Kauri die-back project

As at Day 6, the liquid cultures of Trichodrema and Illyonedra subbed on to Phytophthora agathidicida are growing vigorously.    These will be reviewed more closely by Maj on Friday (Day 9), when we will observe their growth under the microscope.

Illyonedra (2400730) on PTA (17027QU) Q261

Trichoderma (2600210) on PTA (17027QU) Q261

New deposits to the ICMP

As these are new bacteria or yeast to the collection, all seven cultures from the Kombucha project are to be deposited into the International Collection of Microorganisms from Plants (ICMP) under my name:

21922 - BB1 - Brettanomyces bruxellensis
21923 - BB2 - Cyberindnera jadinii
21924 - BB3 - Gluconobacter (oxydans or roseus or new species)
21925 - BB4 - Acetobacter musti
21926 - BB5 - Acetobacter papayae
21927 - BB6 - Pichia manshurica
21928 - BB7 - Gluconobacter (oxydans or roseus or new species)

It was a thrill to complete and sign off the deposit forms.

Signing my name to deposit forms

Page one of the first deposit form

Relevant page from Landcare Research PR pamphlet

DNA sequences - checking against gene bank

Bevan loaded the DNA sequence data of the seven Kombucha samples into a 'Blaster' app, to make comparisons with known specimens held in the National Library of Medicine (NH) database, physically located in North America.  This the world's largest biomedical library.

Bevan confirmed the species, genus and name of each sample, and entered new specimens into the database. Of the seven samples, one yeast and two bacteria were new to New Zealand, with two more bacteria pending.   It was very interesting to see the taxonomy 'trees' denoting species and genus as he conducted his searches.

Bevan, as curator of ICMP, plans to make a submission to the New Zealand Environmental Protection Authority (EPA) on behalf of Landcare Research.  It would appear that the representatives of Kombucha did not disclose their imported sample correctly and that fungi other than the ones declared have been imported.  While there is zero risk to the public, it is important that fungal taxonomy is properly managed in New Zealand.

DNA sequencing - analysing raw data

Printout of Kombucha samples

The DNA printout for each sample is in the format of a chromotogram and a letter code.  Two chromotographs are presented, which superimpose one on the other to align the DNA sequence making one final printout. 

Park looks at the chromotograms for quality; eg, distinct highs and troughs denote high DNA quality; indistinct, 'messy' troughs denote low quality.  The beginning and end of the chromotograph are routinely poor quality as they are the controls.  They are deleted, and no important data is lost.  with his vast experience, Park also checks the chromomotogram against the letter code, and amends it if necessary; eg, a wide peak might denote a double letter and one will be added if absent.

Yeasts have shorter DNA strands (ITS suffix) and bacteria have longer strands (FDI suffix)

DNA sequencing - diagnostic lab

A master mix is made with different primers, dye and agents based on the DNA print out.  BSA and other agents containing animal products are strictly controlled by permit.  Cleaning products and agents that bind up any impurities are wildly expensive, but speed up the process.

Samples are mixed using a multiple pipette, concentrated using a centrifuge and incubated.

The multiple pipette and other equipment used.

Samples are finally placed in the DNA analyser for sequencing.  This takes several hours for fungi.

Genetic analyser

DNA sequencing - prep lab

Park walked me through the steps involved in the prep lab; this is my understanding of the process.
This small run was done manually, but large runs (the norm) are done on the robot machine that can process up to one hundred samples simultaneously.

The robot machine.

Park's notes of explanation.

The  samples were dissolved in a strong alkaline extraction solution and incubated at a high temperature to break down the cell walls  to allow for DNA extraction.  The yeast and bacteria samples, together with a positive and negative control, are incubated separately at different temperatures and cycles.  If it is not known if the sample is a bacteria or yeast, they are incubated in both, both usually scientists can tell by appearance.

The thermal cylinder used for incubating samples.

Tiny amounts of material are used, so the measurements have to be very exact. 

Some of the pipettes used to measure minute amounts.

The samples are pipetted into a BSA buffer solution which starts a polymer chain reaction to amplify the amount of DNA available for analysis.

