Before the war a little Video (sorry no subtitles yet ) =)

This video is about 1 year old, filmed at my original facilitys in Aveiro University. Please don't mind any imprecisions of the reporter and me =).

Launching the chemical war

Rifampicin a possible nitrogen carrier for Olea tissue culture

That's right, but before I go on explaining why i think that Rifampicin might work as a nitrogen carrier let me tell you how i got there.
Like i was saying just a couple of messages before, my last year of work as been mostly devoted to understand why it wasn't possible to scale up the production of the Galega clones at the lab where i was working.
If you were following me from there, you know that my first preocupation was to "close all the windows at the LAB". In other words, increasing the level of certainty in all asseptic procedures, making sure our error or errors wern't having aditional help in spreading the contaminations through the all culture stock. It is important to say that it takes some time to do this. Firstly it is necessary to repeat several cycles of multiplication, and it's related smaller procedures, like medium elaboration, labware cleaning, infrastrucure and equipment cleaning and desinfection, etc, just to understand how rich the culture medium that you are using is for the type of organisms that you are up against, and how vulnerable your actions may reflect in the transfering operations and culture medium distribution.
To simply get the hang of it 6 months would be an acceptable time frame, specially being olive a slow growth species.- I didn't have such time.- This company was thirsty for results and involved in a chronic lack of faith by it's partners and investors.

-After the first statistical surveys on the contamination patterns, it was very obvious that the total impact of fungus was the biggest hazzard for the lab. This was proven by evidence that fungus were continuously making theyr appearence throughout the all process of propagation, wich from early stages, led me to believe that the spores were coming either from the explants and having diferenciated responses along the time of culture, allowing a continuous but  slow germination of spores after first contact with the culture medium. Or simply coming from the outside of the culture vessels in a randomly fashion along time.
Even so:
Statistical results after 3 months were somewhat contradictory.
Changes introduced in several laboratory procedures were still young to give any concrete credible results.
There was a strong conviction of my supervisor that problems were coming from the transfer operations.

After testing PPM (plant preservative mixture) in the cultures, the same antibiotic wich had recently given me  promissing results in the establishment fase for Madural cultivar at my own Lab, it proved ineficient against the fungal contaminations in the Galega stock.
 - It's worth mentioning that PPM has reasonable eficiency against fungus, but it's degradation in the medium along time, makes way for late contaminations. This is what happened basically. After 3 to 4 weeks fungus were back, and since we had a hard time keeping cultures up to date, the situation turned persistently to the same status quo. Continuous loss of explant material.

An extemelly curious observation

As i mentioned in my last post, i spent most of the year of 2010 in a continuous fight againts endogenous and exogenous contaminations of Olea europaea explants, testing all types of desinfection procedures and antibiotics. Some of them i must say quite original but nevertheless effective.- I remember getting to the lab everyday and be overwelmed by the smell of garlic wich reminded me those old portugueses taverns.=) garlic works...
-The explants wich i received almost weekly from one of my faithfull partners, made the work very intensive, more specifically the selection of the right material, wich was frequently a 2 or 3 days journey. Of course, inoculation in the culture medium after desinfection, should be quick, to avoid excessive oxidation of the tissues. This meant long work shifts sometimes night through.
- One of the inumerous substances wich i tested was this dark red powder, with very exquisite solubility in DMSO (dimethylsulfoxide). It's name... RIFAMPICIN.
Rifampicin is a semisynthetic compound produced by the bacteria  Amycolatopsis rifamycinica. A quick search on google points out that it is a type of bacteria that lives in soil. A deeper search relates this genus to nytrogen fixation through symbiosis, and antifungal activity besides is efectiveness in the treatment of comon diseases provoqued by mycobacteria like tuberculosis.
Moreover if we look at the structure of the compound we find four atoms of nytrogen in it.
Check it out:

What is my point?...

The use of antibiotics in Olea europaea tissue culture

As it was demonstrated in earlyer posts in this same blog establishing olive tree explants in vitro is a challenging task.
For more then a year i tried without success to establish Olea europaea cv madural untill i finally got some results. The desinfection procedure itself is cultivar dependent .This means that research with olive starts on the first step for establishment, the desinfection procedure.
 I am also convict that some of the contaminations that i had to face in the maintenance of galega vulgar clones were there almost from te beguining. Process in wich i didn't particiapte.
The multiplication protocol on the other hand was so efective and the plants so vigorous that it was possible to keep cultures actively growing without loosing them definetly. -Even from highly contaminated material i was able manny times to save and recycle clean meristems into new clean cultures.

From the earlyer work with Madural there were many discoverys. This was a time when i was searching undreds of synthetic and organic compounds and i found some really efective antibiotics. Curiously one of them was alycin from garlic, wich method of usage, i have also explained in this blog before as well.

Another one of these incredibly efective substances is PPM that stands for plant preservative mixture. A mix of 2 isothizolones one of them chlorinated.

