Phyto crashed

[pootsnicker]

Woke up this morning to all of my cultures of phyto a yucky brown color. :( Not happy!! this is my second attempt. started these on the 15th just spit 2 days ago. Not sure what went wrong. third times a charm hopefully..

[saxman]

one thing you need to be sure of is to not cross-contaminate your cultures if you are growing more than one species. also, rotifers and other ciliates will crash your cultures as well. don't use anything that touches your tankwater in your culture room. sterilize your equipment between cultures.

[V]

I few of us have done some killer posts on phytoplankton keeping , can we get procedure on to these pages please:up:

[pootsnicker]

I only had one species so far and was waiting to get stable with it before starting the rotifers. so no contamination there. I used a disk from Florida Aqua farms and some fertilizer form West Manicure. I followed the directions on starting the culture from Florida Aqua farms and the set up from Robs Vids. I bleached everything before using. I did have some light on in the room 24hrs so there was not a complete dark period which could have contributed to it I guess???? Maybe??? The bulbs in my lights were also kind of old so maybe not strong enough???? don't know. We just bought a new house so we close at the end of Oct. so Ill be waiting to start over till in the new house and set up. Don't really have anything to feed it to anyway yet as the tank is still cycling. just wanted to get the proses down and get ahead.

[lance]

one thing is do you have a air filter? do you have rotifer's near the phyto? also do you check your phyto for ciliates at all or parasites? most likely rotifers got in there or it got contaminated and crashed phyto culture is really an art form that takes time and precision a good phyto culture will not have any crashes at all but we all don't have the resources and time for that precision but a few step's can put us there.

lance

[pootsnicker]

what kind of air filter are you talking about? I don't have any rotifers going, was trying to just get the phyto stable first. I have a microscope and would do checks every time I split which was twice before the crash, didn't see any rotifers, but I'm not a expert and don't really know what eles to look for.

[CarmieJo]

Some people who grow cultures run a HEPA air filter.

[THEJRC]

the brown color is most likely either dead cells due to poor photosynthesis (many causes for this from a lack amino's and irons to bad lighting cycle). It's also possible you have a contaminate algae that has taken hold. as far as the lighting from what I've read, I think the world has a ton of mis conceptions. As far as photoperiod, so long as 6+ hours of direct photoperiod is examined under the proper water conditions algal bloom will occur with most common species of algaes (nannochloropsis, Isochrysis, etc.). as far as old bulbs and spectrum shift... I'll avoid this argument. Let it be said that I've obtained access to several photonics tools and the idea that bulbs shift as early and often as they do is purely based upon manufacturing process of each and every bulb and cannot truly be predicted. in any case, for microalgaes this shift (which is within only a few 100 kelvin in most cases) does not make much of a difference, in fact in many cases it's beneficiary.

A few questions before we start troubleshooting..... (I'll get into the air filter in these as well)

Did you plop one of those one way valve / air filters on your air supply (trust me, you should) while not hepa, it will stop a lot of larger contaminates

Is your air supply (pump) located above or below the highest fed culture (as to avoid back bleed)

Are you culturing more than one species of phytoplankton or zooplankton within the same area

what is the salinity of your culture medium

what is the formula used by west? is it a soil based medium, guilliards derivitave?? there are many many culture mediums at this point and each will help / hinder different algal species... always good to investigate

What is the algal species, by indicating a FAF disc I assume nannochloropsis but cannot be sure, if it is isochrysis a golden yellow is desired

What does the changed culture smell like, is it earthy ordoes it smell like decaying matter?

What concentration are you performing for the fertilizer medium

Temp statistics? are there fluctuations? whats the average temp of the area?

How large and what shape are the vessels?

are you using diffusers on the air supply? how much pressure is there (count this in turns, as in how often the water turns a full revolution in the vessel... around 80-160 revolutions per minute has worked well for me, anything more and the algal cells get too battered, anything less and the cells tend to settle and lack motility.


Also, I notice you mentioned robs vids (which are updated and accurate) are there any other methods you are using such as melev's (marc levensons) which (while well meant) have not been updated in a long time and may be innacurate???

in this same realm on the topic of bleach.... how quickly are the cultures changing? Two possibilities here. If the culture never takes hold for the first split from the disc your most likely using vessels thatare too large to star with (I start my cultures in 6-12 ounce bottles if starting from a FAF disc.) The other possibility is that of a bad or poor rinse from the bleach. A good way to avoid the issues of bleach rinses is to switch to using 3 litre or 1 gallon water bottles (water can be used for top off or culture media) and rather than cleaning, throw them away every second or third split. you will find that when not on sale the bottles with water are around $1 each at the highest.

[V]

wow, now thats a long @ss post :D

Im going to have to read this thread from front to back before i get in on the action thats for sure!

great to see you on the boards again bud, i think its been awhile yeah?

