Technicians Mailing List Requests

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ACID RAIN

Hi AllCan anyone tell me the rough strength of Acid rain, which I remember vaguely to be fairly dilute sulfuric acid. Sandra McGregor

An article on acid rain.

says it is about the same pH as vinegar and lemon Juice.Nicola Younger

ACIDIFYING POTASSIUM PERMANGANATE

. I want to know how you acidify KMnO4. How much acid to how much solution? Bronwyn Beukes

I use a 1 molar solution of sulfuric acid as the liquid (no water). i.e For a 1M solution of KMnO4, I dissolve 158g of KMnO4 in 1 litre of 1M H2SO4. Lynette Breen

We have stopped acidifying our KMnO4 solution because we found acidifying it produced a brown sediment, presumably some type of Manganese Oxide. We found the un-acidified 0.02M KMnO4 solution was very stable and gave excellent redox titration results in class. We dissolved the KMnO4 in distilled water then brought the solution to the boil and let it sit overnight before filtering it through glass wool, as it reacts slightly with filter paper. I did some research on this early last year after having problems withconsistency in our lab practicals and found that KMnO4 should not be acidified as it is much more stable un-acidified.

When making up the approximately 0.05 mol per litre solution of Fe, we acidify it to approximately 0.8 mol/L H2SO4. We get excellent titration results when titrating this with 0.02 mol per L KMnO4. Stan Augustowicz

Stan is right on the nail with this. I worked as an analyst, and we never added acid to our permanganate, except in the reaction flask. Whereas some reagents are much more stable if acidified e.g. Iron (II) sulfate, permanganate is much less so, and should not be acidified if required to be stored, or for quantitative work.

Later Absolutely! Potassium permanganate should not be made up with acid, as it decreases its stability, whereas the Iron (II) solution will only keep well if it does have acid added. So you have 2 good options: put all the sulfuric acid you need in the titration into your iron (II) solution when you make it up (which gives it a long shelf life of some months at least), or add at least a minimum of sulfuric when you make it up, and add the rest to the titration flask. Generally the approach has been to add extra acid to the titration flask, by adding, say 25ml of 2M H2SO4 each time. However, one of our teachers was keen to put the sulfuric acid all in the iron solution, so I have done that also. It wasn't a solution I enjoyed making up 20L of, I have to say. Ian de Stigter

Our Year 13 Chem students are doing redox titrations. The experiment requires 0.02M potassium permanganate solution which is acidified with sulfuric acid. They are titrating an iron (II) solution accurately weighed for standardisation purposes which is made up to 250mls with 20ml of 1M sulfuric acid included. Is it possible to not acidify the KMnO4, and possibly add some more sulfuric acid to the conical flask prior to the titration? My concern is that the unused KMnO4 solution will go off after awhile and be wasted. Lesley Higham

The recipe I use for acidified potassium permanganate requires it to be made up with equal volumes of water and 2mol.L sulfuric acid. ie if you are making 1 Litre , 500mL of water and 500mL of sulfuric acid

Kay McArdle

CLEAPSS recipe book:

Also known as potassium permanganate. Solutions must be acidic. Solutions are safer made up in dilute sulfuric(VI) acid rather than adding concentrated sulfuric(VI) acid at a later stage.(1M for 0.1M KMnO4, 0.1M for 0.02M or lower)Solutions do not keep well unless the container is scrupulously clean. They slowly reacts with water, forming manganese(IV) oxide which badly stains glass and plastic equipment. Solutions are best kept in dark bottles, shielded from light. Light increases the rate of decomposition and staining of equipment.. Use a 0.002 M solution for carrying out tests for unsaturation in alkenes. It is not possible to make a 1 M solution.

AGAR PLATES

What recipes do people use for making agar?.

Per 100ml water add2g agar, 2g glucose, trace of marmite.

Stand for 10 minutes. Boil until dissolve. Cool to 600C

I make my nutrient agar as follows Agar 3.75g, Peptone 1.25g,

Vegemite 0.75g, salt 1.25g.

Dissolve in 250ml hot water. Place in pressure cooker for 10-15 min. This makes 15-20 plates depending on how thick you pour and I tend to use as little as possible.

Microwave sterilization (I have used this method for the last 2-3 years)

For 500ml agar culture media.

Weigh out your chemicals into 2 x 500ml flasks check that they fit into the microwave. Get 2 stirring rods one for each flask and place them in the flasks. (Leave them there until you are ready to pour the agar the rod helps to prevent bumping in the flask and boil over). Again check that the rod fits inside the microwave or use shorter rods.

Add 250ml water and bung with non-absorbent cotton wool (Glass wool D.C.). Allow to stand for 10 mins, to soften the agar. Microwave each flask separately for 2-3 minutes until boiling, swirl flasks frequently and if necessary, allow to stand hot until all the agar dissolves. Repeat with the other flask. Once all is dissolved and the solution has boiled microwave on the low or defrost setting for 20 minutes check how your microwave treats this work and do not walk away during the process (take some bookwork to do) so that you can keep a close eye on the process. Allow to cool to 500C then pour.

