Thursday, April 14, 2016

April Agar Antics: Culturing microbes

Our April session had two activities using agar; they will be blogged separately.  This one is about cultivating micro-organisms from our environment.

We swabbed surfaces in the our homes, and tried to cultivate microorganisms from the swabs.  

Angie made up nutrient agar using chicken stock as a food source. The recipe used was:


  • 2g agar powder (standard food-grade agar)
  • 2tsp sugar
  • 100ml chicken stock made up to half usual concentration. Don't use stock with herbs - you don't want bits in it!




Nutrient agar sterilised in the pressure cooker.
Very tasty - if you're a microbe!

This was sterilised it in the pressure cooker for 15 minutes, and was poured into petri dishes when it had cooled to 40C - 45C.  If it was poured while too hot, too much condensation developed in the petri dish later. Lids were put on the agar plates immediately, to protect them from contamination by airborne particles.

Another batch was made using beef broth (Bovril), and each petri dish was labelled either BB (Beef broth) or CB (Chicken broth).



Taking swabs from the compost caddy

To take samples, we slightly moistened a clean cotton bud from a freshly-opened pack, and rubbed it on the surface to be tested.  We lifted the lid of the petri dish and wiped the cotton bud across the agar surface in a zig-zag motion. Lids were put back on immediately, and taped in 2 or 3 places around the edge.  Each petri dish was labelled on the back with the date and surface tested. 
Innoculating the agar plate with swab


Labelling petri dishes after innoculation




Everyone took some agar plates home in a ziplock bag with some clean cotton buds, so they could swab some surfaces in their own homes.





Disposal

We practised safe disposal technique.  Once taped shut, the petri dishes would not be opened again.  They were to be kept in the open air, at temperatures below 25C, to reduce the risk of encouraging harmful organisms to grow.  After 3-5 days, people were asked to take some photos and send them to Angie.  After that, the agar plates would be put in ziplock bags and immersed in bleach for a couple of hours at least, then put in the dustbin.  


 Results

One petri dish was kept as a control - that stayed beautifully clear, with only a tiny white spot appearing after 5 days. One left open to the air for 4 hours developed all sorts of fungal and bacterial growths. Swabs taken grew all sorts of impressive-looking nasties - interestingly, while scrapings from under 5 year-old Tommy's fingernails leaped into an early lead alongside swabs from the phone keypad, they were overtaken by swabs from the toilet flush handle. 


The control stayed clear for several days, which was good evidence that the pressure cooker sterilisation and general handling had been effective in minimising contamination.
First set of swabs taken from Angie's house before the session.
Moulds started to out-compete bacterial colonies after a few days, and we saw that some colonies of microbes got an early start and seemed to thrive, but then were overtaken and apparently killed by mould. You can see some more photos of these plates below.

Phone keypad





Swabs from two different children's hands!





Swab from stair banister.

Stair banister swab (being bleached prior to disposal)



Leo's cat's paw - back view

Leo's cat's paw - top view
Leo's cat kindly donated a paw swab, above, and below are swabs Leo took from Angie's door, and his own hand:





Lydia's heating broke down, so her home was cold for this week - which probably inhibited bacterial growth:

Lydia - tap handle

Lydia - sofa arm

Lydia - outside window surface
Angie had a splinter that was suspiciously painful, so she couldn't resist swabbing it. It grew these impressive mustard - yellow bacterial colonies which look like Staphylococcus Aureus. 

Bacteria grown from swabbing a splinter on Angie's finger .:-It looks like Staphylococcus aureus, so we treated this with caution and killed it with bleach after taking photos!


Swab from Angie's splinter, with scale.


Further Reading



 These pages may help you to understand what has grown on your plates :
ScienceBuddies - Interpreting agar plates, and  Identifying unknown bacteria, by Eric Staveney

 The exercise in culturing microbes was a classic based on the Nuffield Practical Science activity, 'Microbes all around us'. 


Extra advice on doing it at home from Robert and Barbara Bruce Thompson 's Illustrated Guide to Home Biology Experiments. The author has placed the full text of the book online, freely available, to help home scientists.

Culture methods - cultivating bacteria at home http://www.gliderspen.net/glidershanger/culture.html


Scientists create works of art using bacteria


April Agar: Diffusion, volume and surface area


Effect of size on uptake by diffusion




We used blocks of Agar as models for different-sized cells.  We wanted to investigate why cells are so small, in the context of diffusion.  Diffusion is passive transport - substances are transported across cell membranes without energy being used.



Angie made up blocks of firm agar with 0.01M (ie very,. very weak) sodium hydroxide and a squirt (technical term there) of phenolphthalein indicator, which made the alkaline blocks turn pink. We cut these blocks into cubes of three different sizes - with sides 2cm, 1cm, and 5mm.  


Leo cutting cubes to size.