[pootsnicker]

THEJRC :(Q):Did you plop one of those one way valve / air filters on your air supply (trust me, you should) while not hepa, it will stop a lot of larger contaminates
(A) I have check valves on my air pump right out of the pump.

(Q)Is your air supply (pump) located above or below the highest fed culture (as to avoid back bleed)
(A)Yes

(Q)Are you culturing more than one species of phytoplankton or zooplankton within the same area

(A) NO only one.

(Q)what is the salinity of your culture medium
(A) 1.019

(Q)what is the formula used by west? is it a soil based medium, guilliards derivitave?? there are many many culture mediums at this point and each will help / hinder different algal species... always good to investigate
(A) It is called Phyto grow It is guilliards derivitave. F2.

(Q)What is the algal species, by indicating a FAF disc I assume nannochloropsis but cannot be sure, if it is isochrysis a golden yellow is desired
(A) nannochloropsis

(Q) What does the changed culture smell like, is it earthy ordoes it smell like decaying matter?
(A) after it lost its green color and became Brownish gray It had a different smell, some what sour.

(Q)What concentration are you performing for the fertilizer medium
(A) following the directions on the bottle 1ml per 2liters. I have a 8 liter culture medium container.

(Q)Temp statistics? are there fluctuations? whats the average temp of the area?
(A) between 72 andd74 air temp.

(Q)How large and what shape are the vessels?
(A) they are 1.8 liter rubber made square jars (same as in Robs vid.)

(Q)are you using diffusers on the air supply? how much pressure is there (count this in turns, as in how often the water turns a full revolution in the vessel... around 80-160 revolutions per minute has worked well for me, anything more and the algal cells get too battered, anything less and the cells tend to settle and lack motility.
(A) air pump is going through a 5 way manifold with valves. I dont have it set up to count right now but I had a steady bubble rate.

I pretty much went buy robs vids only... have seen the others but tried to copy Robs setup for the most part. I would take pictures but have it all tore down getting ready to move to the new house. once setup again ill post pics.

[THEJRC]

Your doing most everything right, the sour smell is due to algal cell decay.

Whats most likely happening is bacterial contaminates are outcompeting the algal cells for nutirents. In such a large vessel to start it often happens as the algae is so sparce it cant take hold.

On your next run start the disc in a much smaller vessel, mix the F2 at 1ml per litre, and mix the medium just before starting the culture (dont let it sit). A good step by step would be to do the following:

1. Mix culture water at 1.019-1.022 (bear in mind that specific gravity is affected by temp, measure with temp at around 72-76*)

2. Add Guilliards at F strength (1ml per litre with the phyto grow medium)

3. Start two cultures from two discs (this doubles your chances) using 16 ounce or smaller bottles (tea or soda bottles work perfect it's the size we're concerned with moreso than shape).

4. Split around day 8-10, moving the entire starter culture into a larger vessel repeating this process until you have your larger cultures running. This way you give the algae a chance to dominate the culture vessel. You can step down to F/2 strength whenever you wish (it's that first bump in the road that we want to make sure nutrients dont get used up).

Some random notes:

Pay close attention to split schedule, often with older set cultures this is the problem. Algae cant survive without nutrients and I've seen a lot of people experience crashes only after moving to much larger vessels. A good rule of thumb is 7-10 days whether it looks darker or not. I hang on an 8 day though I do maintain small 16 ounce "mother" cultures which only receive additional medium and are not split at all most times. Occasionally I will split off the mother cultures but they are more maintained for safety sake.

I typically step my starts from 16 ounce to 1 litre then to 3 liter or gallon water bottles. I pre-mix my culture water (but never with the medium) for the older cultures (in gallon bottles) but always use fresh made culture medium when starting cultures. I only add the medium to the water during splits as it reduces the timeframe for contaminates to get competitive.

I've tried several mediums but have found that the Micro Algae Grow formulation from Florida Aqua Farms has been the most consistent guilliards formulation available (at least to a hobbyist) Naturally I buy it in the unmixed mass packs as I go through a ton of the stuff.

After mixing the mass pack, I pour the Guilliards into multiple opaque bottles that I collect from a friend who receives liquid medicine in, the bottles are dark brown to keep light out and have a topper that a syringe (also supplied with the medicine) to fit into and suck out the contents. Because the syringe has ml markings on it I can easily and quickly nab the appropriate amount of guilliards when mixing cultures. I'll see if I can find these bottles / syringes from someone like us plastics and post a link, it's extremely handy and I've had a TON less contamination problems since I've started using this method.

One of these days I'll update my own howto on the www.copepodgeek.com Home (http://www.copepodgeek.com) site but Rob's instructions are really good and he's done a good enough job that I dont have to (he he thanks rob). The only missing link I think is that most people start cultures in vessels that are too large and the algae cant take hold (at least thats been the bulk of the problems I've seen).

