So you might wonder why after not updating this site for so long I’m putting up a post about Devils Hole pupfish.  Well, they are what have been consuming most of my time.  Besides running AZaquaculture, I’m also a researcher with the USGS Arizona Cooperative Fish and Wildlife Research Unit where I specialize in the captive propagation of endangered fish species, and on the functional genomics of Devils Hole pupfish (Cyprinodon diabolis).  So, instead of more info about breeding marine species, this time I thought I’d share a bit of info about one of my other passions  – the Devils Hole pupfish, and some really cool footage we captured of a mini tsunami in their habitat in Death Valley following a recent earthquake in Baja Mexico.

So, a bit of background about the pupfish:  They are probably the most endangered fish in North America, possibly the world.  Small and silvery blue, Devils Hole pupfish are really an attractive fish that has the smallest geographic distribution of any vertebrate species. Cyprinodon diabolis is found in a 6 foot wide opening in the top of a deep cavern (scuba divers have been down to 440 feet and haven’t seen bottom) that extends into a carbonate aquifer.  The fish live near the limits of their physiological limits.  The water is a near constant 93 degrees Fahrenheit, and often has dissolved oxygen levels of only 2ppm.

Although only about 500 individuals were counted at maximum population levels, the population reached a critical low in 2006 when only 38 fish were counted.  The population has rebounded to about 150 fish, but they are still critically endangered.  No captive populations of the pupfish exist outside of Devils Hole.

While the footage looks like it would be pretty traumatic for the pupfish, it can be beneficial as well.  These occurrences do a nice job sweeping fine silt off of the spawning shelf.

From the USGS press release on the event:

For tiny Devils Hole pupfish, startling video shows it must have felt like a “huge tsunami” when violent water-level oscillations from an earthquake 300 miles away disturbed the small ledge they live on in a single Mojave Desert cavern pool for some 15 minutes.

To see video clips, visit http://gallery.usgs.gov/videos/229

These water-level oscillations in the cavern in Death Valley National Park were caused by the magnitude 7.2 El Mayor – Cucapah earthquake and an immediate aftershock that occurred on April 4.

The video from four U.S. Geological Survey cameras shows significant water-level oscillations, causing great disturbance to the shallow feeding and spawning shelf critical for the continued existence of these fish, said Ambre Chaudoin, a graduate student in fisheries with the USGS Arizona Cooperative Fish and Wildlife Research Unit at the University of Arizona, Tucson.

“The shelf substrate sediment was largely redistributed as a result of the water oscillations,” said Chaudoin. “Such disturbance can be important because the spawning shelf is less than 13 feet long and 7 feet wide, smaller than many walk-in closets.”

Federal and state surveys done within a week after the April 4 earthquakes revealed about 118 individual fish in the pool, an increase from about 70 the year before. Also, biologists saw newly hatched larval fish and evidence that the fish were spawning.

The violent oscillations, though, washed away algae that are essential to the food web of the critically endangered fish, though biologists hope they will grow back quickly. Biologists with the National Park Service, U.S. Fish and Wildlife Service and the Nevada Department of Wildlife will carefully monitor Devils Hole for any harmful after-effects to the fish.

Ambre and fellow USGS researcher Olin Feuerbacher happened to be conducting Devils Hole pupfish behavior surveys on April 4, the day the earthquake struck. They caught the earthquake-induced wave action on video cameras they had just reconnected to their recording position inside the pool 10 minutes before the quake struck.

“The fish begin to move out of the camera’s view as the waves start getting bigger, and then, because of all the sediment being stirred up, you can’t see the fish. As the waves grew stronger, the fish likely moved into deeper waters,” she said.

Paul Barrett, a FWS biologist who leads the Devils Hole Pupfish Recovery Team, said that water-quantity and -quality changes after an earthquake can affect sensitive aquatic environments by changing water levels or by reducing the amount of algae or invertebrates that live on the ledge. But sometimes they can be beneficial as well.

“Earthquakes, such as a 1978 temblor in Mexico, can set up waves that clear the spawning shelf of the algae upon which the pupfish rely, however depending upon the time of year, the algae may regenerate quite rapidly,” said Barrett.  “Furthermore, quakes can serve a useful purpose in shaking silt and other fine particles that have washed into Devils Hole off of the spawning shelf and into the deeper waters.  This frees important space between the substrate particles where the Devils Hole pupfish larvae seek refuge.”

