimation with this method for several years and we get good growth.
Start with cold deionised water and keep an eye on the flask, it will start bublining after 90 sec, and you want to stop start with the bubble until they become large.
Have had no proble with contanimation with this method for several years and we get good growth.
-
Ocean Breeze
- Posts: 798
- Joined: 01 Jun 2006, 10:00
- Job Title: Lab Manager
- State/Location: NSW
yes Sally,I agree.
For years I have only microwaved my agar .
My method is short 30 second bursts. After each burst, I swirl the flask each time to stir and disperse sediment evenly-until it warms up.
When its near to boiling point, plug loosely with cotton wool to prevent frothing up, (and preserve the sterility once its removed from the microwave.)and boil on lower power for a few mins.
I have found that it works well for high school use, and havent had any contamination issues.
No more burnt agar sticking to the bottom of the boiling flask and spoiling the batch, if I am juggling several tasks at once!
Be interested to know what your method is?
For years I have only microwaved my agar .
My method is short 30 second bursts. After each burst, I swirl the flask each time to stir and disperse sediment evenly-until it warms up.
When its near to boiling point, plug loosely with cotton wool to prevent frothing up, (and preserve the sterility once its removed from the microwave.)and boil on lower power for a few mins.
I have found that it works well for high school use, and havent had any contamination issues.
No more burnt agar sticking to the bottom of the boiling flask and spoiling the batch, if I am juggling several tasks at once!
Be interested to know what your method is?
Once again thank you to all for your suggestions I am going to give a few of them a go, particularly the microwave method as this sounds simpler than the pressure cooker.
Also Hi back to Sue S, I had seen your name around the lab and when I saw you on here I mentioned it to Susie so I will definately say Hi to her for you. By the way I am really enjoying working here, great bunch of people and I love getting to work with the kids.
Cheers
Nikki
Also Hi back to Sue S, I had seen your name around the lab and when I saw you on here I mentioned it to Susie so I will definately say Hi to her for you. By the way I am really enjoying working here, great bunch of people and I love getting to work with the kids.
Cheers
Nikki
Hi Y'all!
It's been interesting reading all the feedback on agar plates. I have some other ideas to add, which I've found very successful.
I used to pour the sterilised agar solution into the sterile petri dishes too soon. This causes BIG problems with condensation on the lids. Not a good thing later when you have all sorts of heebie jeebies growing. The thought of some of that dribbling out of the petri dishes makes the blood run cold, doesn't it!
The solution is quite simple. After you remove the flasks of the sterile agar solution from the pressure cooker, rest one of the flasks on the bulb of a thermometer. Pour into petri dishes only when the temperature has dropped to 50[sup]0[/sup] celsius. Voila! No more bad condensation!
Other good ideas to assist with keeping solution as sterile as possible when pouring: after pouring each plate, run the lip of the flask briefly through a bunsen flame before pouring the next one; keep the flask angled (don't hold upright); don't remove petri dish lid any more than you absolutely have to and pour/replace lid as quickly as possible. I also keep windows and doors shut to reduce air flow during the pouring process. I wipe the bench down with bleach and rinse my gloves in bleach as well. These pointers really do work because I almost never get any contamination in the plates.
After the plates have set, turn them upside down, so that any traces of condensation won't spoil the surface of the agar jelly. I seal them in a plastic bag - tightly wrapping the plastic bag, so that, if they are picked up carelessly, the lids won't come off. I seal the plastic bag with a wire twistem, date the bag, and store them in the fridge. I've kept them successfully for months, not days or weeks.
Regards,
DJ.
It's been interesting reading all the feedback on agar plates. I have some other ideas to add, which I've found very successful.
I used to pour the sterilised agar solution into the sterile petri dishes too soon. This causes BIG problems with condensation on the lids. Not a good thing later when you have all sorts of heebie jeebies growing. The thought of some of that dribbling out of the petri dishes makes the blood run cold, doesn't it!
The solution is quite simple. After you remove the flasks of the sterile agar solution from the pressure cooker, rest one of the flasks on the bulb of a thermometer. Pour into petri dishes only when the temperature has dropped to 50[sup]0[/sup] celsius. Voila! No more bad condensation!
Other good ideas to assist with keeping solution as sterile as possible when pouring: after pouring each plate, run the lip of the flask briefly through a bunsen flame before pouring the next one; keep the flask angled (don't hold upright); don't remove petri dish lid any more than you absolutely have to and pour/replace lid as quickly as possible. I also keep windows and doors shut to reduce air flow during the pouring process. I wipe the bench down with bleach and rinse my gloves in bleach as well. These pointers really do work because I almost never get any contamination in the plates.
After the plates have set, turn them upside down, so that any traces of condensation won't spoil the surface of the agar jelly. I seal them in a plastic bag - tightly wrapping the plastic bag, so that, if they are picked up carelessly, the lids won't come off. I seal the plastic bag with a wire twistem, date the bag, and store them in the fridge. I've kept them successfully for months, not days or weeks.
Regards,
DJ.
Hi
Great idea to use thermometer on flask, I have also always let flask cool to reduce condensation, but this much more exact. I use cling wrap to seal and store my up turned petri dishes. I agree with DJ on the time the plates will last. I often make my plates in a number of large batches at the beginning of term and find them still usable at the end of term.
Didee
Great idea to use thermometer on flask, I have also always let flask cool to reduce condensation, but this much more exact. I use cling wrap to seal and store my up turned petri dishes. I agree with DJ on the time the plates will last. I often make my plates in a number of large batches at the beginning of term and find them still usable at the end of term.
Didee