Finally i had discovered as well, a way to convert a fitossanitary antifungal available in a comercial formula into a powerfull antifungal readilly aditionable to the culture medium post autoclaving. The process consists in extracting the active substances of the antifungal switch, fludioxonil and cyprodinil, with a few mililiters of acetone wich is a compatible solvent for both. After this depending on the species you are working with, you will probably have to make trials and see how sensitive your explants are to both molecules as well as to the acetone.

Cefotaxime sodium, gave promissing results in a few of my trials but the explants would not develop after a certain point of the initiation process and a latter experiment with ceftriaxone confirmed toxicity of cefalosporins making the galega explants stop all growth and  very slowly become chlorotic. This is in agreement with some of the bibliography available wich says cefalosporins are toxic and have a narrow spectre of action when used alone.

Quote

A successful man is one who can lay a firm foundation with the bricks others have thrown at him.
David Brinkley 


I add to this quote that i will always rather use the bricks on my own firm foundation, based on honesty and sincerity. 
There's way too many, bricks out of place and brick throwers, in my country.

First moves against the problem

When fighting contaminations of  4 or 5 difrent types of fungus and bacteria, all possibilities must be taken seriously. So, my first, and in my opinion most logical step, was to ensure that all the typical "sneak out's" of spores or viable contamination vectors into the flasks (sneak in's for the matter) were reduced to a minimum.

Looking around in the lab i found some unused contaminated flasks with medium, wich quickly led me to the conclusion that for shure the problem would have more than one origin.

A few changes:

I substituted the petri dishes by autoclaved newspaper squares for explant handling. It seemed more pratical, to work in a constantly renewed absorbant working surface.

I made a small scalpel and forceps stand with stainless steel wire for each worker. Autoclaved petri dishes were being used as the stands.

I made 4 flask double sleeves, with autoclave bags and a vaccum plastic bag sealer. This took a while but allowed us to store autoclaved flasks with more reliability. 

I encouraged operators to reduce they ovements around te lab to pick up materials.

I tried to make bigger batches of medium to allow a small quarentine for each batch before usage.

I made the autoclaving and dosing a more sistematic, quick, and single oriented operation.

why?  because dosing was being done in small batches and sharing space in the laminar flow hood with another worker doing the handling. Acording with the operators themselves shortage of medium was constant.

I tried to improve comunication from the operators with me. They were accounted frequently as the origin of the problem and became fearfull of theyr own activities and change.

I institucioalised the use of foot protection, hair and face masks, gloves and general asséptic procedures as obligatory in all circunstances, although my bosses were still walking around and bringing visitors randomly and unnoticed without any of these worrys. =) not so funny...

I created a small closet for lab coats sterelization with a UV lamp daily after work.

I forbiden any brooming or sweeping in the culture chamber and transfer rooms. Big flash when i saw a person brooming in the culture room.

ffff whatelse have i done...it's late tomorow i will tell you more....

HANDS ON APROACH

After setting all media components for fast handling, mostly making stocks for all added solids, and stocks for stock solutions preparation. -Please ignore excessive stocks =).- My next goal was to start tracing the origin of the existing contaminations. In the original protocol developed for the company, the co-existence of a strain of bacteria with the basal callus of the Galega cv clones was a predisposition for normal growth without signs of chlorosis.

This made things more complicated, specially in what concerns to colony dissociation between diferent bacteria, "good ones" and "bad ones".

A first step was to make frequent (3x per week) surveillance to the culture flasks identifying: type of contamination, fungal or bacterial, place of appearence, medium or explant. Later i asked my co-workers to please identify the flasks with theyr names to see if any sistematic gesture or procedure during the handling was introducing the contaminations. 

As time went by i started to distinguish  a few standard contaminations at the lab and identifying them more accuratly at inicial stage of development.

From the start, the fungal contaminations seemed more frequent than the bacterial infections, wich were also somewhat disguised by the simbiotic bacteria wich had the capacity to develop inside the agar making it cloudy . Later on, the first numbers confirmed that fungus were more than two thirds of the total infection percentage wich oscilated between 20%  and 30% of all newly handled plant material. These percentages were extremelly high for the laboratory ecomical viability, moreover because the explants had to pass by three diferent handlings until a small rooted olive explant was obtained. The result was the previously observed incapacity to produce a large number of plants. It is relevant to say that with such high contamination rate, the laboratory would quicly destroy all cultures if it wasn't for the olive's explants extreme resistence to most of the contaminations, wich in most cases didn't even stop the normal explant development untill critical take over, of the vessels, by the fungus.

BIO-MELHORA, EARLY WORK

Upon arrival at Bio-melhora, a brief period of a few days, was devoted to undersand the laboratory design. It is a fully funccional laboratory with a few basic missing objects, like a washing machine for example but not much else was seriously bad. I tried to adapt some of my routines to the existing material and make a small inventory of whatever was available, together with some tagging and mapping of materials for faster production process.

I firts realized that there was a small batch culture medium production logic, implemented on a weekly basis. For the existing amount of plants this looked like a problem to me. 

First things first i tried to make the medium production quicker, as i had one or two operators available for explant handling and transfer and this would allow me to start devoting more time to the core problem of the lab, A persisting incapacity to increase the volume of explants available for the rooting and aclimatisation steps.