Collecting fungi in Auckland

It has been very interesting, discovering the world of fungi during my time at Landcare Research.  Previously, I had envisaged a mushroom as a discrete organism unto itself.  I now discover that the visible mushroom is merely the fruiting body of a much bigger organism, hidden from view underground, that appears when conditions are favourable for spores to be dispersed.  

Diagram from the bilingual teachers' guide from the Kuru Kaupapa Putaiao (Science) curriculum, kindly shared by Peter Buchanan (and freely available on the publisher's website)

Cultures taken from mushrooms at the Fungal Foray, showing the hyphae that have grown from the original plug.  Chris has incubated these samples in a fruiting chamber which he has developed.  The chamber controls temperature, humidity and carbon dioxide levels, providing  optimum conditions for growth.  He has also developed a medium using molasses which provides a nutritious food source for his Armillaria cultures.

Another revelation was the wealth of mushrooms that can seen, when one is aware enough to look around for them.  I found ten different varieties in Rotorua recently, and discovered twelve on a recent walk through the Auckland Domain.  This was after a wintery cold snap, which I was afraid might have not be conducive to mushrooms.  In general, mushrooms appear in Autumn when humid, damp conditions provide the best conditions for spore dispersal.

A small sample of the colonies of mushrooms found in the Auckland Domain.  Mushrooms are typically found by the base of trees or on dead of rotting wood.

Bio-diversity and Bio-security Seminar

Manaaki Whenua - Landcare Research hosted a graduate seminar for PhD and Masters students to present their research findings to the science community.  There were ten presenters in all, who spoke on a diverse range of scientific topics ranging from weaponery in beetles to light pollution in cities.  

There was a question and answer session after each speaker, and one of the interesting things was the number of times the phrases 'seemingly' or 'seemingly not' were used.  It was a timely reminder to me of the evidence based way of thinking which is inherent in scientific discussion and discourse.

A small selection of the presentations.

DNA sequencing of water microbes

Results from the genetic sequencing carried out on the 31 bacteria found in the Onehunga lagoon show some interesting results. Several new species were found, and in one case a new genus.  Fourteen samples were selected to go into the International Collection of Microorganisms from Plants (ICMP), based on their uniqueness and how they add to the completeness of the collection.

Tree planting at Mangemangeroa Reserve

The first day of winter was (uncharacteristically) gorgeous - sunny and calm, a perfect day for planting at the Mangemangeroa Reserve.  Eco-warriors from Cockle Bay, students from Howick College, Friends of Mangemangeroa, the Gecko Trust NZ, parents and Environmental Services from the Auckland Council all worked together to plant 250 grasses, flaxes and trees.  It is great to see such a sweeping collaboration and Citizen Science in action.  

One of our activities, facilitated by Cate Jessep, the sustainable school adviser from the Auckland Council, was to listen to the sounds of the reserve.  Intermingled in the traffic noise and overhead aeroplanes, was the melodic sounds of tui.  In the future, as our native plantings mature, they will encourage more native birds back into the area. 

A view looking down to plantings on all sides of the fencing.

Staff PD at Cockle Bay School

Gillian Taylor, STLP2017a teacher participant and Science curriculum leader led a science session for staff to learn more about looking teaching science from a Nature of Science viewpoint.  The presentation was supported by myself and the rest of the science team. 

There was a mix of theory about why we do what we do (focusing on the Nature of Science); a hands-on activity with a focus on one of the science capabilities, observation skills; and a think tank using post-it notes, for every member of staff to say what science means to them - this will be incorporated into the school's science vision.  Everyone was very positive and motivated - a great PD session.

Staff worked together in year groups to make a catapult from a small bag of materials.  After much trial and error, there was a sharing time, followed by information about the science behind their construction, involving physical forces.

Finding fungi in Rotorua

On a recent trip to Rotorua, I was amazed at the diverse number of mushrooms we found on family walks under the redwoods near Hamurana Springs forest and around the thermal pools in Kuirau Park.  It is interesting to find so many different varieties of mushrooms, when a few weeks ago before the fungal foray, I would have walked past and seen none.

When I discussed this with scientists at Landcare, it was interesting to discover that there are no published studies of fungi that grow in the warm, humid conditions of the thermal areas around Rotorua and Taupo.

The steam from the thermal pools made my glasses fog up, but not so much as to obstruct my search of mushrooms!

A small selection of the mushrooms we photographed on our walks.

Networking with other STLP teacher participants

As part of the STLP2018 phase one, it is a good idea to network with other participant teachers to learn from their experiences.  

Helen (a STLP2018a fellow participant teacher) and I visited Catherine Shipton at Churchill Park School and Tina Joshua-Bargh at Willowbank Primary School.  We had candid discussions with both teachers, particularly focused around the initiatives that worked well for them in their phase two,  when they returned to the classroom and started to develop/update their school's science strategic plan.

One of Tina's science initiatives was to develop a self watering device using funding from Curious Minds.  She found the organisation very sympathetic and helpful throughout the funding process, which is very encouraging.

The completed self watering device at Willowbank School

I have also visited Helen Armstrong from East Tamaki School, a fellow STLP2018a participant teacher, at her host organisation, the School of Biological Sciences at UOA where she is involved in a project to study the impact of  drought on kauri.