When set invert and store in the fridge in the plastic sleeves the dishes came in (this prevents them drying out if stored for a while.

If only one flask is wanted use a similar flask of water only (250ml) to prevent boil over which is messy to clean up.

I have just made up 3L of agar the old fashioned way (for phenolphthalein bio experiment) – does anyone out there use the microwave for making up this sort of quantity? Could you please give me the procedure and also the maximum recommended volume that you would microwave? Diane Cooper

I make 250ml of agar at a time in the microwave.

Use a 500ml conical flask and 4.5g of Bacteriological agar (nutrient agar for plates works as well) and add 250ml water mix, and let stand for 10minutes or so.

Cook on High for 2min, mix

Cook on Med low for 2min, mix

Cook for a further 5min approx on low

Ours is an old microwave so you will have to try out for the correct settings. Defrost could be the same as the low setting

I haven't tried it out on bigger quantities but should be no problem if the times are altered accordingly. I find the 500ml conical easy as you just need to swirl it around to mix, and bigger flasks don't fit.

These times suit my microwave and I don't have the problem of the agar frothing up over the top. Different microwaves might need a little less time to avoid this.

Lesley Higham

Here is what we use for 1 litre of agar -

3.5g oxo or bovril

4g table salt

10g peptone

15g agar powder

Weigh the above into a 1L beaker, add water to 1L mark, stir well and pressure cook for 15 minutes. Pour into plates when cool enough to handle. (I leave it for 1 hour after turning off the pressure cooker.) As for the condensation, it will happen whatever you do so I just ignore it. Greg Ryan

0g glucose

5 g peptone

7g agar

5g vegemite

add 500mL hot water. stir 5 min.

pour into 3 x 250mL beakers - sterilise in pressure cooker. Pour.

Helen Given

Try a Mannitol salt agar plate.

They are good for growing Staph (gram positives) and yeast.

20 plates: 1L distilled H20

1.0g Beef extract(powder or marmite)

75g NaCL

10.0g mannitol

0.025g phenol red powder

15.0g Agar powder

If you have these, then you can add them as well: 5.0g digest casein and 5.0g digest of animal tissue

The indicator(red) will turn yellow when growth occurs and then you can see the zone around the colonies very easily. Sam Kemp

Fungus probably grew because there was too much air movement as you were pouring them. If you make them too long before you need them, there is more chance of contamination.

I make 400 ml Schott blue top bottles full, boiled in microwave (carefully) and then lid screwed down tight while still hot. I store these for ages... months even, in the fridge, and melt in microwave and pour on morning of use in a room with the doors and windows shut, and into sterile plates. They can be easily ready by period 2, if you have a cool place to let them set.

Don't worry about condensation... The only way I reckon is to have the agar quite cool, before you pour it.

I use: 6 g Agar, 1.2 g marmite smeared on tiny bit of paper (it dissolves off, and the paper stays in the bottle when the plates get poured) and 400ml dist water. Lizzy McAlinden

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I make up a litre at a time. It takes about 5-7 minutes and I keep an eye on it. When it froths it is usually done. The trick is to catch it before it goes over the top of your flask. Sharon Zwart

I make 1 litre at a time in a 2L Pyrex type jug (or Arcoroc) available from the supermarket.

The reason for this is that 2L beaker is too tall for my microwave, and the jug has the added convenience of having a handle. The downside is that the jug is heavy. Elizabeth Smith

I don’t have the luxury of the use of a microwave in my lab so I make AGAR in a beaker on the magnetic stirrer hot plate. I heat the solution to over 80o and then pour this into the plates. It sets just fine. Gaye B

Old style pressure cooker fits 2 x 500ml conical flasks, I use non-absorbent cotton wool in the top of the flask covered by tin foil and get the pressure cooker up to pressure then leave it on that pressure for 5 minutes then turn the heat off and wait for the pressure to reduce before removing the lid.Rosemary Chatfield

A word of warning - just in case anyone isn’t aware – near-boiling agar solutions can froth up very violently if swirled or otherwise handled. I had a nasty burn on my hand once from this, so now am careful to stir with a glass rod to “defuse” it before swirling. I wouldn’t like anyone else to get such a painful surprise! Janice Deverick

I back up Janice’s comment -be very careful heating anything in the microwave.

I recently severely burned 2 fingers when the starch solution I was boiling super-heated in the microwave. The beaker did have a stirring rod in it but before I removed the beaker from the microwave I gave it a stir and when the contents of the beaker boiled over in a steaming mess I was unable to move my hand away far enough as I hit the roof of the microwave.

It took keeping my fingers in iced water until 2pm to stop the burning (accident happened at 8.10am and serves me right for doing 4 things at once). Needless to say not everything that needed doing got done that day .A case of less haste more speed. Beryl McKinnell

I make up agar in a conical flask on a heated stirrer and heat to boiling (keeping an eye on it near boiling stage).

The magnetic stirrer stops it from getting burnt on the bottom.