 A loading dye, GelRed was added that has a heavier molecular weight than DNA allowing it to sink into the DNA.  Most chemicals used for DNA extraction comes form America or Germany, and are extremely expensive.
  
A process called Electrophoresis was run on this equipment.  DNA is negatively charged, so when a charge in run through the solution, each sample will make a distinct DNA ladder, which can be compared to others to help with  analysis.

Printout of the DNA ladders for my samples: bright bands denote bacteria;  lighter bands denote yeasts;  the two indistinct bands at either end are the negative and positive controls.

Samples are then placed in a air lock between the prep lab and the diagnostic lab to avoid contamination.

Specimens ready to sequence!

Seven sub cultures have been grown from the three Kombucha samples, Sample 1 - SCOBY bought in New Zealand, sample 2 - drink marketed by LoBros in New Zealand, and sample 3 - drink marketed by Remedy in Australia.

The cultures were pure, except of Sample 1D, which showed signs of contamination. Specimen 1D was re-plated, as only pure strains can go for genetic sequencing.  The seven cultures, 3 yeasts and 4 bacteria, were each given a unique codes: BB1 to BB7.

Fungal Foray prep

As part of preparing to the Fungal Foray in early May, I am reading and making notes of recommended books on fungi.  This book was kindly given to be by Jessica.  It is one used in the field by the late Frank Newhook, a noted authority on fungi.  The PC2 lab, The Newhook Laboratory, is named in his honour.  

Liquid cultures

 On Day 12, all the liquid cultures of Illyonederma,  Trichoherma and Fortini had grown sufficiently.  Maj directed that the 'lawn' cultures of Phytophthora Agathicicida  grown earlier (9 March and 16 March) are to be cultured with the concentrated liquid culture.

The cloudiness of the liquid indicates growth.

Using the centrifuge, I watched Maj layer the liquid cultures so that the concentrate gravitated to the bottom of the tube, and she was able to discard the clear liquid at the top.  

Loading up the centrifuge - there were three samples, so a tube of water was used for balance.

The three concentrated samples were cultured on to PTA lawn, and incubated at 18 degrees C.  After observing Maj, I was able to prepare one but it was difficult to do, as the concentrated cultures were quite viscous.  These cultures will be incubated at 18 degrees C and the growth monitored and recorded digitally as before.

Inhibition cultures - kauri die back

The inhibition assays were checked daily and images taken as a record.   It was important to trial growth rates against Phytophthera Agathidicida as it has been identified as a causal agent in kauri die-back disease, but scientists don't know exactly how it is attacking kauri.  They are looking for a potential agent for biocontrol of this disease.

A sample of the inhibition assays on Day 9.  You can see the relative growth of the two fungi.
The images below were taken on the lab camera as a permanent record.

Illyonectia (orange) and Phytophthora Agathidicidia

Fortini (black) and Phytophthora Agathidicida

Tricholderma (white) and Phytophthora Agathidicida

Kauri die-back school resource

On the last day of term, I visited school to wish everyone well for the holidays, and was surprised to see a new MOE resource - 'Keep Kauri Standing - kauri die-back school resource'.  When I inquired at Landcare Research, scientists said that they had been sent digital copies before its distribution to schools.

Kombucha - isolating bacteria

When checking Kombucha subcultures on Day 4, Megan confirmed that that some of the cultures were pure isolates.  I subbed these again twice - one for ID through genetic sequencing and the other for the ICMP collection (if the sequencing confirmed its status as a new bacteria or yeast).  A wonderfully exciting moment in the lab!

Under Megan's guidance, I made wet slides to look at some of the bacteria and yeast in the Kombucha samples under 40x magnification, to see the differences in shape and size between the two cultures.  I found focusing very difficult, lots of practice required.  

This image using the lab camera shows a pure isolate - these images are kept on file.

This shows contamination, so it was subbed again.

Visit to UOA School of Biological Sciences

Helen A, a fellow STLP2108a participant teacher is working with the School of Biological Sciences at the Tamaki Campus - we are practically neighbours.   When I visited, it was interesting to see how we are both working on kauri - Helen's work involves the effect of long term drought on kauri, and Landcare Research's work involves kauri die back disease.  