Hope that helps people out there, sorry for the long winded post but... eh I love this subject!!

[THEJRC]

I few of us have done some killer posts on phytoplankton keeping , can we get procedure on to these pages please:up:
At this request:

Procedures for Phytoplankton culture – Generic culture methodology

This procedure assumes use of seed cultures in disc form from Florida Aqua Farms and Guillards F formulation “Micro Algae Grow” also provided from Florida Aqua Farms. This procedure is written as generic as possible with aims at general culturing of species such as Nannochloropsis, Isochrysis, and Tetraselmis. Adjustments to procedure for varying species, culture sizes, and environments will most likely be desired.

Topic One: Culture Medium Preparation

For the sake of this article, culture medium is described as prepared salt water with the addition of Guillards formulation at any strength.
It is best to prepare culture medium immediately prior to starting / splitting cultures, while some keep prepared culture medium on hand in storage containers there is substantial increase in contaminate possibility as medium sits. This method allows for preparation at different strengths as needed.

Use RO (or better) water and desired salt, mix to 1.018-1.024 specific gravity or 23-28 ppt. salinity, it is important to remember that the salinity is a better measure of salt content as specific gravity measurements are affected by temperature. Once salt water is prepared store in vessel that can easily be tapped such as a water jug with a petcock, you can prepare this ahead of time, it is merely the addition of fertilizer (guillards) that we wish to avoid until the last minute.

When preparing culture medium for starter cultures I recommend using full F strength, this is achieved by adding micro algae grow to your culture at 2ml per liter. For split cultures, use F/2 strength achieved by mixing 1ml per liter. If you expect a longer split time, or are running CO2 injection you may want to mix at full F strength to avoid depletion of nutrients in the culture.

Utilize a syringe to add the Guillards directly to the culture vessel and be careful to avoid allowing the syringe to touch the culture or culture vessel as cross contamination will occur. When culturing multiple species, it is a good practice to utilize multiple syringes and formula sources (one for each species) to further avoid cross contamination.

[THEJRC]

Topic Two: Culture Vessels

Culture vessels can play a large role in success of species growth. When starting cultures it is best to utilize a smaller vessel (8-16 ounces) and through splits slowly build up to the targeted final vessel. For this document I will explain my vessel methodology and recommend that everyone examine their own needs and tune to suit. I utilize disposable vessels to avoid the need to sanitize and clean them. If you wish to utilize re-usable vessels I recommend examining the sterilization processes documented by Rob Weatherly at the Talking Reef. Talkingreef (http://www.talkingreef.com) (coincidentally this whole document is being written due to a thread posted there so you are probably already here!).

For starter cultures I utilize soda or water bottles in the 8-16 ounce range, after draining / drinking the contents I wash the bottle thoroughly using Alconox soap (available in powder form at Florida Aqua Farms, it is important to note that I use the Alconox soap to clean all utensils and have had excellent luck with it. I use a ¼” drill bit to put a hole in the cap of the vessel for air supply (more details under Air Supply Considerations).

At the first “split” date (8-12 days after culture start) I move the entire contents of the cultures to a 1 Liter soda bottle prepared following the same procedures. For splits from the 1 Liter I move to either a 3 Liter or 1 Gallon water jug (available at the grocery stores with water of course for around $1 each) which I have adopted as my standard culture vessel size. If larger cultures are needed I will move from a 1 gallon water jug to a 3 gallon carboy, and from the 3 gallon carboy to a 5 gallon carboy. For most the 1 gallon or 3 Liter vessels will work fine unless a great amount of culture is needed. I will re-use vessels for 3-5 splits before discarding and replacing dependant on how fouled they are.

After medium has reached its final “grow out” vessel during splits I will harvest approximately two thirds of the culture and replace with the same amount of prepared salt water. Guillards is added to the culture after the refill and prior to capping and placing back on the rack.

[THEJRC]

Topic Three: Air Supply Considerations

A great deal of success or failure will hinge on the air supplied to aerate the vessels. Most algae’s cultured are non motile and will not stay in suspension in still waters. With the advent of introducing air bubbles into the water we also allow ourselves a great possibility to introduce contaminates that may kill or compete with the algae for nutrients so it is important to have as clean of a supply as possible. I typically follow the following procedure.

My air pump is placed above the culture vessels; this helps to avoid “bleed back” of air or water in the event of a pump outage, one way “duck” valves are also utilized as a second method of bleed back prevention. Separate pumps and manifolds are used for each species of plankter cultured to help isolate cultures from each other and avoid cross contamination. Small airline filters (available at any pet/aquarium shop) are installed either at the main supply to the manifold or before each culture (dependent on need) to help filter out larger contaminations pulled in from the air.

I replace the rigid tubing used in each vessel every two vessels or if excessive fouling of the tubing has occurred. Airlines are replaced in full every 6 months and should a crash occur from the same feed line twice in a row that line will be replaced.