In fact, said Barrett, after the April 4 Mexicali quake, the National Park Service recorded a slight increase in larval abundance as compared to a similar survey a few weeks before the earthquake occurred.

The phenomenon of earthquakes and corresponding seismic effects on water wells, streams, springs, seeps and lakes is well known. Generally, large earthquakes (greater than magnitude 6.0) with epicenters hundreds to thousands of miles away can cause hydrologic responses in water wells and surface-water bodies. Hydrological effects from the April 4 earthquake were also noticed in places as far away as Virginia.

Devils Hole pupfish populations remained about 400-500 individuals until the late 1960s when the water level in the pool dropped in response to pumping of nearby irrigation wells. Pupfish numbers declined precipitously, and though water in Devils Hole is now maintained at a minimum level, the pupfish are still greatly imperiled.  With intensive management efforts, pupfish numbers are increasing from a critical low of just 38 individuals in 2006 to about 118 in the 2010 spring survey after the recent temblor.

Although Chaudoin and USGS researcher Olin Feuerbacher said they would have liked to have seen first-hand the effects of the quake on the hole, they were fortunate in having removed the tiny viewing platform they had been sitting on right above the pool before the quake hit. “Our viewing platform is about 50 feet below ground level, only a few inches above the water surface of the pool,” said Chaudoin. The earthquake caused water to hit our cameras about 4 feet above the platform, so it probably would have been a rather unpleasant experience if we had been on the platform,” she said.

The USGS scientists are using video to help them assess relationships between environmental conditions and spawning in the pupfish to help managers better understand the habitat and spawning requirements and ultimately help in captive propagation. This study is being conducted by the USGS Arizona Fish and Wildlife Cooperative Research Unit and is funded by the U.S. Fish and Wildlife Service, in partnership with the National Park Service and Nevada Department of Wildlife.

About Devils Hole Pupfish

Contained deep within a limestone cavern in the Mojave Desert, Devils Hole is a constant temperature, 10 by 50 foot pool of water that provides a window into the extensive carbonate aquifer within the Amargosa Valley groundwater basin. Devils Hole pupfish live only in Devils Hole, dependent on a tiny spawning shelf less than 13 feet long and 7 feet wide. There, these colorful fish – the males a sparkling blue, the females a more subdued grey-blue or silvery-blue – have made their home for thousands of years near the upper limit of its temperature tolerance – the water remains around 93 degrees — and near the lower limit of its oxygen tolerance. Adult fish average only about one inch in length and are unique among pupfish in lacking pelvic fins.

Each spring, just enough sunlight penetrates Devils Hole to allow food in the form of algae and small invertebrates to grow in the spring. This food supports a new generation of pupfish before the previous year’s fish die of old age. No one knows how deep the pool is, but the pupfish live in the upper 80 feet of the pool.

You can find the press release here

In the first section of this guide, we looked at an overview of the general process of raising clownfish at home.  In the next few sections we will cover broodstock care in more detail, particularly housing, species selection, feeding, and maturation and spawning behaviors.

Most hobbyists don’t get interested in clownfish breeding because they have set out to become clownfish breeders, rather they realize that their clowns have spawned in a display tank and are curious about how to rear the eggs.  In this case, the considerations of species selection and housing are already taken care of.  As we discussed previously, the young will have an abysmal chance of survival left to their own devices in the adult’s tank.  Instead, they will need to be removed to a small larval rearing tank and fed appropriately sized plankton.  If you don’t have your plankton cultures ready, don’t despair, well fed and healthy clownfish pairs will typically spawn every two weeks or so for many years to come.  You will have plenty of opportunities to try your hand at rearing clownfish.

One of the goals of this series is to pass along the tricks and tips we have acquired along the way to make your breeding efforts as simple and as little work as possible.  Here is one tip:  When transferring the larvae from the parental tank, there are two methods – capturing the larvae after they hatch, or moving the nest in its entirety just before hatch.  If you are just planning on raising a hatch or two, then siphoning or other methods of manual capture of the larvae is just fine.  If however, you are planning on raising clownfish on anything beyond a casual basis, it is worth your while to “train” your clownfish to spawn on a removable substrate so you can move the eggs before they hatch.