I let it cool for a while, covered with a watchglass, to avoid spores and bacteria getting in and contaminating the agar plates. I pour the plates, let them cool and put them back in the plastic bag and tape it up. Then store in the fridge, upside down, to avoid condensation making the agar wet.

I have a question too, I use nutrient agar and need to order more. Does anyone know of the best source, as it is very expensive, I noticed. Irma Steenhuis

Irma you can make up your own nutrient agar.

Add 10g Lemco beef extract, I believe Oxo cubes also work but I haven’t tried this. 10g peptone

5 g Sodium chloride

18g agar and make up to 1 litre with distilled water. David Cook

A really cheap way to make the nutrient agar is use. Marmite and 3 grams of peptone, in the agar this will grow fungi and some bacteria that is not pathogenic. Rosie Chatfield

An infinitely safer way to make agar is to do so in a container that can be sealed - not that you would seal it completely when heating it since the pressure needs to escape. One idea is to prepare set batches in Schott bottles or Pyrex bottles with caps - the caps are then not tightened during the heating process. Such containers should never be more than half-filled, because of the risks of overflow, as has been mentioned before. You also need to be careful about the volumes used in preparation - larger volumes (>500ml) need a much longer time to be sterilized. if you're preparing agar in open containers on the bench, you run the risk of contamination, particularly from fungi, as well as the burning risk. An often used method is to prepare and sterilise agar in sealed container batches in advance (old pressure cookers work well) and simply melt in the microwave or in a double-boiler prior to use. Agar prepared in this way can last many months.

Agar does not need to be stirred - the boiling process does the stirring. In fact, it is extremely dangerous to do so because of the high risk of boiling over and scalding.Hope this helpsBarry WardsMAF Biosecurity New Zealand

We use the prepared plates from Fort Richard - what a treat!. They very efficient and usually deliver to us overnight.

On the diffusion prac - one of our teachers prefers to start with the agar pink (use 0.1M NaOH to "pink" it). She says it gives a much better result as you can see it turning clear as the acid diffuses through the cube and the kids find it easier to see the difference without having to cut the cubes in half. Lynette Breen

Another option is to use Calcium carbonate (0.05mol.L-1) instead of the sodium hydroxide same result but safer. Christine Bird

I am preparing the Agar plates for the 'Bacterial growth practical' by the following method:

"Dissolve Agar (15-20 g) in distilled water (1L)...then boil it for 10-15 minutes...then pour this into dishes...and once solidified, place these into the refrigerator until use".

Is it the right method?

Yes. Let the agar sit in cold water for 10 minutes before you boil it

Let it cool to 600 before you pour it into disposable plates

Store them upside down in the fridge when setSylvia Carter

I always add teaspoon of glucose and knob of vegemite.Shelley.

If you don’t have an autoclave/ pressure cooker you need to boil then cool for 3 days to make sterile this is called the Tyndallisation method.

Just doing it the once does not kill the spores just the bacteria, the spores then germinate and you are back to square one, that is why you do it 3 times. Nic Younger

I always dissolve the nutrient agar powder in water, dispense into bottles with loose lids, and sterilise in a pressure cooker for 15 mins at 115oC. These plates keep for months in the fridge without growing anything, so they seem pretty sterile. Does just boiling the agar for 15 mins do as good a job? Robyn. Mckay

Hi allWhen I first started making agar for micro pracs. I was advised to use the microwave. I had a terrible problem with contaminants right throughout the agar. Bought a second hand pressure cooker off a staff member (unused wedding pressie…) and has been plain sailing to whip up a few batches of agar.

300ml in a 500ml conical flask – boil 10 -15 mins – 15 -17 plates. Haven’t tried putting two flasks in at a time, but sounds worth like a good idea!!

Pressure cookers are a little scary at first, but once you get used to them it’s easy and safe.

Agar for the phenolphthalein expt. I just boiled some water and dissolved the agar into it – like making jelly.Didn’t have any problems with contaminants with that one. Toni Renalson

My recipe is for 9g agar to 500ml water, I get this dissolved first with heat, using a hotplate, and then add 2.5g salt, 2.5g peptone and a spatula of marmite. I bring this all to the boil and remove, allowing to cool slightly, and pour into agar plates. This recipe has never let me down yet so hopefully you will find success also. Sandra Timpany

We use 20g agar (and 20g glucose + 2g marmite) to 1L

Mix

Leave the mixture for 10-15 minutes and boil for 1 minute Then leave to cool to 60deg before pouring. Sylvia Carter

MILK AGAR

Hi all, I have been asked to source some milk agar plates, I have asked Derek at Delta and he is looking into it for me too. Just thought I would ask all you lovely people too, if you have any idea where I could get these? Thanks Nicola Younger.

Fort Richard laboratories are most helpful and quick; customer

phone 09 2765569 – OtahuhuJeanette Price

Thanks all for your replies, I have called them and they are $8.25 for 20, which I thought was a bit of a bargain. Nic Younger

STARCH AGAR

Does anyone know the best concentration of starch to put into starch agar please? I have found everything from 0.4 – 1.5%. Your advice is much appreciated, Robyn Eden