To avoid using too much (or too little) air, watch and “time” your cultures turnover rate. Do this by tilting your rigid tubing to one end of the vessel and count how many revolutions your water does within 15 seconds, multiply that by 4 and you now have your turnover per minute rate. I like to keep this between 60 and 90.

[THEJRC]

Topic Four: Lighting

Lighting as well will play a huge role, without delving into spectrum and multiple species needing multiple spectrums I will throw my general considerations out as one could easily spend a lifetime delving into what specific Kelvin each species of algae prefers.

I vertically light my cultures; this allows me to set up quite a large amount of cultures in a compact space. I use four 4 ft fixtures mounted vertically to the wall in an even spread behind a 4 foot shelf unit. This gives me 4 shelves to set cultures on with each shelf having a standard capacity of 4-6 culture vessels. For lights I use normal output T8 florescent bulbs in the 5400 Kelvin spectrum. I have been known to change spectrums for other species however have found that this is the most versatile (and easily obtained) bulb. Keep cultures a minimum of 2” away from bulbs to avoid heat problems. Light cycle is another arguable variable but I have found a simple 16 on 8 off cycle has been most versatile and productive.

[THEJRC]

Topic Five: Split Timing

Split timing is pretty simple to figure out but can be a thorn in the side for many. The goal here is to keep cultures as dense as possible without allowing them to outcompete themselves. I will avoid a lecture here and recommend that anyone serious about culturing should read the Plankton Culture Manual released by Florida Aqua Farms as it delves into the theories and ideas of keeping cultures in “logarithmic” state for higher nutritional value and cell counts.

When starting cultures, split between 8 and 12 days, it is fine to give the culture a little extra time at this stage. For subsequent splits it is very important to keep as rigid and repeatable schedule as possible to avoid shocking the cultures. Subsequent splits should typically be performed every 8 days, if extended time between a particular split is necessary it is a good idea to add a small additional amount of Guillards to keep the culture from depleting nutrients.

While many preserve harvested cultures through refrigeration or freezing I prefer to utilize the culture as soon as possible to maintain its viability.

[THEJRC]

Broad level: Step by Step guide and additional information

1. Prepare appropriate amount of saltwater to 1.019

2. Put a few drops of the saltwater in the culture starter Petri dish and set for 12-24 hours

3. Fill small (8-16 ounce) vessel with saltwater and add approximately 1ml of Micro Algae Grow

4. Use sterile swab to swab culture from dish into vessel

5. Set vessel on culture rack with air supply at 60-90 circulations per minute

6. Run 16 on /8 off light cycle for culture, visually inspecting color daily

7. On day 10, transfer culture to 1 liter bottle, adding 1ml of Micro Algae Grow and fresh salt water to top off

8. Allow culture to grow for an additional 8 days

9. Transfer from 1 Liter bottle to 1 gallon bottle, adding 3.75ml (4ml is fine) of micro algae grow and topping off with fresh saltwater

10. Let grow for 8 more days

11. Harvest approximately two thirds of the culture, refilling the vessel with fresh salt water and adding 3.75ml of micro algae grow

12. Repeat steps 10 and 11, carefully monitoring vessel, replace vessel every 5 splits or when fouling occurs, replace rigid tubing every time vessel is replaced.

I know it’s long winded and probably glosses over a ton of missed stuff but that’s the gist of how I run my cultures. I do perform some advanced tasks on other cultures such as CO2 injections and working with other strange species with different temperature / lighting / fertilizer requirements but those topics are beyond the scope of this document. If you wish to learn more I would recommend the following (which I use as constant references):

The Plankton Culture Manual – Book by Frank Hoff and Terry Snell from Florida Aqua Farms www.florida-aqua-farms.com

For sources of other algae’s check out the University of Texas algae bank UTEX - The Culture Collection of Algae (http://www.utex.org)

For other supplies (including more algae cultures) head to Carolina Biological Supply Carolina Biological Supply: Science Supplies, AP Kits, Chemistry Supplies, Microscopes (http://www.carolina.com)

For other culture medium formulas, more cultures, as well as a whole wealth of information take a good look at the Provasoli-Guillard National Center for Culture of Marine Phytoplankton http://ccmp.bigelow.org

and my apologies for the thread spam he he!

[sunny d polyp]

Wow JRC, as usual you are of a great asset to these forums, I know you have done much to get me started on the proper techniques of phyto culturing. I still only culture phyto (tet, nano and T.iso) and 2/gals of roti's, but thats more than enough to supply family and friends with phyto and trade a few litres of it a week to the LFS for supplies. I'm still cope challenged a bit, not sure why but the Acartia God was probably going to get me if I crashed any more cultures of it so I stopped to regroup. Thx again for everything you and others have done for the forum.