Training your clownfish to a removable substrate is (usually) a simple endeavor.  It can be as simple as putting a 4 inch clay flowerpot or ceramic tile where the egg mass was deposited after it hatches.  Often, the fish will take to their new substrate right away, but it may take a few tries of  “chasing the spawn” around the tank, placing substrates wherever the breeding clownfish move their spawning site.  Eventually they will settle and accept their new substrate.  If you don’t like the look of tiles or clay pots in your beautiful reef tank, check with your LFS and see if they can provide you with an appropriately sized empty shell from a Tridacna spp. clam.  Clowns love spawning on the concave interiors of these shells and these substrates make for a more natural display.   Larval siphoning and egg transfer methods will be described in further detail later.

Nutrition of the adults plays a critical role in the quality of eggs produced and the survival chances of the juveniles.  Normally in reef tank systems, the adults have a well-balanced and healthy diet.  However, on occasion there will be a nutrient deficiency that leads to poor egg quality.  If you are experiencing difficulty in getting eggs to hatch, or large larval die-off particularly in the first 72 hours, adult nutrition should be considered.  Supplementing the adult clownfish diet with a variety of high quality foods can be a deciding factor in your success as a breeder.  Don’t neglect nutrient export especially on a reef system. Corals will love the extra feedings as much as the clownfish, but a good protein skimmer will help in removing waste products before they can fuel nuisance algae growth.

In the next section we will look at how to set up a dedicated aquarium for clownfish breeding.

Contents

Part 1 – A General Overview of Clownfish Breeding

Part 2 – Broodstock

Part 3 – Larval Care

Part 4 – Plankton Culture

Part 5 – Metamorphosis and Juvenile Growout

One of the most common questions that we get asked at AZaquaculture is “My clownfish just laid eggs – what do I do?”  Well, there are a lot of answers to that question.  What this guide will do is present a simplified version of the methods we use at AZaquaculture to spawn clownfishes and rear their young, while trying to eliminate as many of the energy intensive steps and pitfalls that may put new breeders off of the idea.  These are certainly not the only methods available, but we have found these to be time-tested and allow prospective breeders to still have a life while enjoying successes with marine fish breeding.  We hope that we can help as many hobbyists as possible to “give it a go”.  Many of the advancements in ornamental fish culture industry occur not in laboratories, but in the homes of hobbyists.  By encouraging the home culture of marine fishes, we hope that we can help spur the advancement of exploration in this field while at the same time, reducing the dependence on wild-caught fishes, creating a more eco-friendly and sustainable hobby for all to enjoy.

Breeding Clownfish – An Overview

Rearing clownfish can be a challenging, time-consuming, and sometimes frustrating endeavor, but for those wishing to expand their knowledge and try their hand at clownfish rearing, it can also be immensely rewarding. There is nothing like seeing your first rearing tank fill up with hundreds of tiny versions of their parents.  So, to begin with, we will present an overview of clownfish breeding and what you can expect along the way to that point.

Clownfishes were among the first ornamental species to be commercially bred in the 1980’s.  They remain the most popular marine fish and fortuitously are relatively simple to breed in captivity.  There are several reasons for this; they are demersal spawners (lay their eggs on the bottom or other fixed substrate), and these eggs give rise to comparatively large larvae.  They are also sequential hermaphrodites, meaning that they begin their lives as males, but as they pair up, the larger or more dominant fish will change sexes to become a female.   So, if purchased as juveniles, any two clownfish of the same (or similar) species can become a fertile pair.

Clownfishes typically reach sexual maturity at an age of 8-18 months.  As they approach spawning age, they will vie for dominance, which can include some chasing, fighting, tail nipping, and a general bullying attitude by the victor.  Once dominance has been established, courtship behaviors will commence.  This includes vigorously fanning the bottom as they clean potential spawning sites, side-by-side swimming with the typical clownfish “waggle”, and a decreased (although not eliminated) level of aggression.
Spawning occurs during daylight hours and a small clutch of eggs is laid usually on the underside of a rock or other solid base.  The nest is usually compact, measuring 1-3 inches in diameter, circular, and comprised of small red or orange eggs, each smaller than a match head.  A typical nest may contain 100-300 eggs for the smaller species such as percula, Amphiprion percula, and false percula, Amphiprion ocellaris species, or up to several thousand eggs for maroon clownfish, Premnas biaculeatus .

Clownfish eggs will hatch after dark, 6-9 nights after they are deposited, releasing a swarm of small 3-5mm silvery larvae.  Clownfish fry will not survive in the parental tank.  Filters, corals, and even hungry parents will make short work of these delicate hatchlings.  Newly hatched fry can be moved with a siphon to a larval rearing tank (usually a bare bottomed 10 gallon with only an airstone and heater inside), or the entire nest can be moved to the larval tank on the night of hatch.