We worked out the surface area, volume, and surface area to volume ratio of these cubes:




A
Length of side (cm)
B=A2
Area of one side (cm)
C
=6B
Total surface area
of cube (cm2)
D
=A3
Volume of cube (cm3)
E
=C/D
Surface area to
volume ratio
2 4cm224cm2 8cm3 3
1 1cm2 6cm21cm3 6
0.5 0.25 cm2 1.5cm2 0.125cm3 12

If you are a bit unsure about how to calculate volume and surface area, this diagram may help:
It is very easy to see the importance of the surface area to volume ration as a factor limiting cell size.
Surface area and volume diagram from BBC Bitesize (link at foot of page)


We dropped our cubes in very dilute (0.1M) hydrochloric acid . We removed them after 5 minutes, patted them dry with paper towels, then cut them in half and measured how far the colour had changed. 




As soon as we dropped the cubes into the acid solution, the pink colour started to disappear from the outside, leaving it colourless.  We could see the amount of pink shrinking before our eyes as the acid diffused into the agar block. 



The surprising thing about this was that the diffusion rate really was so exact and precise - the coloured area of the blocks shrank at the same rate all round and they looked really symmetrical when sliced in half.  The rate of colour change was consistent.  






After 5 minutes, the smallest block had lost all its pink colour - but the largest one still had a substantial pink area unchanged in the middle.



We measured the distance from the outside of the cube to the start of the remaining pink area.  The most common result was that the acid diffused 4mm into each block in 5 minutes - so, for the cube with sides of 0.5mm, it diffused right into the centre.  The rate of diffusion was approximately 0.8mm/minute. We would need to run more tests before we had enough data to be reliable.

Discussion


If we think of oxygen diffusing into a cell, we can see that a large surface area to volume ratio means that it takes longer for substances to reach the centre of the cell.

From a useful handout - No Brain Too Small:

Cells need to be small because they rely on diffusion for getting many substances into and out of their centres. When a cell grows, there is relatively less membrane for the substances to diffuse through, resulting in the centre of the cell not receiving the substances that it needs.  Diffusion is less efficient, cell processes slow down, and the cell stops growing.  The cell then needs to divide into two smaller cells, which each have a larger surface area: volume ratio, and can diffuse materials more efficiently again.

This is why large organisms can't rely on diffusion to meet their needs for oxygen and nutrient transport.  They need specialised organ systems (and also something called active transport, which uses energy to move substances - that that's another topic).

From BBC Bitesize on Organ Systems:

All living organisms rely on exchanges with the environment to survive. However, diffusion only works efficiently if the distance over which the substances have to diffuse is small and the organism has a large surface areacompared to its volume. This is the case for small organisms.
For larger, more complex organisms – which have a small surface area:volume ratio and a bigger distance from the surface to the cells inside the body - diffusion alone is insufficient to meet the needs of all cells.
As larger organisms evolved, specialised organ systems - with surfaces across which substances could be exchanged efficiently - also evolved. These specialised organ systems were needed in order to transport substances around the organisms.

Apart from keeping cells small, what else can change to increase the surface area to volume ratio of a tissue?

Some of us cut different shapes, to see what happened if we increased the ratio of surface area to volume by cutting a wavy or convoluted surface:


These convoluted shapes quickly lost their colour.


This is one of the ways that exchange surfaces in organisms have adapted - by developing folded surfaces which increase the surface area relative to the volume.  Examples are the alveoli in the lungs, and villi in the intestines.


Alveoli in lungs








Further Reading


Our activity was based on the classic  "Effect on size of uptake by diffusion " from the Nuffield Foundation 

BBC Bitesize: Organ Systems
BBC Bitesize on Diffusion

Sourcing Materials :

I used food grade agar powder, which is cheaper than laboratory-grade. For activities at this level, you don't necessarily need expensive specialist materials. 

The agar powder cost £5.95 for 100g,which was plenty -as you use only 2g per 100ml of water to make up approx 100ml of agar jelly.
 See Special Ingredients Agar Agar Premium Quality Powder 100 g https://www.amazon.co.uk/dp/B00EZMPMNE/ref=cm_sw_r_other_apa_MGgjybJMNG576

Phenolphthalein indicator can be bought on eBay or Amazon. 

Sodium Hydroxide is caustic soda. It  can be bought from the high street, eg Poundland or any large supermarket . We wanted very dilute solution  (0.1M) so that it was low-hazard; dilute to one-hundredth of the strength suggested for cleaning drains etc.. 

CLEAPSS Student Safety Sheet for sodium hydroxide : http://www.cleapss.org.uk/attachments/article/0/SSS31.pdf

CLEAPSS Recipe Book : guidance on safety in science education. 
http://www.cleapss.org.uk/attachments/article/0/RBPrint.pdf