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.

Making media

The first thing I need to do is make different media on which I can incubate the three water samples.
I made eight different media.  These each contain different nutrients, and so will encourage the growth of different microbes dependent on what they consume; eg, BHI is highly nutritious and encourages the cultivate of certain fungi; marine broth, with its high salt content, helps to simulate sea water and is used to cultivate heterotrophic marine bacteria

• Reasoner's 2A agar (R2A)
• MacConkey agar (MAC)
• Yeast nutrient agar (YNA)
• Marine broth (MAR broth - liquid)
• Marine agar (MAR)
• Potato dextrose agar (PDA)
• King's B (King's B)
• Brain Heart Infusion (BHI)

Key to distinguish different media by making plates

Different powders, in different combinations, are mixed with distilled water to make media solutions.

Electronic scales are used to measure powders.

Magnetics 'bullelts' are added to the solution to aid optimum mixing.

The magnets are removed with a magnetic wand after mixing is completed.

 

The media solutions are labelled and taped, ready for auto-claving.

It is necessary to used leather gloves as they are removed from the auto-clave.

As an built-in alert, the lines in the tape go black when auto-claving is completed.

My research project

Now that I have some rudimentary scientific skills, it is time to start and manage my own research project, under my mentor's guidance  This will be good for me because it will make me more aware of what is required to work like a scientist and run my own mini project: including scientific techniques that I need to perfect or learn; research that I need to conduct; pitfalls I will face and need to overcome; scientific discussions that I will have to clarify my thinking and to learn.

After some discussion with Megan around suitability and viability, I proposed a study of the microbes in the water in the Onehunga Lagoon.  This is our local designated off-the-lease area for dogs, as well as a wakeboard park, a kayaking area for the local sea scouts, a sailing site for the local model yacht club.  In short, it is a multi use expanse of water which is widely used for water sports even though it displays permanent 'swimming is not advised' signage.  There is so much discussion in the media about water pollution around the Auckland coastline, that I am interested in investigating the water quality in the lagoon, and to ascertain whether the water odour (our dog often smells 'swampy' after her habitual swim) is a reliable indication of the presence of microbes. 

In my proposal, I have listed specific aims, the method I will use and the research techniques I will learn and become familiar with.  This may be amended as I proceed and find shortfalls in my initial proposal, as part of my (rather steep) scientific learning curve.

A study of microbes in the water of the Onehunga Lagoon

Onehunga Bay Lagoon

It was decided to choose three collection sites, so as to have a manageable number of plates in my study.

Collection point 1 - mid way along dog swimming beach.

Collection point 2 - by storm water pipe by wake board station

Collection point 3 - midway along main beach by water run-off pipe

Nature Watch to the Rescue!

One of the scientists from Landcare Resources was looking for a particular fungi on the foray but, despite his best efforts, was not lucky enough to find a specimen.   However, he was able to locate it through Nature Watch New Zealand.  

Nature Watch NZ is a website available to the general public, where people can use the database to identify specimens, or upload photos and details their finds.  If they unsure of the identification,  they can leave it blank or query it; the communal nature of Nature Watch allows experts in the field to help with, or correct their ID.  

An excerpt of the home page of Nature Watch New Zealand

The aims of Nature Watch New Zealand, from their webpage.

I accompanied Chris as he used Nature Watch NZ to assist his research - it is a fabulous example of citizen science at work.  

A member of the public had uploaded information about the sought after fungus in April 2018: a photo, common name, Latin name, host and location with GPS co-ordinates.

It was a matter of travelling down to Christchurch in sufficient time to search for the specimen before our flight.  This was made even more eventful in that there were two more sites to check - one in Springfield and another one further out in the Christchurch area.  Coverage was an issue with both these sites and, without a definitive GPS, our searches were unsuccessful.  With time starting to run out, we tried the last site and found the specimen relatively quickly.  It was a young sample, but Crhis has since been able to culture it back at the lab at Manaaki Whenua, Auckland.  It was an exciting introduction to the workings of Nature Watch NZ!