Larval clownfish feed on plankton of very specific sizes.  The standard first food for clownfishes is marine rotifers Brachionus plicatilis.  These are tiny gelatinous creatures that have little nutritional value.  Rotifers must be “gut loaded” with microalgae to make them nutritious enough to be used as a clownfish prey item.  Depending on the species of clownfish, larvae will consume rotifers exclusively for the first 4-8 days of its life.  Once the clownfish babies grow large enough, they are transitioned onto a second food, Artemia spp. (brine shrimp).  Atremia are much larger and more nutritious than rotifers.  Artemia are purchased as dry cysts that can be hatched into nauplii and fed to the larvae.  During this time clownfishes can also begin their transition onto frozen and dry feeds.

At an age of 10-14 days clownfishes will undergo metamorphosis.  This is when clownfish “earn their stripes” and will begin to look like real clownfish.  Metamorphosis can be a tough time for the fish, and losses can be heavy if they are not managed carefully.  Once they have made it through metamorphosis, the hard work is over.  Filters can be added and the fish can be completely weaned onto prepared foods.  Then it’s time to relax and watch your new babies grow into a “herd” of clowns.  By an age of four months, clownfish should reach a size that is safe to sell to fish stores, trade with hobbyists, and help to feed your aquarium addiction.

In the next part of this series we will look at selecting your broodstock and preparing a suitable tank for clownfish breeding.  In the meantime, here are some links to additional sources for information on clownfish and marine fish breeding.

Clownfishes by Joyce D. Wilkerson
- A fantastic and thorough guide to keeping and breeding clownfishes.

Conditioning, Spawning and Rearing of Fish With Emphasis on Marine Clownfish by Frank H. Hoff
- For those interested in some deeper science of clownfish culture, or for those interested in opening a commercial hatchery, this is indispensable.

Plankton Culture Manual – Sixth Edition by Frank H. Hoff
- The companion to Hoff’s clownfish book, this covers in great detail the culture of many marine plankton species from microalgae to rotifers, artemia, copepods, and even mysids and daphnids.

Due to extensive out-of-state research commitments this month, we will be unable to accept or proccess orders from the public, although our wholesale operations will continue as normal.  We will begin accepting public orders again Aug 1.  In the meantime we will be posting our new series Breeding Clownfish – A Guide for Hobbyists.  We hope you enjoy it, and we look forward to serving all of your aquaculture needs again this Fall!

With several of our staff presenting at the Western Division of the American Fisheries Society meeting in Albuquerque, we will be shutting down shipments from May 3-9.  As usual, we will be able to answer emails (somewhat slowly perhaps), and take orders, but we will not be able to make any shipments until Monday, May 11.  Thanks for your understanding, and we hope to put up some highlights from the meeting that may benefit the reef tank community!

Thanks to the amazing support of our friends and customers, we have outgrown our current laboratory facilities!  Over the course of the next week we will be moving our operations to a much larger facility.  During this time we will not be shipping orders.  Order shipments will resume April 21.  We appreciate your patience during the move, and look forward to being able to bring you many new and exciting lines of aquacultured organisms as our new systems come online. Thanks again for your support and patience!

Biologists from AZaquaculture will be attending the annual meeting of the Desert Fishes Council in Cuatrociénegas, Coahuila, México.  Only a skeleton staff will remain in Tucson.  As such, we will be closed during the period of November 10-20.  Phones will not be answered, but you can leave a message and we will do our best to get back to you in a timely fashion.  Emails should be answered, but this will be limited by the availability of wi-fi or other internet accessability in Cuatrociénegas, and we won’t know that for sure till we arrive.

Thanks for your patience and understanding, and we look forward to serving your aquaculture needs upon our return!

It seems only fitting that our first creature feature focuses on one of the first marine ornamentals to have been raised successfully in captivity, the Neon Goby, Elacatinus oceanops, (formerly Gobiosoma oceanops). First reared in the 1970’s the neon goby is a popular aquarium fish owing to its general hardiness, attractive appearance, and abilities in picking ectoparasites off of other fishes.

Size and Appearance

Neon gobies are small and typical gobiioid in shape. Most Elacatinus spp. are less than 5cm (2 inches) total length. Neon gobies are black overall with a neon blue stripe extending from front of eye to the base of caudal fin.