In addition, I hosted Sophia Douglas at Manaaki Whenua for a morning.   Sophia is another STLP2018a participant teacher from Terrace End School, Palmerston North.  We had a walk through of the Landcare Research laboratories, fungi fruiting chamber and New Zealand arthropod collection.  We also had morning tea with a group of scientists from the Mycology Team  who were interested to hear more about the programme, and offer their ideas. 

Sequencing the bacteria

Altogether there were 31 bacteria found in the Onehunga Lagoon.  They were selected for genetic sequencing, as this is the only way to identify them absolutely.  There were 14 from collection site 1, 9 from collection site 2 and 8 from site 3.  Of these 8 were bacteria that had been found in the mud collected at the water/mud interface and 23 from sea water.  However, this project was not a quantitative study and no conclusions can be drawn from the numbers at each location; for instance, some were discarded because they were too similar to another sample from a different location.

There were 43 samples in all to sequence: my water microbes, fungi from Chris and quarantined (Q) cultures for the ICMP from Rose.  The Q cultures are held in the Newhook Laboratory under very strict conditions, laid out in their permit.  Q cultures can not be moved from their containment facility, unless they are first de-natured, and no longer viable.  To do this, Park had to temporarily relocate one of the thermal cyclers, and incubate the Q cultures at 95oC for ten minutes.  Then it was safe for them to leave the Newhook Laboratory for the Genetics Laboratory to complete the DNA sequencing.

Park adds an extracting solution; it is very alkaline which helps break down the cell walls.

Park keys in an individual cycle of temperatures for specific samples.

Park adds different primers for bacteria and fungi.

With Park's guidance, I made and poured the running gel for the electrophoresis unit.  Once the gel was poured, the spacer racks were inserted at even distances apart.  When the gel set, the spacer racks were carefully removed, and samples were first coloured with dye, and then added to each space,

When the electrophoresis unit is turned on, the negatively charged DNA will be attracted to the positive charge.  Over time, this will form characteristic bands or ladders, like the marker chart below.

Here is a print out of the ladders for these samples.  It gives a visual representation (or marker) of the DNA present in each sample.  The plate with the DNA ladders is then placed in the molecular imager.  The resulting ladder map is read to ascertain whether the samples are sufficiently robust for the final stages of the sequencing process.

Isolating pure cultures

It was possible to see growth in the  water microbe cultures within 24 hours, although some were more slow growing.  It was important to monitor the cultures to check that there were no contaminants, and that only one bacteria was present.  Here are a few samples:

Colonies growing in marine agar, (MAR).  This has a high salt content and is
used to cultivate marine bacteria.

Colonies growing in MacConkey (MAC) Agar.  MAC provides essential 

nutrients and vitamins for the growth of micro-organisms.

Colonies growing in Yeast Nutrient Agar (YNA).  YNA supports the

growth of wide range of organisms, and is used to isolate rod bacteria.

There were two plates where I could observe two different colonies, so these had to be re-subbed carefully, streaking the two different strains on two different plates, and incubating them.

Once the cultures are established, I subbed the cultures again to more clearly define the nature of the bacteria.  They were plated on to Blood Heart Infusion (BHI) or YNA, as these media are both highly nutritious and are used to cultivate a wide range of bacteria.  

All environmental samples are incubated at 25oC.

It was amazing how many plates I had to manage!  First, the initial samples from three different collection sites were subbed on to eight different media; later, when the colonies grew, the isolated bacteria were re-subbed onto general purpose media to more clearly define their nature.  Finally, each colony was subbed again, this time on duplicate plates, one for the ICMP collection and one for DNA sequencing.

I devised  this spreadsheet to keep track of all the plates.  This seemed impossibly daunting at the beginning of my time in the lab when three Masters students were conducting a project related to kauri die-back.  However, I was able to methodically label, sort and manage the plates for this project.  It is a clear indication of my developing technical skills in the lab.  

Incubating samples

The next step is to incubate the samples from the three different collection points.  There were three samples from each collection point: 

• Water sample, 100%
• Water sample diluted to 1 in 10 with sterile water to provide a better population spread
• Mud collected from the mud/water interface

Water and mud samples from the 3 collection points.

As there were three different samples and seven different media, not counting the marine broth which was inoculated in test tubes, it was necessary to label and streak 63 plates.  This was another part of my learning curve.  I was not familiar with plating solutions and by adding too much liquid,  I swamped the media with culture.  All 63 plates had to be redone.  Luckily I had prepared sufficient plates!

Streaked plates ready to be incubated.

I'm very proud to have been allocated my own incubator for the length of the project. 

 Environmental samples are incubated at 25 degrees C.

As the project progresses, it will be necessary to manage more and more plates,  to keep clear, systematic records and to be able to identify  trends.  When a bacteria flourishes on a particular medium and a colony can be clearly seen, it is necessary to sub it, to further isolate the culture until, eventually, a pure strain is isolated.    I have developed an XL sheet to record this.                                                                                                                                                                

     

Pouring media

Once the media have been auto-claves, it is necessary to pour them into plates as soon as possible, as the agar in the medium will solidify upon cooling.    One of my first mistakes was to make too many different media at once.  It was difficult to pour them all in a time, some cooled too much so that the bottom of the flask congealed.  

These plates are marked with a black/green/red line to denote the R2A medium.

A medium made with 500ml of water will make 20 plates.

Plates are poured under sterile conditions under a hood, and left to solidify.

This was a whole day's work.  10 test tubes of marine broth and 140 petri dishes, 20 of each of 

the seven different media, marked with their specific ID stripes to distinguish them easily.