The page which led us to the fungi we were looking for - with the common name, Latin name, description 

of the host (growing on dead crack willow), location (Travis Wetland, Christchurch) and GPS co-ordinates.

The entry to Travis Wetlands, Christchurch.

Keas in the Wild

When we parked near Arthur's Pass on the the way back to Christchurch, 
this kea very confidently made himself familiar with our car.  

Public information on display to keep the kea, New Zealand's mountain parrot, safe.  

They are curious birds, and often frequent this car park, so it is vital that the public 

behave appropriately when they are close by.

The view of Arthur's Pass from the car park.

Map of our route from Moana to Christchurch.

Fungal Foray Wrap-up

Here is a group photo of all the participants at the Fungal Foray 2018a, taken on our last day.  Even the photographer managed to slide into view with only seconds to spare.

The group photo was accompanied by this recommendation, that you might be interested in watching.  I have certainly become fascinated by fungi since the foray, and even members of my family are seeing them in our local park and out bike riding, where we have never noticed them before.

For fellow foray participants, mycophiles, and anyone interested in biodiversity, I highly recommend: “The Kingdom - How Fungi Made Our World”

 The Kingdom - How Fungi Made Our World

Since the dawn of life, fungi have been the driver of evolution on land.  This makes them both powerful allies and, given the chance, formidable foes.

An expansive documentary which leads us on a fascinating journey from a billion years into the past and forward to an uncertain future, The Kingdom consults many of the world’s leading experts on fungi, and tells the remarkable story of evolution driven by a spore-based life form that literally ate rocks to kickstart Earth’s ecology.

Winner of the Best Film at the 2018 SCINEMA International Science Film Festival, “The Kingdom - How Fungi Made Our World” is a 50 minute documentary released last month, directed by Annamaria Talas and Simon Nasht and produced by Susan MacKinnon (Australia), Anne Pick and Bill Spahic (Canada). A joint venture by Real To Reel Productions (R2R; an award-winning Canadian independent production company, specializing in documentaries, helmed by husband and wife team Anne Pick and Bill Spahic) and Smith&Nasht (a creative partnership between Australian entrepreneur and philanthropist Dick Smith and documentary filmmaker Simon Nasht). 

For an Overview, see:  https://popj.ca/news/fungi-science-documentary-kingdom

Day Five of the Fungal Foray

First, and early morning breakfast of porridge and scrambled eggs, before deciding on the collection site of the day.

Our first collection site for the day was Prossers Bush Walk in Hokitika.  This council reserve
houses a small remnant of the Kahikatea forest that would have originally covered the area.

Some specimens from Prossers Bush Walk.  It was enlightening to see that even a small, 

ten minute walk contains such interesting diversity of fungi.

Our second collection area was in the Hokitika Gorge Scenic Reserve.  This walk 

follows the Hokitika River, descending through a podocarp/hardwood forest.

Sometimes mushrooms can be hard to spot.

Photos without catalogue numbers denote mushrooms that have been collected by someone else in the party.

Over the course of the week, I have found tinfoil has been a great tool for collecting,

especially in heavy rain.  It is easy to fold the specimen, together with the collection

slip, into a compact waterproof parcel.  While experienced foragers used fold away 

penknives, I found my embroidery scissors stood up to the job, and were less problematic

at Aviation Security.

My bank of specimens on Day Five.  I am becoming more familiar working through all the
identification features and using the reference books to identify the mushrooms I collect.

As always, the Pop Up Lab was operating, with banks of microscopes freely available for use.

Temporary photographic booths were set up for taking high quality images.

Spore prints were taken of some specimens.

A lovely momento of the foray - a mushroom house illuminated by a candle from the silent auction. 
The organisers use the funds raised for expenses in future forays, including sponsoring students.