Broodstock Care

Omnivorous and hardy they will do well in nearly any species-only or reef aquarium situation, but due to their size should not be kept with larger predatory fishes. Neon gobies do best in water temperatures below 26.5 decrees C (80 degrees F). Foods should include a variety of grated frozen shrimp, squid and fish, as well as commercial gelatin or pellet diets. Multiple feedings daily will condition neon gobies for spawning.

Pair Formation

Hermaphroditic sexual patterns are common in the family Gobiidae. I am not aware of a definitive classification of Elacatinus oceanops, but experience in our lab suggests that they are sequential hermaphrodites rather than simultaneous hermaphrodites.
Males are often larger and more slender. Females will possess a swollen abdomen, particularly when ripe with eggs.

Neon Gobies may be kept as groups of 6 or more individuals when provided with sufficient hiding spots, as these gobies can be quite quarrelsome. If they are to be kept as a pair, they should be observed closely during the first week after introduction. If fighting is excessive, one member of the pair should be swapped until marital harmony ensues. Groups of fewer than 6 individuals are not suggested, as pairs will begin to try to “evict” other gobies in their territory. Larger groups dampen and disperse these aggressive tendencies.

Spawning and Hatching

Spawning can occur as often as every fourteen days with plenty of feeding and warm water conditions. In their natural environment, demersal eggs are laid in small holes and crevices in the reef and under discarded bivalve shells. In captivity, small Tridacna sp shells serve well, as do halved clay flowerpots and short sections of half inch PVC pipe. Both parents tend eggs. Depending on temperature, hatching will commence in 6-8 days. Hatching occurs after dark.

Larval Rearing

Neon goby larvae are slightly shorter and substantially slimmer than clownfish larvae. The larval period ranges from 18-25 days depending on temperature and food type. The first diet is rotifers, followed by Artemia nauplii. The transition period is variable between these foods. Elacatinus larvae can be transitioned to Artemia as early as day 6, and while growth is more rapid, mortality is often high. Waiting until day 12-15 to begin Artemia feedings will delay metamorphosis by a few days, but will also significantly increase survivorship. We have successfully reared batches of neon goby hatchlings through metamorphosis only on rotifers, but metamorphosis took 30-35 days.

A critical factor in the success of a plankton culture system is the proper sterilization of both culture vessels and any solutions that go into them. Such sterilization prevents the overgrowth of the target species with microbial contaminates. Such contaminates may include undesirable bacterial, fungal, and protozoan species. Microbial contaminates may exert deleterious effects on the target species via predation, release of toxins, or through secretion of harmful metabolic byproducts and competition for nutrients and space. Contamination of rotifer and microalgae cultures by certain species of dinoflagellates has been shown to be a major factor in the mortality of clownfish larvae in hatchery settings. At the very least, maintaining a clean and sterile culture system will go a long way in producing reliable production levels of planktonic or larval organisms and will help speed the troubleshooting process when things go wrong.

Below are descriptions of various methods used in aquaculture hatcheries, as well as an explanation of their typical applications, limitations, and in some cases, links to protocols for implementation.

STERILIZATION BY MEMBRANE FILTRATION:

Primarily utilized for small culture volumes, membrane filtration provides a high level of sterility while being extremely gentle to water chemistry. Sterilization is accomplished by forcing liquid through a filter that has a defined pore size, typically either 0.45 or 0.22 microns. This allows for the elimination of bacteria and fungi, which are too large to fit through the pores, without modification of the chemical constituents of the culture media. Effective for bacterial, fungal, and protozoan species, these filters are not generally effective against viruses as these are small enough to pass easily through the pores.

Membrane filters typically come in two flavors:
Syringe filters are handy for sterilizing very small volumes of liquid, less than 100ml. These filters come in convenient disposable cassettes that attach to the tip of syringes. Liquid is forced from the syringe through the filter and the exiting solution is sterilized.
Vacuum filtration setups are effective for larger volumes, up to a liter. These allow the culture media to be pulled through a larger filter membrane via a vacuum pump. The sterilized culture media is collected in a receiver vessel, often a side-arm Erlenmeyer flask. It is important to note that for either of these methods, all downstream vessels and apparatus that contact the sterilized media must be sterile themselves. Thus, it is common to use these methods only for making small stocks of solutions such as f/2 that are sensitive to other forms of sterilization, and may be stored in pre-sterilized, disposable containers.