We both thought this poster in Helen's SBS office would be a great one for our schools.

Inhibition assays

The inhibition assays show the relative growth of two different cultures over time.  After plating these assays, I monitored their growth, and recorded it digitally.

Inhibition assays are incubated at 18 degrees C. Q261 must always be prominently displayed to indicate it is held under the strict condition of its permit.

The relative growth of each specimen is monitored, as shown in this plate held up to the light.  Photos were taken of each assay on Day 4, and this will be repeated on Day 7 after the weekend.

Insect awards

The next draft of the information sheet to accompany New Zealand native insect display case is finished, except for photos.  Images have to come from Landcare Resource's database for copyright reasons, and some of the photos I had selected were from other databases. LandCare has vast catalogues, but specimens numerically, not by name, which made it difficult for me to be certain that I had selected the correct photo.  Sarah will help me with this.  

It was decided the make up the display cases for school in themes -  I entitled this one 'The Inaugural Insect Awards' as each specimen was chosen for a specific reason: the longest, heaviest, stinkiest, noisiest, brightest, and so on.

I visited Science Club at Cockle Bay as the guest speaker.  After looking at some display cases, students decided what themes they would be interested in for insect displays:

I will take this information back to Landcare and work on some more themed displays!

Kauri die back in the media

Kauri die back disease is in the media, with newspaper articles about the closing of walking tracks in the Waitakere Ranges.

Checking Kombucha sub cultures

By Day 3,  all the subcultures of Kombucha showed some evidence of growth, some more than others.  The cultures growing on BHI were the slowest growing. 

After some instruction, I took photos of all the subcultures and kept a record of photos digitally.

The camera was a very sophisticated piece of equipment.

When recording growth, the convention is that the day when cultures are plated is Day 0, then numerically after that.  Days can be recorded as a minus number to indicate the days of preparation before plating, eg when making and concentrating liquid culture before plating.

Kombucha project

Megan has tasked me with isolating the bacteria in three samples of Kombucha, one bought in New Zealand, one from Australia and one from the drink itself.  Under her direction, I plated the three samples of five different media: half PDA, brain, heart infusion (BHI), chocolate (horse blood medium),  Kings Bee and AC.  

On Day 4, after the weekend, I looked to see what colonies were starting to form in which medium.
Some sample plates:

Under Megan's guidance, I make subcultures of 'interesting looking' colonies, ie those that were more likely to be bacteria rather than yeast.  It was a case of looking for the unusual, ones that were not too glossy like yeast.   I prepared plates with 3 or 4 unusual looking subcultures (from the same medium) on the same plate, to make efficient use of the media available.   These cultures are being incubated at 25 degrees C.  

I am relishing having a project to manage, with Megan available to answer questions and guide me when necessary.  It is making me feel more directly involved, and accelerating my learning about what it feels like to be a scientist.  I am learning about lab procedures and the methodical recording of data but more importantly, the wider picture of being a scientist - starting with a question - do we know what it is Kombucha? Could some of the bacteria be new to New Zealand?  How came we find out?  What scientific investigations do I need to conduct?  How will I record my data? What are the ramifications of this investigation?  It is authentic hands-on science, as opposed to observational science, and I am very excited to be involved.

Introducing Kombucha

Kombucha is a fermented tea made from SCOBY - Symbiotic Colony of Bacteria and Yeast.  Megan is endeavouring to isolate the bacteria to ascertain that there are no strains of bacteria present that are new to New Zealand.  It is proving difficult to isolate the bacteria as the yeast is so abundant in the cultures.  

Megan showed me the yeast and bacteria on slides. she had prepared.

Inhibition assays

I prepared inhibition assays for the kauri die-back project by taking little plugs of the fungal subcultures, two at a time,  so that they oppose each other on the petri dish.  This is a way to observe which fungus grows the strongest and thus inhibits the other's growth.  Over time, it will provide valuable data as scientists work towards a potential biocontrol of the disease.

Instructions from Marj written in my notebook as diagrams for clarity.

It is necessary to work in the PC2 hood when working on Q cultures.  It has a 20 minute cycle of ultra violet light to sterilise surfaces before and after work is carried out.