HEAT STERILIZATION:

Heat sterilization, when properly performed, can be among the best methods of sterilization. However, many desirable constituents of culture media may be temperature sensitive and can be destroyed by heat. Most notable are vitamins, fertilizers, and antibiotic solutions, which are typically filter sterilized and added to heat sterilized media after it cools. In addition to being damaging to additives, high temperatures can cause undesirable precipitation of a variety of constituents of seawater, especially as temperatures approach boiling. Such precipitation may or may not have adverse effects on the culture, depending on the species and conditions that are utilized.

Autoclaving is the most common and effective method for sterilizing moderate amounts of material, especially in a laboratory setting. Requiring specialized equipment, material is heated under pressure in the presence of steam. Given an adequately long exposure time, this is an effective method of destroying bacteria, fungi, spores, and viruses. The most common exposure conditions are 121 degrees C at 15psi. Similar levels of sterility can be attained in a pressure cooker without the expense of an autoclave.
Boiling is a moderately effective method of sterilization. It does a good job of killing most bacteria, viruses, and fungi. However, it often is not successful at destroying the environmentally resistant spores produced by some species. To ensure complete killing of spores, it may be necessary to boil the medium on 2 or 3 consecutive days, allowing the medium to cool between treatments.
Pasteurization is not quite as assured a method for complete destruction of microorganisms as autoclaving, but properly executed can reach nearly the same kill rates without the problems of precipitation that may occur with boiling or autoclaving. Pasteurization can be accomplished by heating the solution to 80 degrees Celsius, allowing the solution to cool naturally, then heating again, generally 24 hours later. This may be repeated a third time for extra safety.

CHEMICAL STERILIZATION

The most economical and convenient method for sterilizing moderate to large volumes of water is chemical sterilization. This is most often accomplished through the addition of strong oxidizing agents such as chlorine, or by dropping the pH below 4 through the use of hydrochloric or muriatic acid. Highly effective, these methods are affected by factors such as temperature, contact time, dissolved organics etc., therefore, sterilization parameters will need to be adjusted in response to these conditions.
It is important to return the pH to normal before using the media if acid-sterilization was employed, and chlorine solutions must be neutralized, generally through additions of sodium thiosulfate. Chemical sterilization may destroy additives such as vitamins, antibiotics, and fertilizers, so sterile stocks of these should be added only after neutralization of the chlorine or acid.

The Easy Bleach Method
For sterilizing seawater of average cleanliness, this protocol will yield water of acceptable sterility and quality for most microalgae (greenwater) and larval culture.

**Be sure to familiarize yourself with proper chemical handling techniques before attempting these procedures. Always wear protective goggles and gloves when handling any chemicals**

1.  Collect a known quantity of water to be sterilized.  If water is turbid, it may be necessary to prefilter for clarity.  This can be accomplished by dripping the water through a few coffee filters.

2.  Add 0.5ml unscented laundry bleach per liter of water.

3.  Seal the vessel and swirl the media so that all internal surfaces are wet.

4.  Allow the vessel to sit at room temperature for at least 6 hours, preferably 12-24.  The vessel may be stored for long period of time with the bleach inside, provided the vessel remains uncontaminated.

5) Prepare a 1M stock solution of Sodium Thiosulfate.  The most commonly available crystalline form is the pentahydrate, and should be added at 248 grams per liter.  This solution may be filter sterilized or autoclaved to ensure sterility.

6.  To de-chlorinate the water, add 0.1ml of 1M sodium thiosulfate per liter of water.

7.  Aerate at least 2 hours.

8.  Confirm that no residual chlorine exists before using the medium.  Dip tests for pools work well, as do the DPD reagent available in convenient liquid or powder pillow-packets.

Acid Sterilization

**If you are not familiar with the safety hazards associated with handling concentrated acids, DO NOT attempt this method**

1. Collect a known quantity of water to be sterilized.  If water is turbid, it may be necessary to prefilter for clarity.  This can be accomplished by dripping the water through a few coffee filters.

2.  Add sufficient hydrochloric acid or muriatic acid to reduce the pH of the solution to 3.6.  The amount needed will vary depending on the concentration of the acid stock, the initial pH of the water, and its buffering capacity, which may also be affected by residual calcium deposits in the vessel.  A good starting point for muriatic acid (about 32% concentration) is 2ml/L.

3.  Confirm pH with a pH meter or narrow range test strips.

4.  Swirl the sterilized solution so that all internal surfaces are moistened.

5.  Allowing a sufficient sterilization time (2-4 hours).

6.  Return the pH to normal with sodium bicarbonate (baking soda).  A good starting point is 1/8 tsp/L.

6.  Confirm pH is appropriate for culture with a pH meter or narrow range test strips.